# Patent

## Title
Thermostable C. bescii enzymes

## Abstract

The disclosure provides thermostable enzymes isolated from Caldicellulosiruptor bescii and fragments thereof useful for the degradation of cellulose and/or hemicellulose, including thermostable cellulases and hemicellulases. The disclosure further provides nucleic acids encoding the thermostable enzymes of the disclosure. The disclosure also provides methods for the conversion of cellulose and hemicellulose into fermentable sugars using thermostable enzymes of the disclosure. The disclosure also provides enzyme cocktails containing multiple enzymes disclosed herein. The enzymes can be used to release sugars present in cellulose or hemicellulose for subsequent fermentation to produce value-added products.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a U.S. National Phase patent application of PCT/US2011/066272, filed Dec. 20, 2011, which claims the benefit of U.S. Provisional Application No. 61/425,623, filed Dec. 21, 2010, and U.S. Provisional Application No. 61/532,060, filed Sep. 7, 2011, both of which are hereby incorporated by reference in their entirety.


FIELD
The present disclosure relates to compositions and methods for the degradation of cellulose, hemicellulose, and cellulose and/or hemicellulose-containing materials. In particular, the disclosure provides thermostable enzymes for the degradation of cellulose, nucleic acids encoding the enzymes, and methods of use thereof. The disclosure also provides thermostable enzymes for the degradation of hemicellulose, nucleic acids encoding the enzymes, and methods of use thereof. The disclosure further provides thermostable enzymes that enhance the activity of thermostable cellulase and/or hemicellulases, nucleic acids encoding the enzymes, and methods of use thereof.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 335032000700SubSeqListing.txt, date recorded: Feb. 25, 2016, size: 524 KB).
BACKGROUND
Microorganisms that are currently being used to ferment sugars to biofuels such as ethanol usually cannot utilize complex polysaccharides such as cellulose and hemicellulose. As a result, a significant bottleneck occurs in the conversion of lignocellulosic materials to biofuels.
Cellulose, a major component of plants and one of the most abundant organic compounds on earth, is a polysaccharide composed on long chains of β(1-4) linked D-glucose molecules. Due to its sugar-based composition, cellulose is a rich potential source material for the production of biofuels. For example, sugars from cellulose may be fermented into biofuels such as ethanol. In order for the sugars within cellulose to be used for the production of biofuels or other commodity chemicals, the cellulose must be broken down into smaller molecules.
Cellulose may be enzymatically hydrolyzed by the action of cellulases. Cellulases include endoglucanases, exoglucanases, and beta-glucosidases. The actions of cellulases cleave the 1-4 β-D-glycosidic linkages in cellulose, and result in the ultimate release of β-D-glucose molecules. During the breakdown of cellulose into individual sugar molecules, glucose polymers of various lengths may be formed as intermediate breakdown products. Glucose polymers of approximately 2-6 molecules in length derived from the hydrolysis of cellulose are referred to as “cellodextrins” or “cellooligosaccharides.”
Hemicellulose constitutes the second largest component of polysaccharides in many plants, such as the perennial grasses switchgrass and Miscanthus. Hemicellulose is a complex polysaccharide that has a xylose-linked backbone, with side chains of arabinose, glucuronyl, and acetyl groups. A structural model of a hemicellulose illustrates the xylose backbone residues joined together in beta-1,4-linkages (FIG. 1). Several functional groups decorate the backbone, including esters of acetyl (Ac) groups, arabinose, glucuronic acids, and esters of feroryl groups. The feroryl groups link the entire structure to lignin. Enzyme cocktails that hydrolyze hemicellulose into its major component sugars such as xylose (a 5-carbon sugar) and arabinose (a 5-carbon sugar) will significantly increase the fermentable sugars for biofuel production from lignocellulose-based feedstock. Enzymatic removal of hemicellulose by hemicellulases will also increase accessibility of cellulases to the cellulose component of plant cell walls or lignocellulosic feedstocks. Thus, the degradation of hemicellulose is a critical step in the utilization of lignocellulose feedstock for biofuel production.
Thermostable enzymes are particularly desirable for the efficient degradation of cellulose and hemicellulose, because thermostable enzymes are more compatible than non-thermostable enzymes with other processes involved in converting lignocellulose-based materials into biofuels. For example, treatments of lignocellulose-based materials to decrease the crystallinity of cellulose may require high temperatures that inactivate non-thermostable enzymes.
In addition, thermostable enzymes are desirable for the degradation of cellulose and/or hemicellulose because they may have a higher specific activity as compared to their mesophilic counterparts, and because they can operate at high temperatures that reduce or eliminate the risk of microbial contamination.
Accordingly, there is a need for thermostable enzymes and enzyme cocktails capable of degrading cellulose and/or hemicellulose.
BRIEF SUMMARY
This disclosure provides enzymes and enzyme cocktails which satisfy the need for thermostable enzymes capable of degrading cellulose and/or hemicellulose. In some aspects, the disclosure provides enzymes having cellulase activity. In some aspects, the disclosure provides truncated enzymes having cellulase activity. In some aspects, the disclosure provides improved enzyme mixtures for the degradation of cellulose-containing materials. In some aspects, the disclosure provides enzymes having hemicellulase activity. In some aspects, the disclosure provides improved enzyme mixtures for the degradation of hemicellulose-containing materials. The disclosure further provides enzyme cocktails containing one or more cellulases and one or more hemicellulases with improved activity on materials containing both cellulose and hemicellulose, wherein cellulase and hemicellulase mixtures have synergistic activity. The disclosure further provides polypeptides that enhance the activity of enzymes having cellulase or hemicellulase activity, and/or mixtures thereof. The disclosure further provides nucleotide sequences encoding the polypeptides disclosed herein. The polypeptides disclosed herein can be utilized alone, in combination, or with other enzymes.
In one embodiment, the disclosure provides a host cell, comprising two or more recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38. In another embodiment, a host cell comprising three recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38 is provided. In another embodiment, a host cell comprising four recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38 is provided. In another embodiment, a host cell comprising five recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38. In another embodiment, a host cell comprising six recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38 is provided.
In another embodiment, the disclosure provides a host cell, comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide].
In another embodiment, the disclosure provides a host cell, comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide], wherein the host cell further comprises one or more recombinant nucleic acids encoding one or more cellulases.
In another embodiment, the disclosure provides a host cell comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide], wherein the host cell is selected from the group consisting of Escherichia spp., Pseudomonas spp., Proteus spp., Ralstonia spp., Streptomyces spp., Staphylococcus spp., Lactococcus spp., Bacillus spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Aspergillus spp., Chrysosporium lucknowense, or Trichoderma reesei. 
In another embodiment, the disclosure provides a method for producing at least two of the enzymes selected from the group consisting of endoxylanase, α-arabinofuranosidase, α-glucuronidase, β-xylosidase, and acetyl xylan esterase, comprising: culturing a host cell comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide] in a culture medium, under suitable conditions to produce the endoxylanase, α-arabinofuranosidase, α-glucuronidase, β-xylosidase, and acetyl xylan esterase.
In another embodiment, the disclosure provides a host cell, comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide] and culture medium.
In another embodiment, the disclosure provides a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37.
In another embodiment, the disclosure provides a composition comprising six recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37.
In another embodiment, the disclosure provides a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein the composition further comprises one or more recombinant cellulases.
In yet another embodiment, the disclosure provides a method of converting biomass to fermentation product comprising contacting the biomass with a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.
In another embodiment, the disclosure provides a method of converting biomass to fermentation product comprising contacting the biomass with a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product, and wherein the biomass is subjected to pretreatment prior to being contacted with the composition comprising two or more recombinant proteins, wherein the pretreatment comprises one or more of the treatments selected from the group consisting of: ammonia fiber expansion (AFEX), steam explosion, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid.
In another embodiment, the disclosure provides a method of converting biomass to fermentation product comprising contacting the biomass with a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product, wherein the biomass comprises a plant material.
In another embodiment, the disclosure provides a method of converting biomass to fermentation product comprising contacting the biomass with a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product, wherein the biomass comprises a plant material selected from the group consisting of Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine, birch, willow, aspen, poplar wood, and energy cane.
In another embodiment, the disclosure provides a method of converting biomass to fuel comprising contacting the biomass with the composition a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel.
In another embodiment, the disclosure provides a method of converting biomass to fuel comprising contacting the biomass with the composition a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel, and wherein the biomass is subjected to pretreatment prior to being contacted with the composition comprising two or more recombinant proteins, wherein the pretreatment comprises one or more of the treatments selected from the group consisting of: ammonia fiber expansion (AFEX), steam explosion, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid.
In another embodiment, the disclosure provides a method of converting biomass to fuel comprising contacting the biomass with the composition a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel, wherein the biomass comprises a plant material.
In another embodiment, the disclosure provides a method of converting biomass to fuel comprising contacting the biomass with the composition a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel, wherein the biomass comprises a plant material selected from the group consisting of Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine, birch, willow, aspen, poplar wood, and energy cane.
In another embodiment, the disclosure provides a method of degrading biomass comprising contacting the biomass with the composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution.
In another embodiment, the disclosure provides a method of degrading biomass comprising contacting the biomass with the composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution, and wherein the biomass is subjected to pretreatment prior to being contacted with the composition comprising two or more recombinant proteins, wherein the pretreatment comprises one or more of the treatments selected from the group consisting of: ammonia fiber expansion (AFEX), steam explosion, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid.
In another embodiment, the disclosure provides a method of degrading biomass comprising contacting the biomass with the composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution, wherein the biomass comprises a plant material.
In another embodiment, the disclosure provides a method of degrading biomass comprising contacting the biomass with the composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution, wherein the biomass comprises a plant material selected from the group consisting of Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine, birch, willow, aspen, poplar wood, and energy cane.
In yet another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said one or more functional groups are selected from the group consisting of arabinose, glucuronyl, and acetyl.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said treating is conducted at a temperature between 40 and 80° C.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said treating is conducted at a temperature between 60 and 80° C.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with a transgenic host cell that secretes two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with a transgenic host cell that secretes two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said one or more functional groups are selected from the group consisting of arabinose, glucuronyl, and acetyl.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with a transgenic host cell that secretes two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said treating is conducted at a temperature between 40 and 80° C.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with a transgenic host cell that secretes two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said treating is conducted at a temperature between 60 and 80° C.
In another embodiment, the disclosure provides a host cell, comprising two or more recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38, wherein at least one of the two or more recombinant nucleic acids is selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 8, 14, 20, 28, and 34. In another embodiment, a host cell comprising three recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38, wherein at least two of the three recombinant nucleic acids are selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 8, 14, 20, 28, and 34, is provided. In another embodiment, a host cell comprising four recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38, wherein at least three of the four recombinant nucleic acids are selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 8, 14, 20, 28, and 34, is provided. In another embodiment, a host cell comprising five recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38, wherein at least four of the five recombinant nucleic acids are selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 8, 14, 20, 28, and 34, is provided In another embodiment, a host cell comprising six recombinant nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38, wherein at least five of the six recombinant nucleic acids are selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 8, 14, 20, 28, and 34, is provided.
In another embodiment, the disclosure provides a host cell, comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide], wherein at least one of the two or more recombinant nucleic acids is selected from the group consisting of: a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], and a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)].
In another embodiment, the disclosure provides a host cell, comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide], wherein at least one of the two or more recombinant nucleic acids is selected from the group consisting of: a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], and a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], and wherein the host cell further comprises one or more recombinant nucleic acids encoding one or more cellulases.
In another embodiment, the disclosure provides a host cell comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide], wherein at least one of the two or more recombinant nucleic acids is selected from the group consisting of: a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], and a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], and wherein the host cell is selected from the group consisting of Escherichia spp., Pseudomonas spp., Proteus spp., Ralstonia spp., Streptomyces spp., Staphylococcus spp., Lactococcus spp., Bacillus spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Aspergillus spp., Chrysosporium lucknowense, or Trichoderma reesei. 
In another embodiment, the disclosure provides a method for producing at least two of the enzymes selected from the group consisting of endoxylanase, α-arabinofuranosidase, α-glucuronidase, β-xylosidase, and acetyl xylan esterase, comprising: culturing a host cell comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide], wherein at least one of the two or more recombinant nucleic acids is selected from the group consisting of: a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], and a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)] in a culture medium, under suitable conditions to produce the endoxylanase, α-arabinofuranosidase, α-glucuronidase, β-xylosidase, and acetyl xylan esterase.
In another embodiment, the disclosure provides a host cell, comprising two or more recombinant nucleic acids selected from the group consisting of: a) a nucleic acid encoding the polypeptide of SEQ ID NO: 3 [Caldicellulosiruptor bescii endoxylanase (Cb193)], b) a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], c) a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], d) a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], e) a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], f) a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], g) a nucleic acid encoding the polypeptide of SEQ ID NO: 37 [Caldicellulosiruptor bescii endoxylanase (Cb193) lacking signal peptide], wherein at least one of the two or more recombinant nucleic acids is selected from the group consisting of: a nucleic acid encoding the polypeptide of SEQ ID NO: 7 [Caldicellulosiruptor bescii endoxylanase (Cb195)], a nucleic acid encoding the polypeptide of SEQ ID NO: 13 [Caldicellulosiruptor bescii α-arabinofuranosidase (Cb1172)], a nucleic acid encoding the polypeptide of SEQ ID NO: 19 [Caldicellulosiruptor bescii α-glucuronidase (Cb909)], a nucleic acid encoding the polypeptide of SEQ ID NO: 27 [Caldicellulosiruptor bescii β-xylosidase (Cb2487)], and a nucleic acid encoding the polypeptide of SEQ ID NO: 33 [Caldicellulosiruptor bescii acetyl xylan esterase (Cb162)], and culture medium.
In another embodiment, the disclosure provides a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33.
In another embodiment, the disclosure provides a composition comprising six recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein five of the six recombinant proteins are selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33.
In another embodiment, the disclosure provides a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition further comprises one or more recombinant cellulases.
In yet another embodiment, the disclosure provides a method of converting biomass to fermentation product comprising contacting the biomass with a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.
In another embodiment, the disclosure provides a method of converting biomass to fermentation product comprising contacting the biomass with a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product, and wherein the biomass is subjected to pretreatment prior to being contacted with the composition comprising two or more recombinant proteins, wherein the pretreatment comprises one or more of the treatments selected from the group consisting of: ammonia fiber expansion (AFEX), steam explosion, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid.
In another embodiment, the disclosure provides a method of converting biomass to fermentation product comprising contacting the biomass with a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product, wherein the biomass comprises a plant material.
In another embodiment, the disclosure provides a method of converting biomass to fermentation product comprising contacting the biomass with a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product, wherein the biomass comprises a plant material selected from the group consisting of Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine, birch, willow, aspen, poplar wood, and energy cane.
In another embodiment, the disclosure provides a method of converting biomass to fuel comprising contacting the biomass with the composition a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel.
In another embodiment, the disclosure provides a method of converting biomass to fuel comprising contacting the biomass with the composition a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel, and wherein the biomass is subjected to pretreatment prior to being contacted with the composition comprising two or more recombinant proteins, wherein the pretreatment comprises one or more of the treatments selected from the group consisting of: ammonia fiber expansion (AFEX), steam explosion, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid.
In another embodiment, the disclosure provides a method of converting biomass to fuel comprising contacting the biomass with the composition a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel, wherein the biomass comprises a plant material.
In another embodiment, the disclosure provides a method of converting biomass to fuel comprising contacting the biomass with the composition a composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution; and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fuel, wherein the biomass comprises a plant material selected from the group consisting of Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine, birch, willow, aspen, poplar wood, and energy cane.
In another embodiment, the disclosure provides a method of degrading biomass comprising contacting the biomass with the composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution.
In another embodiment, the disclosure provides a method of degrading biomass comprising contacting the biomass with the composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution, and wherein the biomass is subjected to pretreatment prior to being contacted with the composition comprising two or more recombinant proteins, wherein the pretreatment comprises one or more of the treatments selected from the group consisting of: ammonia fiber expansion (AFEX), steam explosion, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid.
In another embodiment, the disclosure provides a method of degrading biomass comprising contacting the biomass with the composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution, wherein the biomass comprises a plant material.
In another embodiment, the disclosure provides a method of degrading biomass comprising contacting the biomass with the composition comprising two or more recombinant proteins, the recombinant proteins selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 3, 7, 13, 19, 27, 33 and 37, wherein at least one of the two or more recombinant proteins is selected from the group consisting of the polypeptide sequences of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein the composition may further comprise one or more recombinant cellulases, to yield a sugar solution, wherein the biomass comprises a plant material selected from the group consisting of Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine, birch, willow, aspen, poplar wood, and energy cane.
In yet another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein at least one of the two or more enzymes is selected from the group consisting of the polypeptide of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein at least one of the two or more enzymes is selected from the group consisting of the polypeptide of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said one or more functional groups are selected from the group consisting of arabinose, glucuronyl, and acetyl.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein at least one of the two or more enzymes is selected from the group consisting of the polypeptide of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said treating is conducted at a temperature between 40 and 80° C.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein at least one of the two or more enzymes is selected from the group consisting of the polypeptide of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said treating is conducted at a temperature between 60 and 80° C.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with a transgenic host cell that secretes two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein at least one of the two or more enzymes is selected from the group consisting of the polypeptide of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with a transgenic host cell that secretes two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein at least one of the two or more enzymes is selected from the group consisting of the polypeptide of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said one or more functional groups are selected from the group consisting of arabinose, glucuronyl, and acetyl.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with a transgenic host cell that secretes two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein at least one of the two or more enzymes is selected from the group consisting of the polypeptide of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said treating is conducted at a temperature between 40 and 80° C.
In another embodiment, the disclosure provides a method for degrading hemicellulose, said method comprising the steps of: a) providing plant material comprising hemicellulose, wherein said hemicellulose comprises a xylose backbone comprising β-1,4-linkages and one or more functional groups; and b) treating said hemicellulose with a transgenic host cell that secretes two or more enzymes selected from the group consisting of the polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, wherein at least one of the two or more enzymes is selected from the group consisting of the polypeptide of SEQ ID NOs: 7, 13, 19, 27, and 33, and wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose, wherein said treating is conducted at a temperature between 60 and 80° C.
In one aspect, provided herein is a host cell containing one, two, three, four, five, six or more recombinant nucleic acids, wherein the recombinant nucleic acids encode one, two, three, four, five, or six polypeptides selected from: Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides.
In another aspect, provided herein is a host cell containing one, two, three, four, five, six or more recombinant nucleic acids, wherein the recombinant nucleic acids encode one, two, three, four, five, or six polypeptides selected from: Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides, and wherein the Cb1952 polypeptide has a sequence selected from SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide has a sequence selected from SEQ ID NOs: 60, 61, and 111. wherein the Cb1954 polypeptide has a sequence selected from SEQ ID NOs: 74, 121, and 76; wherein the Cb1946 polypeptide has a sequence selected from SEQ ID NOs: 86, 87, and 113; wherein the Cb629 polypeptide has a sequence selected from SEQ ID NOs: 98, 119, and 100; and wherein the Cb486 polypeptide has a sequence of SEQ ID NO: 106.
Also provided herein is a host cell containing one, two, three, four, five, six or more recombinant nucleic acids, wherein the recombinant nucleic acids encode one, two, three, four, five, or six polypeptides selected from polypeptides having the sequence of: SEQ ID NO: 46, 111, 76, 113, 100, and 106.
Also provided herein is a host cell containing one, two, three, four, five, six or more recombinant nucleic acids, wherein the recombinant nucleic acids encode one, two, three, four, five, or six polypeptides selected from: Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides, and wherein the recombinant nucleic acid encoding a Cb1952 polypeptide has a sequence selected from SEQ ID NOs: 45, 115, 125, 127, 129, and 47; wherein the recombinant nucleic acid encoding a Cb1953 polypeptide has a sequence selected from SEQ ID NOs: 62, 63, and 110; wherein the recombinant nucleic acid encoding a Cb1954 polypeptide has a sequence selected from SEQ ID NOs: 116, 75, and 77; wherein the recombinant nucleic acid encoding a Cb1946 polypeptide has a sequence selected from SEQ ID NOs: 88, 89, and 112; wherein the recombinant nucleic acid encoding a Cb629 polypeptide has a sequence selected from SEQ ID NOs: 99, 120, and 101; and, wherein the recombinant nucleic acid encoding a Cb486 polypeptide has the sequence of SEQ ID NO: 107.
Also provided herein is a host cell containing one, two, three, four, five, six, or more recombinant nucleic acids, wherein the recombinant nucleic acids encode one, two, three, four, five, or six polypeptides selected from: Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides, and wherein the recombinant nucleic acids have a sequence selected from SEQ ID NOs: 47, 110, 77, 112, 101, and 107.
Also provided herein host cell containing six recombinant nucleic acids, wherein the nucleic acids have the sequences of SEQ ID NOs: 47, 110, 77, 112, 101, and 107.
Any of the host cells provided herein may also contain one or more recombinant nucleic acids encoding a hemicellulase, wherein the hemicellulase has a sequence selected from SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37. In some aspects, a nucleic acid encoding a hemicellulase has a sequence selected from SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38. In some aspects, host cells provided herein may contain recombinant nucleic acids having the sequences of SEQ ID NOs: 8, 14, 20, 28, 34, and 38, or recombinant nucleic acids having the sequences of SEQ ID NOs: 8, 14, 20, 28, and 38.
Further provided herein is a composition containing one, two, three, four, five, six, or more recombinant polypeptides, wherein the recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides.
In another aspect, provided herein is a composition containing one, two, three, four, five, six, or more recombinant polypeptides, wherein the recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides, and wherein the Cb1952 polypeptide has a sequence selected from SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide has a sequence selected from SEQ ID NOs: 60, 61, and 111. wherein the Cb1954 polypeptide has a sequence selected from SEQ ID NOs: 74, 121, and 76; wherein the Cb1946 polypeptide has a sequence selected from SEQ ID NOs: 86, 87, and 113; wherein the Cb629 polypeptide has a sequence selected from SEQ ID NOs: 98, 119, and 100; and wherein the Cb486 polypeptide has a sequence of SEQ ID NO: 106.
Also provided herein is a composition containing one, two, three, four, five, six, or more recombinant polypeptides, wherein the recombinant polypeptides have a sequence selected from SEQ ID NOs: 46, 111, 76, 113, 100, and 106.
Also provided herein is a composition containing six recombinant polypeptides, wherein the recombinant polypeptides have the sequences of SEQ ID NOs: 46, 111, 76, 113, 100, and 106.
Also provided herein is a composition containing one or more recombinant polypeptides, wherein the one or more recombinant polypeptides are selected from the group consisting of the polypeptides of SEQ ID NOs: 46, 111, 76, 113, 124, 126, 128, and 100.
Any of the compositions provided herein may also contain one or more hemicellulase polypeptides, wherein the hemicellulase has a sequence selected from SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37. In some aspects, compositions provided herein contain polypeptides having the sequences of SEQ ID NOs: 7, 13, 19, 27, 33, and 37 or polypeptides having the sequences of SEQ ID NOs: 7, 13, 19, 27, and 37.
In another aspect, provided herein is a method for producing one or more cellulases, the method including: a) culturing any of the host cells disclosed herein which contain one or more recombinant nucleic acids encoding one or more Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides, in culture media under conditions sufficient to support the expression of the recombinant nucleic acid(s), and collecting one or more cellulases from said media and/or said host cell.
In another aspect, provided herein is a method for degrading a cellulose-containing material, the method including: a) contacting the cellulose-containing material with any host cell or composition disclosed herein, and, b) incubating the host cell or composition and cellulose-containing material under conditions that support cellulose degradation.
Cellulose-containing material may be pretreated prior to being contacted with a composition or host cell disclosed herein. Pre-treatment steps may include one or more of the treatments of: ammonia fiber expansion (AFEX), steam explosion, treatment with alkaline aqueous solutions, treatment with acidic solutions, treatment with organic solvents, treatment with high pressure, treatment with high temperature, treatment with ionic liquids (IL), treatment with electrolyzed water, and treatment with phosphoric acid.
Also provided herein is a method of reducing the viscosity of a pretreated cellulose-containing material, the method including contacting pretreated cellulose-containing material with any host cell or composition provided herein.
Also provided herein is a method of converting a cellulose-containing material to fermentation product, the method including: a) contacting the cellulose-containing material with any host cell or composition provided herein, to yield a sugar solution, and culturing the sugar solution with a fermentative microorganism under conditions sufficient to produce a fermentation product.
Also provided herein is a method for degrading a cellulose-containing material, the method including: a) contacting the cellulose-containing material with one or more polypeptides selected from SEQ ID NOs: 46, 111, 76, 113, 124, 126, 128, and 100, and b) incubating the one or more polypeptides and cellulose-containing material under conditions that support cellulose degradation.
In some aspects, cellulose-containing material provided herein is a plant material. Plant material may include, without limitation, Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, rye hulls, wheat hulls, sugarcane bagasse, copra meal, copra pellets, palm kernel meal, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine wood, birch wood, willow wood, aspen wood, poplar wood, energy cane, waste paper, sawdust, forestry wastes, waste paper, and crop residues.
In some aspects, at least a portion of any of the methods provided herein may be conducted at a temperature above 50° C. In some aspects, at least a portion of any of the methods provided herein may be conducted at a temperature between 40° and 80°, 50° and 80°, 60° and 80°, 70° and 80°, 45° and 55°, 50° and 60°, 55° and 65°, 60° and 70°, 65° and 75°, 75° and 85°, or 80° and 90° C.
In some aspects, in any host cells disclosed herein that contain two or more recombinant nucleic acids, two or more of the recombinant nucleic acids may be present in a contiguous polydeoxyribonucleotide chain.
In any of the compositions or methods above, a Cb1581 polypeptide may be provided in the composition or the method with the cellulases and/or hemicellulases. In some aspects, the Cb1581 polypeptide is a polypeptide containing the sequence of SEQ ID NO: 146. Also provided herein are any of the above host cells that further contain a nucleic acid encoding a Cb1581 polypeptide. In some aspects, a nucleic acid encoding a Cb1581 polypeptide is a nucleic acid containing the sequence of SEQ ID NO: 147.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a model of a typical hemicellulose such as xylan.
FIGS. 2A to 2E: FIG. 2A shows the putative domain architecture of the Cb193 and Cb195 proteins. FIG. 2B shows an SDS-PAGE of purified Cb193 and Cb195; the molecular markers are in the lane marked M. The proteins were purified by metal affinity chromatography, followed by ion exchange chromatography and then gel filtration. The predicted molecular masses of Cb193 and Cb195 were 77.7 kDa and 42.0 kDa, respectively. FIG. 2C shows the enzymatic activity of Cb193 on natural substrates using TLC analysis. Various substrates were tested: soluble wheat arabinoxylan (SWAX), oat-spelt xylan (OSX), Birchwood xylan (BWX), carboxymethyl cellulose (CMC), lichenan, glucomannan, 1,4 β-mannan, and arabinan. In the case of SWAX, OSX, and BWX, in the presence of Cb193 (+), short xylose chains were released. In the minus (−) lanes, no enzyme was added and therefore no products of hydrolysis were released. X1 (xylose monomer), X2 (xylose dimer or a disaccharide), X3 (trisaccharide), X4 (tetrasaccharide), and pentasaccharide (X5) were loaded in the first lane (M) as markers. The results showed that this enzyme releases shorter chains or oligosaccharides from the complex substrates (SWAX, OSX, and BWX). FIG. 2D shows the enzymatic activity of Cb195 on natural substrates using TLC analysis. Various substrates were tested: SWAX, OSX, BWX, CMC, lichenan, glucomannan, 1,4 β-mannan, and arabinan. In the case of SWAX, OSX, and BWX, in the presence of Cb195 (+), short xylose chains were released. In the minus (−) lanes, no enzyme was added and therefore no products of hydrolysis were released. X1 (xylose monomer), X2 (xylose dimer or a disaccharide), X3 (trisaccharide), X4 (tetrasaccharide), and pentasaccharide (X5) were loaded in the first lane (M) as markers. The results showed that this enzyme releases shorter chains or oligosaccharides from the complex substrates (SWAX, OSX, and BWX). FIG. 2E shows the enzymatic activity of Cb193 and Cb195 on natural substrates from a reducing sugar assay. In this experiment, a different assay for reducing sugars was used to determine the release of products from the substrates. A standard was made based on known glucose concentrations and their absorbance (color development) in the presence of para-hydroxy-benzoic acid hydrazide (Cann et al. 1999. J. Bacterial. 181:1643-1651 and other reference above-Laver, M. 1972.). Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents.
FIGS. 3A and 3B: FIG. 3A shows the thermostability of Cb193, and FIG. 3B shows the thermostability of Cb195. 5 nM of Cb193 and Cb195 were incubated at different temperatures ranging from 65 ̃90° C. For Cb193, the enzymes were incubated at 70° C., 75° C., 80° C., 85° C., and 90° C.; for Cb195, the enzymes were incubated at 65° C., 70° C., 75° C., and 80° C. The incubated enzymes were taken out at certain time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h, 16 h, and 24 h) as indicated, and immediately incubated with wheat arabinoxylan (final 1%, w/v) to measure the enzyme activity. The initial velocity of reaction was calculated. The residue activity (%) was calculated by dividing the activity of each samples by the initial activity at zero time. Bars are shown with standard errors for three independent experiments.
FIGS. 4AA to 4BC show the kinetic data of Cb193 on hydrolysis of wheat arabinoxylan (FIGS. 4AA and 4BA), oat spelt xylan (FIGS. 4AB and 4BB), and birchwood xylan (FIGS. 4AC and 4BC). The Km, kcat, and kcat/Km are indicated as well. In FIGS. 4AA, 4AB, and 4AC, the experiment was conducted at 75° C. with 50 mM citrate buffer (pH 6.0). In FIGS. 4BA, 4BB, and 4BC the experiment was conducted at 85° C. with 50 mM citrate buffer (pH 6.0). Xylan substrates (final 2.5-50 mg/mL) were incubated with Cb193 (final 5 nM for wheat arabinoxylan and final 50 nM for oat spelt xylan and birchwood xylan). The initial velocity of reaction was calculated. The initial velocities were then plotted against the concentrations of xylan substrates. The Km and kcat were calculated by non-linear fit using the Graphpad software. Bars are shown with standard errors for three independent experiments.
FIGS. 5AA to 5BC show the kinetic data of Cb195 on hydrolysis of wheat arabinoxylan (FIGS. 5AA and 5BA), oat spelt xylan (FIGS. 5AB and 5BB), and birchwood xylan (FIGS. 5AC and 5BC). The Km, kcat, and kcat/Km are indicated as well. In FIGS. 5AA, 5AB, and 5AC, the experiment was conducted at 75° C. with 50 mM citrate buffer (pH 6.0). In FIGS. 5BA, 5BB, and 5BC, the experiment was conducted at 75° C. with 50 mM sodium phosphate buffer (pH 6.5). Xylan substrates (final 2.5-50 mg/mL) were incubated with Cb195 (final 5 nM for wheat arabinoxylan and final 50 nM for oat spelt xylan and birchwood xylan). The initial velocity of reaction was calculated. The initial velocities were then plotted against the concentrations of xylan substrates. The Km and kcat were calculated by non-linear fit using the Graphpad software. Bars are shown with standard errors for three independent experiments.
FIGS. 6A to 6E: FIG. 6A shows an SDS-PAGE of purified Cb1172. FIG. 6B shows the enzymatic activity of Cb1172 on natural substrates from a reducing sugar assay. Five different hemicellulosic substrates were tested: arabinan (sugar beet), SWAX, rye arabinoxylan (RAX), OSX and debranched arabinan. Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of arabinose equivalents. Hydrolysis of arabinan was higher than hydrolysis of other natural substrates. FIG. 6C shows the enzymatic activity of Cb1172 on natural substrates using HPLC analysis. Five different hemicellulosic substrates were tested: arabinan (sugar beet), SWAX, RAX, OSX and debranched arabinan. In each case, in the presence of Cb1172, arabinose was released. In the absence of Cb1172, only minor amount of arabinose was observed for debranched arabinan; no products of hydrolysis were released for other natural polysaccharides. The results showed that this enzyme releases arabinose from complex substrates (arabinan, SWAX, RAX, OSX and debranched arabinan). FIG. 6D shows the domain architecture of the Cb1172 protein; it has a glycoside hydrolase (GH) family 51 catalytic domain. FIG. 6E shows the thermostability of Cb1172. Cb1172 has 57%, 45%, 35% and 22% activity after incubation at 70° C., 75° C., 80° C. and 85° C. for 24 h, respectively. Fifty nM Cb1172 was kept at different temperatures (70° C., 75° C., 80° C., 85° C. and 90° C.). The samples were taken out at the following time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately applied to enzyme activity measurement.
FIGS. 7A and 7B show the kinetic data of Cb1172 on hydrolysis of pNP-α-L-arabinofuranoside. The Km, kcat, and kcat/Km are indicated as well. In FIG. 7A, the experiment was conducted at 90° C.; in FIG. 7B, the experiment was conducted at 75° C. One hundred μl pNP-α-L-arabinofuranoside substrate of different concentrations was kept at 85° C. for three minutes to equilibrate. Then twenty five μl of the protein sample (fifty nM) was added to the substrate and mixed by pipetting up and down for several times. The optical density at 400 nm was recorded by a Cary 300 UV-Visible spectrophotometer for 2.5 minutes. The initial velocity of reaction in the first minute was calculated. The initial velocities were then plotted against the concentrations of pNP-α-L-arabinofuranoside. The Km and kcat were calculated by non-linear fit using the Graphpad software.
FIGS. 8A to 8EC: FIG. 8A shows putative domain architecture of Cb909. FIG. 8B shows SDS-PAGE of purified Cb909. FIG. 8C shows the activity of Cb909. The substrate is aldouronic acids, that is a mixture of xylo-oligosaccharides decorated with MeGlcA. After incubation with Cb909 at 75° C. for 60 minutes, MeGlcA group was cleaved by Cb909 from aldouronic acids to release undecorated xylose, xylobiose, xylotriose and xylotetraose as products. The condition of the reaction was as follows: 6 nM Cb909, 50 mM Phosphate buffer pH 6.0, 150 mM NaCl, 1 mg/ml aldouronic acids. FIGS. 8DA, 8DB, and 8DC show the results of a pH optimization assay. The maximum activity was detected at pH 5.5. This assay was carried out as follows: 1 mg/ml aldouronic acids solution was incubated with 6 nM Cb909 for 10 minutes at 75° C. at each pH. 50 mM citrate buffer containing 150 mM NaCl was used in the range from pH 5 to pH 6.50 mM phosphate buffer containing 150 mM NaCl was used in the range of pH 6 to pH 7. After the reaction, the temperature was quickly increased to 100° C. to terminate the reaction. The amounts of products were detected by HPLC. FIGS. 8EA, 8EB, and 8EC show the results of optimum temperature assay. The maximum activity of Cb909 was detected at 75° C. (xylobiose and xylotriose). Xylose was produced most efficiently at 70° C. but the amounts of produced xylose at 70° C. and 75° C. were almost the same. This assay was carried out as follows: 1 mg/ml aldouronic acids solution was incubated with 6 nM Cb909 for 10 minutes in 50 mM citrate buffer pH 5.5 that contained 150 mM NaCl. After the reaction the temperature was quickly increased to 100° C. to terminate the reaction. The amounts of products were detected by HPLC.
FIGS. 9A to 9H: FIG. 9A shows the putative domain architecture of Cb2487. The putative conserved domains of Cb2487 were analyzed through the NCBI Conserved Domains Database search tool. FIG. 9B shows SDS-PAGE of purified Cb2487. FIG. 9C shows a biochemical assay to determine the optimum pH of Cb2487. For pH optimum assay, para-nitrophenyl-beta-D-xylopyranoside (pNP-X, 0.8 mM) was incubated with Cb2487 (concentration 10 nM) at 75° C. in different buffers: pH 4.0-6.0 (citrate buffer, 50 mM, 150 mM NaCl), pH 6.0-8.0 (phosphate buffer, 50 mM, 150 mM NaCl), pH 8.5-9.0 (Tris-HCl, 50 mM, 150 mM NaCl). FIG. 9D shows a biochemical assay to determine the optimum temperature of Cb2487. For temperature optimum assay, pNP-X (0.8 mM) was incubated with Cb2487 (10 nM) in citrate buffer (50 mM, pH 6.0, 150 mM NaCl) at different temperatures (40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100° C.). FIGS. 9EA and 9EB show the kinetic parameters of Cb2487 with pNP-β-D-xylopyranoside as substrate. For the left side panel (FIG. 9EA), the kinetic parameters were determined at 90° C., pH 6.0. For the right side panel (FIG. 9EB), the kinetic parameters were determined at 75° C., pH 6.0. For these assays, different concentrations of pNP-X (0.08-24 mM) were incubated with Cb2487 (10 nM) in citrate buffer (50 mM, pH 6.0, 150 mM NaCl) at 75 and 90° C. FIG. 9F shows hydrolytic activity of Cb2487 on xylo-oligosaccharides. Cb2487 (0.5 μM) was incubated with different xylo-oligosaccharides (X2-6) at 75° C. for 15 hr and then the products were separated by TLC. FIG. 9G shows thermostability assay for Cb2487. Cb2487 was incubated in citrate buffer (pH 6.0, 50 mM) at different temperatures (70, 75, 80, 85, 90, 95° C.) without substrate addition, the protein was taken at different times (0, 10 min, 30 min, 1 h, 3 h, 4, 8 h, 12 h, 24 h) and the residual activity was assayed with pNP-X as substrate. FIG. 9H shows synergism of β-xylosidase (Cb2487) and α-glucuronidase (Cb909). Aldouronic acids were incubated with Cb2487 (0.5 μM) and Cb909 (0.5 μM) in citrate buffer (pH 6.0) at 75° C. overnight, then assayed with HPLC. Adding Cb909 cleaved off the methylglucuronic acid decorations in aldouronic acids to release xylose and xylo-oligosaccharides. Adding Cb2487 cleaved available beta-1,4-xylosidic linkages to release more xylose. Mixing the two enzymes led to the conversion of the xylo-oligosaccharides released by Cb909 to xylose by Cb2487.
FIGS. 10A to 10F: FIG. 10A shows the domain structure of Cb162; the protein has a single domain of acetyl xylan esterase. FIG. 10B shows an SDS-PAGE of purified Cb162. FIG. 10C shows the pH profile of Cb162 on pNP-acetate using para-nitrophenol adducted acetate (pNP-acetate) as a substrate. The released pNP was monitored continuously at an absorbance of 400 nm using Synergy 2 Microplate reader (BioTek). The initial rate of hydrolysis was adopted as an enzyme activity. The pH effect on the Cb162 was examined at 50° C. in the presence of 50 mM citrate-NaOH (pH 4.0 to 6.0) or 50 mM Na2HPO4—HCl (pH 6.0 to 8.0), with 150 mM NaCl. 0.1 μM of purified Cb162 and 2 mM pNP-acetate were used for this assay. FIG. 10D shows the temperature profile of Cb162 on pNP-acetate. The temperature profile was performed in 50 mM Na2HPO4—HCl, pH 7.0 and 150 mM NaCl, at temperatures between 40° C. and 75° C. with 5° C. increments. 0.04 μM of purified Cb162 and 2 mM pNP-acetate were used for this assay. FIG. 10E shows the thermostability profile of Cb162 on pNP-acetate; 0.02 μM of purified Cb162 in 50 mM Na2HPO4—HCl, pH 7.0 and 150 mM NaCl was incubated for 0 to 24 hours at temperatures between 60° C. and 80° C. with 5° C. intervals, and the residual activities were measured. FIG. 10F shows a kinetic study of Cb162. 0.04 μM of purified Cb162 in 50 mM Na2HPO4—HCl, pH 6.0, and 150 mM NaCl was incubated with various concentrations of pNP-acetate, and the initial rate of hydrolysis was plotted on the graph. The kinetic parameters were determined by Michaelis-Menten equation utilizing Graph Pad Prism v5.01 (GraphPad Software).
FIGS. 11A and 11B show synergy of C. bescii hemicellulolytic enzymes on soluble wheat arabinoxylan (SWAX) hydrolysis. SWAX (8.0%, w/v) was incubated with different hemicellulase mixes at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar [FIG. 11A] and HPLC [FIG. 11B] analysis. The hemicellulases applied include Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM).
FIGS. 12A and 12B show synergy of C. bescii hemicellulolytic enzymes on oatspelt xylan (OSX) hydrolysis. OSX (8.0%, w/v) was incubated with different hemicellulase mixes at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar [FIG. 12A] and HPLC [FIG. 12B] analysis. The hemicellulases applied include Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM).
FIGS. 13A to 13C: FIG. 13A shows soluble wheat arabinoxylan hydrolysis with hemicellulase cocktail at different temperatures. SWAX (8.0%, w/v) was incubated with Cb193 (0.5 μM), Cb2487 (4 μM), Cb1172 (0.5 μM), Cb162 (0.5 μM), and Cb909 (0.5 μM) at 65° C., 70° C., 75° C., 80° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay. FIG. 13B shows birch wood xylan hydrolysis with hemicellulase cocktail at different temperatures. BWX (8.0%, w/v) was incubated with Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM) at 65° C., 70° C., 75° C., 80° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay. FIG. 13C shows oat spelt xylan hydrolysis with hemicellulase cocktail at different temperatures. OSX (8.0%, w/v) was incubated with Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM) at 65° C., 70° C., 75° C., 80° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay.
FIGS. 14A to 14C: FIG. 14A shows SWAX hydrolysis was improved by adding two xylanases (Cb195 and Cb193) in the hemicellulase mixture. SWAX (8.0%, w/v) was incubated with different hemicellulase mixes at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar analysis. Different hemicellulase mixtures were applied in the hydrolysis: Mix I) Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); Mix II) Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); or Mix III) Cb195 (0.25 μM), Cb193 (0.25 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM). FIG. 14B shows BWX hydrolysis was improved by adding two xylanases (Cb195 and Cb193) in the hemicellulase mixture. BWX (8.0%, w/v) was incubated with different hemicellulase mixes at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar analysis. Different hemicellulase mixtures were applied in the hydrolysis: Mix I) Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); Mix II) Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); or Mix III) Cb195 (0.25 μM), Cb193 (0.25 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM).
FIG. 14C shows OSX hydrolysis was improved by adding two xylanases (Cb195 and Cb193) in the hemicellulase mixture. OSX (8.0%, w/v) was incubated with different hemicellulase mixes at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar analysis. Different hemicellulase mixtures were applied in the hydrolysis: Mix I) Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); Mix II) Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); or Mix III) Cb195 (0.25 μM), Cb193 (0.25 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM).
FIGS. 15A and 15B show soluble wheat arabinoxylan hydrolysis with hemicellulase cocktail of Caldicellulosiruptor bescii. Different concentrations of SWAX (1.0, 2.0, 4.0, 6.0, 8.0%, w/v) were incubated with Cb193 (0.5 μM), Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb162 (0.5 μM), and Cb909 (0.5 μM) for 15 hr at 75° C. in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay. FIG. 15A shows reducing sugar in the control and hydrolysis mixtures, and FIG. 15B shows comparison of calculated and average of actual reducing sugar in hydrolysis mixtures with different substrate concentrations.
FIGS. 16A and 16B show birch wood xylan hydrolysis with hemicellulase cocktails of Caldicellulosiruptor bescii. Different concentrations of BWX (1.0, 2.0, 4.0, 6.0, 8.0%, w/v) were incubated with Cb193 (0.5 μM), Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb162 (0.5 μM), and Cb909 (0.5 μM) at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay. FIG. 16A shows reducing sugar in the control and hydrolysis mixtures, and FIG. 16B shows comparison of calculated and average of actual reducing sugar in hydrolysis mixtures with different substrate concentrations.
FIGS. 17A and 17B show oat spelt xylan hydrolysis with hemicellulase cocktail of Caldicellulosiruptor bescii. Different concentrations of OSX (1.0, 2.0, 4.0, 6.0, 8.0%, w/v) were incubated with Cb193 (0.5 μM), Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb162 (0.5 μM), and Cb909 (0.5 μM) at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay. FIG. 17A shows reducing sugar in the control and hydrolysis mixtures, and FIG. 17B shows comparison of calculated and average of actual reducing sugar in hydrolysis mixtures with different substrate concentrations.
FIG. 18: Schematic structures of wild-type Cb1952 and its truncation mutants. The signal peptide is shown in filled rectangle. GH9: family 9 glycoside hydrolase domain; GH5: family 5 glycoside hydrolase domain; CBM3c: family 3 type C carbohydrate binding module; CBM3b: family 3 type B carbohydrate binding module.
FIG. 19: SDS-PAGE of Cb1952 wild-type and its truncation mutants. Lane 1: protein molecular mass marker; lane 2: Cb1952 wild-type; lane 3: Cb1952TM1; lane 4: Cb1952TM2; lane 5: Cb1952TM3; lane 6: Cb1952TM4; lane 7: Cb1952TM5; lane 8: Cb1952TM6; lane 9: Cb1952TM7. Two μg of each enzyme was resolved on a 12% SDS polyacrylamide gel.
FIG. 20: Enzymatic activity of Cb1952WT on natural substrates from a reducing sugar assay. Twelve different substrates were tested: Avicel, phosphoric acid swollen cellulose (PASC), sodium carboxymethyl cellulose (CMC-Na), lichenin, mannan, locust bean gum (LBG), guar gum, konjac glucomannan (KGM), wheat arabinoxylan (WAX), birchwood xylan (BWX), oat-spelt xylan (OSX) and xyloglucan. Incubation of enzymes with Avicel, PASC, CMC-Na, lichenin, mannan, LBG, guar gum, KGM, WAX and OSX substrates led to release of products that were quantified as a concentration of glucose equivalents. The Cb1952WT mainly hydrolyzes glucose- and mannose-configured substrates, but not xylose-configured substrates.
FIG. 21: Enzymatic activity of Cb1952TM1 on natural substrates from a reducing sugar assay. Twelve different substrates were tested: Avicel, phosphoric acid swollen cellulose (PASC), sodium carboxymethyl cellulose (CMC-Na), lichenin, mannan, locust bean gum (LBG), guar gum, konjac glucomannan (KGM), wheat arabinoxylan (WAX), birchwood xylan (BWX), oat-spelt xylan (OSX) and xyloglucan. Incubation of enzymes with Avicel, PASC, CMC-Na, lichenin, mannan, LBG, guar gum, KGM, WAX, BWX, OSX and xyloglucan substrates led to release of products that were quantified as a concentration of glucose equivalents. The results show that Cb1952TM1 mainly hydrolyzes glucose-configured substrates. It also has some activities on mannose-configured substrates. It has low activities on xylose-configured substrates.
FIG. 22: Enzymatic activity of Cb1952TM5 on natural substrates from a reducing sugar assay. Twelve different substrates were tested: Avicel, phosphoric acid swollen cellulose (PASC), sodium carboxymethyl cellulose (CMC-Na), lichenin, mannan, locust bean gum (LBG), guar gum, konjac glucomannan (KGM), wheat arabinoxylan (WAX), birchwood xylan (BWX), oat-spelt xylan (OSX) and xyloglucan. Incubation of enzymes with CMC-Na, lichenin, mannan, LBG, guar gum and KGM substrates led to release of products that were quantified as a concentration of mannose equivalents. The Cb1952TM5 mainly hydrolyzes mannose-configured substrates, but does not have obvious activity on glucose- or xylose-configured substrates.
FIG. 23: Thin Layer Chromatography (TLC) analysis of enzymatic activity of Cb1952WT, Cb1952TM1 and Cb1952TM5 on glucose and cellooligosaccharides. G1, G2, G3, G4, G5, and G6 refer to glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, respectively. Cb1952WT and Cb1952TM1 hydrolyze cellotriose, cellotetraose, cellopentaose and cellohexaose into glucose and cellobiose, but have no activity on cellobiose. Cb1952TM5 has no activity on glucose and any of the cellooligosaccharides tested. None of the enzyme has transglycosylation activity on glucose and cellooligosaccharides.
FIG. 24: Thin Layer Chromatography (TLC) analysis of enzymatic activity of Cb1952WT, Cb1952TM1 and Cb1952TM5 on mannose and mannooligosaccharides. M1, M2, M3, M4, M5, and M6 refer to mannose, mannobiose, mannotriose, mannotetraose, mannopentaose and mannohexaose, respectively. Cb1952WT and Cb1952TM5 hydrolyze mannotriose, mannootetraose, mannopentaose and mannohexaose into mannose and smaller mannooligosaccharides, but have no hydrolyzing activity on mannobiose. Cb1952TM1 hydrolyzes mannopentaose and mannohexaose into smaller oligosaccharides but has no hydrolyzing activity on mannobiose, mannotriose, mannotriose and mannotetraose. None of the enzyme has transglycosylation activity on mannose and mannooligosaccharides.
FIG. 25: HPLC analysis of enzymatic activity of Cb1952TM1 on cellulose substrates. Three different cellulosic substrates were tested: Avicel, CMC-Na and PASC. In each case, in the presence of Cb1952TM1, glucose and cellobiose were released. In the absence of Cb1952TM1, neither glucose nor cellobiose was observed for all the substrates. The results showed that this part of the enzyme or polypeptide (Cb1952) cleaves glucose and cellobiose as end products from cellulosic substrates (Avicel, CMC-Na and PASC).
FIG. 26: HPLC analysis of time-course hydrolysis of PASC by Cb1952TM1. 100 nanomolar of Cb1952TM1 was incubated with 2.5 mg/ml PASC at 75° C. At different time intervals (0, 0.5 min, 2 min, 10 min, 1 h, 4 h and 24 h), samples were taken out and immediately boiled for 10 min to inactivate the enzyme. After centrifugation, the supernatants of the samples were appropriately diluted with water and applied to HPLC analysis. The results show that Cb1952TM1 initially releases glucose, cellobiose, cellotriose and cellotetraose. With increasing time, only glucose and cellobiose were left in the reaction mixture.
FIG. 27: Thermostability of Cb1952WT using PASC as substrate for activity measurement. Cb1952WT has 75%, 43%, 17% and 12% activity after incubation at 70° C., 75° C., 80° C. and 85° C. for 24 h, respectively. 500 nM Cb1952WT was kept at different temperatures (70° C., 75° C., 80° C. and 85° C.). The samples were taken out at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately used for enzyme activity measurement. The enzyme activity was measured at pH 5.5 and at 85° C. on a thermomixer. 2.5 mg/ml final concentration of PASC was used for measurement, and 8.31 μl of the protein sample was added to the substrate and mixed by pipetting up and down for several times. The total volume was 100 μl. The reducing ends corresponding to glucose equivalents were measured according to the methods of Lever, M. (A new reaction for colorimetric determination carbohydrates. Anal. Biochem. 1972: 47; 273-279). The velocity of reaction in 10 minutes was calculated. The velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the velocity of reaction at time 0, then multiplied by 100, respectively.
FIG. 28: Thermostability of Cb1952TM1 using PASC as substrate for activity measurement. Cb1952TM1 has 94%, 76%, 18% and 13% activity after incubation at 70° C., 75° C., 80° C. and 85° C. for 24 h, respectively. 500 nM Cb1952TM1 was kept at different temperatures (70° C., 75° C., 80° C. and 85° C.). The samples were taken out at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately used for enzyme activity measurement. The enzyme activity was measured at pH 5.5 and at 85° C. on a thermomixer. 2.5 mg/ml final concentration of PASC was used for measurement, and 8.31 μl of the protein sample was added to the substrate and mixed by pipetting up and down for several times. The total volume was 100 μl. The reducing ends corresponding to glucose equivalents were measured according to the methods of Lever, M. (supra). The velocity of reaction in 10 minutes was calculated. The velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the velocity of reaction at time 0, then multiplied by 100, respectively.
FIG. 29: Domain architecture of wild-type (WT) Cb1953, Cb1953TM1 and Cb1953TM2.
FIG. 30: SDS-polyacrylamide gel with purified wild-type Cb1953, Cb1953TM1 and Cb1953TM2 proteins.
FIG. 31: A zymogram of Cb1953WT, Cb1953TM1, Cb1953TM2 on carboxylmethyl cellulose (CMC). The gel was prepared as in standard dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with CMC substrate (final 0.1%, w/v). After electrophoretic fractionation of the proteins, gels were washed twice in distilled water and incubated in 30 mL of refolding buffer (20 mM citrate buffer, pH 6.0, 2.5% Triton X-100, 2 mM dithiothreitol, 2.5 mM CaCl2) for 1 hour at 25° C. and then held overnight in fresh buffer at 37° C. The gel was washed twice in 50 mM Citrate buffer (pH 6.0) and then the results were visualized by staining with 0.1% Congo red and destaining with 1M NaCl. As shown in FIG. 31, Cb1953WT and Cb1953TM2 showed significant white bands at the positions of their expected sizes indicating cellulase activity, but not Cb1953TM1 protein.
FIGS. 32 and 33: Enzymatic activity of Cb1953WT, Cb1953TM1, and Cb1953TM2 on natural substrates from a reducing sugar assay. Seven different substrates were tested: Avicel, Phosphoric acid swollen cellulose (PASC), carboxylmethyl cellulose (CMC), wheat arabinoxylan (WAX), lichenin, konjac glucomannan, and mannan. Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents. The tubes were incubated with constant mixing in a Thermomixer R (Eppendorf) at 75° C. for 16 h. The tubes were centrifuged at 10,000 rpm for 5 min at 4° C. 50 μL of sample supernatant was transferred to a clean 1.5 mL centrifuge tube for the pHBAH assay. The wavelength at 410 nm was measured for the standards and samples. The A410nm and glucose concentrations were plotted against each other, and linear regression was used to fit a line to the data. The reactions were resolved by thin layer chromatography (TLC), The mobile phase consisted of n-butanol:acetic acid:H2O, 10:5:1 (vol/vol/vol) and 10 cm×20 cm plates were used. The reducing sugar assay (FIG. 32) and TLC (FIG. 33) results show that Cb1953WT and Cb1953TM2 have cellulase activity whereas Cb1953TM1 has mannanase activity.
FIG. 34: HPLC analysis of time course of enzymatic activity of Cb1953TM2 on PASC. For analysis of the products of hydrolysis, the samples were analyzed by high performance anion-exchange chromatography (HPAEC). For HPAEC analyses, 100 μL of each diluted sample was injected onto a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.). For the analysis, glucose and five different cellooligosaccharides (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose) were used as standards. In the reaction, Cb1953TM2 started to release cellooligosaccharides (C2-C4) and then glucose was released later. The results showed that this enzyme releases mainly cellobiose from PASC.
FIGS. 35 and 36: Thermostability of Cb1953WT (FIG. 35) and Cb1953TM2 (FIG. 36) on PASC. Fifty nM Cb1953WT and Cb1953TM2 were kept at different temperatures (70° C., 75° C., 80° C., 85° C. and 90° C.). The samples were taken out at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately used in enzyme activity measurement. The enzyme activity was measured at 85° C. using Cary 300 UV-Vis spectrophotometer (Varian). The initial velocity of reaction in the first minute was calculated. The initial velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the initial velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the initial velocity of reaction at time 0, then multiplied by 100. From the results, Cb1953WT (FIG. 35) and Cb1953TM2 (FIG. 36) were quite stable at 70° C. and 75° C., maintaining activity of 75 ̃90% of heat non-treated proteins.
FIGS. 37A and 37B: Kinetic studies of Cb1953WT (FIG. 37A) and Cb1953TM2 (FIG. 37B) on PASC. 0.05 μM of purified Cb1953WT or Cb1953TM2 in 50 mM Na2HPO4—HCl, pH 6.0, and 150 mM NaCl was incubated with various concentrations of phosphoric acid swollen cellulose (PASC), and the initial rate of hydrolysis was plotted against substrate concentration. The kinetic parameters (Km: 7.603 mg/mL, kcat: 7.513 s−1 and kcat/Km: 0.988 s−1 mL/mg for Cb1953WT and Km: 3.032 mg/mL, kcat: 5.411 s−1 and kcat/Km: 1.785 s−1 mL/mg for Cb1953TM2) were determined by fitting the data to the Michaelis-Menten equation (Graph Pad Prism v5.01).
FIG. 38: Domain architecture of wild-type (WT) Cb1954, Cb1954TM3 and Cb1954TM5 polypeptides.
FIGS. 39A and 39B: FIG. 39A: SDS-polyacrylamide gel with purified Cb1954TM3 protein. FIG. 39B: Enzymatic activity of Cb1954TM3 on natural substrates from a reducing sugar assay. Three different cellulose substrates were tested: Avicel, sodium carboxymethyl cellulose (CMC-Na) and phosphoric acid swollen cellulose (PASC). Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents. Hydrolysis of PASC was higher than hydrolysis of other substrates.
FIG. 40: HPLC analysis of enzymatic activity of Cb1954TM3 on cellulosic substrates. Three different cellulosic substrates were tested: Avicel, CMC-Na and PASC. In each case, in the presence of Cb1954TM3, glucose and cellobiose were released. In the absence of Cb1954TM3, neither glucose nor cellobiose was observed for all the substrates. The results showed that this enzyme releases glucose and cellobiose, and also longer chain oligosaccharides as end products from cellulosic substrates (CMC-Na and PASC).
FIG. 41: Thermostability of Cb1954TM3. Cb1954TM3 has 75%, 87%, 64% and 7% activity after incubation at 70° C., 75° C., 80° C. and 85° C. for 24 h, respectively. 500 nM Cb1954TM3 was kept at different temperatures (70° C., 75° C., 80° C. and 85° C.). The enzyme activity was measured at pH 5.5 and at 95° C. on a thermomixer. 2.5 mg/ml final concentration of PASC was used for measurement, and 10 μl of the protein sample was added to the substrate and mixed by pipetting up and down for several times. The total volume was 100 μl. The reducing ends corresponding to glucose equivalents were measured according to the methods of Lever, M. (supra). The velocity of reaction in 10 minutes was calculated. The velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the velocity of reaction at time 0, then multiplied by 100, respectively.
FIG. 42: Domain architecture of wild-type (WT) Cb1946, Cb1946TM1 and Cb1946TM2 polypeptides.
FIG. 43: SDS-polyacrylamide gel with purified wild-type Cb1946, Cb1946TM1 and Cb1946TM2 proteins.
FIG. 44: Zymogram of Cb1946WT, Cb1946TM1, and Cb1946TM2 on carboxylmethyl cellulose (CMC) agar plate. The agar plate was prepared with CMC substrate (final 0.25%, w/v). After spotting 1 μg of each protein on agar-CMC plates, the plate was incubated at 37° C. overnight and then the gel was visualized by staining with 0.1% Congo red and destaining with 1M NaCl. As shown in FIG. 44, Cb1946WT and Cb1946TM2 showed significant halos on the agar plate indicating cellulase activity, but not Cb1953TM1 proteins.
FIGS. 45 and 46: Thin Layer Chromatography (TLC) (FIG. 45) and High Performance Liquid Chromatography (HPLC) (FIG. 46) analysis of enzymatic activity of Cb1946WT, Cb1946TM1, Cb1946TM2 on phosphoric acid swollen cellulose (PASC). Each enzyme (final 0.5 μM) was reacted with phosphoric acid swollen cellulose (PASC) at 1% final concentration in 50 mM citrate-150 mM NaCl, pH 6.0 at 75° C. for 16 hours. The reactions were resolved by thin layer chromatography (TLC) (FIG. 45). The mobile phase consisted of n-butanol:acetic acid:H2O, 10:5:1 (vol/vol/vol) and 10 cm×20 cm plates were used. In FIG. 45, C1, C2, C3, C4, and C5 refer to glucose, cellobiose, cellotriose, cellotetraose and cellopentaose, respectively. For more quantitative analysis of the products of hydrolysis, the samples were analyzed by high performance anion-exchange chromatography (HPAEC) (FIG. 46). For HPAEC analyses, 100 μL of each diluted sample was injected into a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.). For the TLC and HPLC analysis, glucose and five different cellooligosaccharides were used: cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose as standards. Based on the results of TLC and HPLC, Cb1953WT and Cb1953TM2 showed significant release of products such as glucose, cellobiose, cellotriose, and cellotetraose from PASC substrate, indicating that Cb1946WT and Cb1953TM2 have cellulase activities, but not Cb1953TM1.
FIG. 47: Domain architecture of wild-type Cb629 and Cb629TM1 polypeptides.
FIG. 48: SDS-polyacrylamide gel with purified Cb629TM1 protein.
FIG. 49: Enzymatic activity of Cb629TM1 on substrates with products determined through a reducing sugar assay. Three different cellulose substrates were tested: Avicel, sodium carboxymethyl cellulose (CMC-Na) and phosphoric acid swollen cellulose (PASC). Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents. Hydrolysis of PASC was higher than hydrolysis of the other substrates.
FIG. 50: HPLC analysis of enzymatic activity of Cb629TM1 on substrates. Three different cellulosic substrates were tested: Avicel, CMC-Na and PASC. In each case, in the presence of Cb629TM1, glucose and cellobiose were released. In the absence of Cb629TM1, neither glucose nor cellobiose was observed from all the substrates. The results showed that this enzyme releases glucose and cellobiose as end products from cellulosic substrates (Avicel, CMC-Na and PASC).
FIG. 51: TLC analysis of enzymatic activity of Cb629TM1 on cello-oligosaccharides. G1, G2, G3, G4, G5, and G6 refer to glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose respectively.
FIG. 52: Thermostability of Cb629TM1. Cb629TM1 has 109%, 99%, 96%, 83% and 34% activity after incubation at 60° C., 65° C., 70° C., 75° C. and 80° C. for 24 h, respectively. 500 nM Cb629TM1 was kept at different temperatures (60° C., 65° C., 70° C., 75° C. and 80° C.). The samples were taken out at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately used for enzyme activity measurement. The enzyme activity was measured at pH 5.5 and at 70° C. on a thermomixer. 2.5 mg/ml final concentration of PASC was used for measurement, and 8.31 μl of the protein sample was added to the substrate and mixed by pipetting up and down for several times. The total volume was 100 μl. The reducing ends corresponding to glucose equivalents were measured according to the methods of Lever, M. (supra). The velocity of reaction in 10 minutes was calculated. The velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the velocity of reaction at time 0, then multiplied by 100, respectively.
FIGS. 53A and 53B: FIG. 53A: Domain architecture of wild-type Cb486. FIG. 53B: SDS-polyacrylamide gel with purified wild-type Cb486 protein.
FIG. 54: TLC analysis of enzymatic activity of Cb486 on xylo-oligosaccharides (X2-X6). The following xylo-oligosaccharides (X2-X6) were tested: xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose. This was done by an overnight hydrolysis of the xylo-oligosaccharides followed by resolving of the products with TLC. In each case, in the presence of Cb486, xylose and xylobiose were released. In the absence of Cb486, only minor amount of xylose was observed for xylobiose; no products of hydrolysis were released for other xylo-oligosaccharides. The results showed that this enzyme releases xylose and xylobiose from xylo-oligosaccharides (xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose). X1, X2, X3, X4, X5, and X6 refer to xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose, respectively.
FIG. 55: TLC analysis of enzymatic activity of Cb486 on glucose and cellooligosaccharides. Glucose and five different cellooligosaccharides were used for the assay: cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose. C2, C3, C4, and C5 refer to cellobiose, cellotriose, cellotetraose and cellopentaose, respectively.
FIGS. 56A and 56B: FIGS. 56A and 56B show the pH and temperature profiles, respectively of the activity of Cb486. For these assays, the enzyme concentration of Cb486 was 10 nM. For pH profiling, the reactions were carried out in two buffers: 50 mM sodium citrate, 150 mM NaCl (pH 4.0-pH 6.0) and 50 mM Na2HPO4—NaH2PO4, 150 mM NaCl (pH 6.5-pH 8.0). The enzyme was incubated with 1 mM pNP-β-D-galactopyranoside in each buffer at a given pH at 75° C., and the activities in a 30 min assay were determined. For determination of optimal temperature, 10 nM of Cb486 was incubated with 1 mM pNP-β-D-galactopyranoside at pH 5.5 at different temperatures ranging from 40° C. to 95° C. with a 5° C. interval. The releasing of pNP was recorded by monitoring the increase of optical density at 410 nM with a Cary 300 UV-Visible spectrophotometer (Agilent, Santa Clara Calif.).
FIGS. 57A and 57B: Domain architecture (FIG. 57A) and SDS-polyacrylamide gels containing purified proteins (FIG. 57B) of a cellulase mixture composed of Cb629TM1, Cb486, Cb1946TM2, Cb1952TM1, Cb1953TM2, and Cb1954TM3 cellulases.
FIG. 58: SDS-polyacrylamide gels containing purified proteins of the hemicellulases Cb193, Cb195, Cb1172, Cb909, Cb2487, and Cb162.
FIGS. 59 and 60: TLC (FIG. 59) and HPLC (FIG. 60) analysis of samples of microwave-pretreated Miscanthus that were treated with a cellulase mixture containing Cb629TM1, Cb486, Cb1946TM2, Cb1952TM1, Cb1953TM2, and Cb1954TM3 cellulases and/or a hemicellulase mixture containing Cb193, Cb195, Cb1172, Cb909, and Cb2487 hemicellulases. FIG. 59 shows analysis of assays with samples containing 2%, 5%, or 8% Miscanthus, and FIG. 60 shows analysis of an assay with a sample containing 8% Miscanthus. In FIG. 59, C1, C2, C3, C4, and C5 refer to glucose, cellobiose, cellotriose, cellotetraose and cellopentaose, respectively. X1, X2, X3, X4, and X5 refer to xylose, xylobiose, xylotriose, xylotetraose and xylopentaose, respectively. A1 refers to arabinose. For FIG. 60, the 8% substrate reaction samples were analyzed by high performance anion-exchange chromatography (HPAEC). For HPAEC analyses, 100 μL of each diluted sample was injected onto a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.).
FIGS. 61 and 62: TLC (FIG. 61) and HPLC (FIG. 62) analysis of samples of autoclave-pretreated Miscanthus that were treated with a cellulase mixture containing Cb629TM1, Cb486, Cb1946TM2, Cb1952TM1, Cb1953TM2, and Cb1954TM3 cellulases and/or a hemicellulase mixture containing Cb193, Cb195, Cb1172, Cb909, and Cb2487 hemicellulases. FIG. 61 shows analysis of assays with samples containing 2%, 5%, or 8% Miscanthus, and FIG. 62 shows analysis of an assay with a sample containing 8% Miscanthus. In FIG. 61, C1, C2, C3, C4, and C5 refer to glucose, cellobiose, cellotriose, cellotetraose and cellopentaose, respectively. X1, X2, X3, X4, and X5 refer to xylose, xylobiose, xylotriose, xylotetraose and xylopentaose, respectively. For FIG. 62, the 8% substrate reaction samples were analyzed by high performance anion-exchange chromatography (HPAEC). For HPAEC analyses, 100 μL of each diluted sample was injected onto a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.).
FIGS. 63 and 64: TLC (FIG. 63) and HPLC (FIG. 64) analysis of samples of microwave-pretreated 8% Miscanthus samples that were treated with a cellulase mixture containing Cb629TM1, Cb1946TM2, Cb1952TM1, Cb1953TM2, and Cb1954TM3 cellulases (the mixture lacks the β-glucosidase Cb486), and/or a hemicellulase mixture containing Cb193, Cb195, Cb1172, Cb909, and Cb2487 hemicellulases. In FIG. 63, C1, C2, C3, C4, and C5 refer to glucose, cellobiose, cellotriose, cellotetraose and cellopentaose, respectively. X1, X2, X3, X4, and X5 refer to xylose, xylobiose, xylotriose, xylotetraose and xylopentaose, respectively. For FIG. 64, the reaction samples were analyzed by high performance anion-exchange chromatography (HPAEC). For HPAEC analyses, 100 μL of each diluted sample was injected onto a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.).
FIGS. 65 and 66: TLC (FIG. 65) and HPLC (FIG. 66) analysis of samples of autoclave-pretreated 8% Miscanthus samples that were treated with a cellulase mixture containing Cb629TM1, Cb1946TM2, Cb1952TM1, Cb1953TM2, and Cb1954TM3 cellulases (the mixture lacks the β-glucosidase Cb486), and/or a hemicellulase mixture containing Cb193, Cb195, Cb1172, Cb909, and Cb2487 hemicellulases. In FIG. 65, C1, C2, C3, C4, and C5 refer to glucose, cellobiose, cellotriose, cellotetraose and cellopentaose, respectively. X1, X2, X3, X4, and X5 refer to xylose, xylobiose, xylotriose, xylotetraose and xylopentaose, respectively. For FIG. 66, the reaction samples were analyzed by high performance anion-exchange chromatography (HPAEC). For HPAEC analyses, 100 μL of each diluted sample was injected onto a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.).
FIGS. 67A and 67B show the pH and temperature profiles, respectively of the activity of Cb1952TM1. For pH profiling, the reactions were carried out in two buffers: 50 mM sodium citrate, 150 mM NaCl (pH 4.0-pH 6.0) and 50 mM Na2HPO4—NaH2PO4, 150 mM NaCl (pH 6.5-pH 8.0). The enzyme concentration of Cb1952TM1 was 0.5 μM. The enzyme was incubated with 2.5 mg/ml PASC in each buffer at a given pH at 75° C., and the activities in a 10 min assay were determined. The reducing sugars released were measured using the pHBAH assay. For determination of optimal temperature, 0.5 μM of Cb1952TM1 enzyme was incubated with 2.5 mg/ml PASC at pH 5.5 at different temperatures ranging from 40° C. to 95° C. with a 5° C. interval.
FIGS. 68A and 68B: Cleavage products resulting from the incubation of Cb1953TM2 with cellohexaose. FIG. 68A: TLC analysis of reaction products; FIG. 68B: HPLC analysis of reaction products. The data indicates that Cb1953TM2 hydrolyzes cellohexaose randomly.
FIGS. 69A and 69B show the pH and temperature profiles, respectively of the activity of Cb1954TM3.
FIGS. 70A and 70B show the pH and temperature profiles, respectively of the activity of Cb629TM1.
FIG. 71: HPLC analysis of time course of enzymatic activity of Cb629TM1 on PASC. For analysis of the products of hydrolysis, the samples were analyzed by high performance anion-exchange chromatography (HPAEC). For HPAEC analyses, 100 μL of each diluted sample was injected onto a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.). For the analysis, glucose, cellobiose, and cellotriose were used as standards.
FIG. 72: Substrate specificity analysis of Cb486. 50 nM of Cb486 was incubated at 75° C. in its optimal buffer (50 mM sodium citrate, 150 mM NaCl, pH5.5) with 1 mM each of pNP-α-L-arabinopyranoside, pNP-β-D-fucopyranoside, pNP-β-D-galactopyranoside, pNP-β-D-glucopyranoside, pNP-βD-xylopyranoside, and pNP-β-D-cellobiose, respectively, for 30 min. The release of pNP was recorded by monitoring the increase in optical density at 410 nM with a Cary 300 UV-Visible spectrophotometer (Agilent, Santa Clara Calif.).
FIGS. 73A to 73D: Hydrolysis of treated miscanthus with cellulase and/or hemicellulase mixtures. FIG. 73A: 0.5 μM enzyme; 2% substrate; FIG. 73B: 0.5 μM enzyme; 5% substrate; FIG. 73C: 0.5 μM enzyme; 8% substrate; FIG. 73D: 1.0 μM enzyme; 10% substrate. Different concentrations (2%, 5%, and 8%) of Miscanthus pre-treated using two different methods (autoclaving or microwaving) were incubated with either the cellulase mix (containing 0.5 μM each of Cb1946TM2, Cb1952TM1, Cb1953TM2, Cb1954TM3, Cb629TM1, and Cb486) or the hemicellulase mix (containing 0.5 μM each of Cb193, Cb195, Cb1172, Cb2487, and Cb909), or both enzyme mixtures in a total volume of 500 μl at 75° C. with an end-over-end shaking manner for 15 hours. Further, increased concentration (10%) of pretreated Miscanthus of both pretreatment types was incubated with an increased enzyme concentration of 1.0 μM at 75° C. with an end-over-end shaking manner for 15 hours. The reducing ends were measured using the pHBAH method.
FIG. 74: Hydrolysis of treated miscanthus with enzyme mixtures lacking Cb486. For these assays, reaction mixtures with 0.5 μM enzyme and 8% substrate were used. Pre-treated Miscanthus (8%) using two different methods (autoclaving or microwaving) was incubated with either the cellulase mix (containing 0.5 μM each of Cb1946TM2, Cb1952TM1, Cb1953TM2, Cb1954TM3, and Cb629TM1, but without Cb486) or the hemicellulase mix (containing 0.5 μM each of Cb193, Cb195, Cb1172, Cb2487, and Cb909), or both enzyme mixtures in a total volume of 500 μl at 75° C. with an end-over-end shaking manner for 15 hours. The reducing ends were measured using pHBAH method.
FIGS. 75A and 75B: FIG. 75A: Domain architecture of Cb1952TM2, Cb1954TM3, Cb629TM1, and Cb486 polypeptides. FIG. 75B: Analysis of samples of pretreated Miscanthus (AC=autoclaved; MW=microwaved) that were treated with Cb1952TM2, Cb1954TM3, Cb629TM1, Cb486, or a mixture containing Cb1952TM2, Cb1954TM3, Cb629TM1, and Cb486 cellulases. Pre-treated Miscanthus, using two different methods (autoclaving or microwaving), at a final concentration of 2% was incubated with an individual cellulase (Cb1952TM2, Cb1954TM3, Cb629TM1, or Cb486, 0.5 μM each) or a mixture containing all four cellulases in a total volume of 500 μl at 75° C. with an end-over-end shaking manner for 15 hours. The reducing ends were measured using pHBAH method.
FIG. 76: Reducing sugar assay with Cb1946WT, Cb486, or a mixture containing Cb1946WT and Cb486 cellulases. The reactions were carried out using 0.5 μM of Cb1946WT, Cb486 or both enzymes in a phosphate buffer (50 mM sodium phosphate, 150 mM NaCl, pH 6.5) at a total volume of 500 μl in a 16-hours incubation with an end-over-end shaking manner at 75° C.
FIGS. 77A and 77B: Analysis of PASC (FIG. 77A) or Avicel (FIG. 77B) samples treated with Cb1946WT, Cb486, or a mixture containing Cb1946WT and Cb486 cellulases. The reactions were carried out using 0.5 μM of either Cb1946WT or Cb486 or both enzymes in a phosphate buffer (50 mM sodium phosphate, 150 mM NaCl, pH 6.5) in a total volume of 500 μl in a 16-hours incubation with an end-over-end shaking manner at 75° C. Seven μl of the hydrolysis products were applied to TLC analysis.
FIGS. 78A to 78C: Time course hydrolysis of PASC by Cb1952 WT (FIG. 78A), TM1 (FIG. 78B), and TM5 (FIG. 78C). Two point five mg/ml PASC was incubated with 0.5 μM Cb1952 WT, TM1, and TM5 at 75° C. At different time intervals (0 min, 2 min, 10 min, 60 min, 4 h, and 24 h), samples were taken out and applied to HPAEC-PAD analysis.
FIG. 79: Amino acid sequence alignment of the GH9 domain of Cb1952 (SEQ ID NO: 150) with those of CloceCel9G (Clostridium cellulolyticum Cel9G, GenBank accession number: AAA73868, (SEQ ID NO: 151)) (26) and ThefuCel9A (Thermobifida fusca Cel9A, GenBank accession number: AAB42155, (SEQ ID NO: 152)) (34). CloceCel9G (non-processive) and ThefuCel9A (processive) represent the two types of family 9 theme B1 endoglucanases whose enzyme-cello-oligosaccharide complex structures have been resolved. The asterisks indicate the identical or similar amino acid residues within the three sequences. The filled triangles indicate non-conserved residues. The numbers under a specific amino acid residue indicate the subsites of the cello-oligosaccharides interacting with this amino acid residue based on the CloceCel9G and ThefuCel9A enzyme-substrate complex structures.
FIGS. 80A and 80B: Qualitative binding of Cb1952 wild-type and its truncation mutants with Avicel (FIG. 80A) and phosphoric acid swollen cellulose (PASC) (FIG. 80B). Thirty micrograms of each protein were incubated with 40 mg/ml Avicel cellulose or 2.5 mg/ml PASC in 50 mM Tris buffer, 150 mM NaCl (pH 7.5). The mixture was shaken end-over-end at 4° C. for 1 h. Then the bound and unbound proteins were separated by centrifugation of the mixture at 16,400 rpm for 3 min. The cellulose pellet was washed with 1 ml buffer (50 mM Tris buffer, 150 mM NaCl, pH 7.5) for 4 times. Then the pellet was added with 70 μl of 1×SDS-PAGE loading buffer and boiled for 5 min. The protein corresponding to one tenth volume of each fraction was applied to a 12% SDS-PAGE.
FIGS. 81A to 81C: Thermostability of Cb1952 and its truncation mutants harboring cellulase activities. FIG. 81A: Cb1952 TM2; FIG. 81B: Cb1952 TM3; FIG. 81C: Cb1952 TM4. The enzymes were incubated at 75° C., 80° C., and 85° C. (WT, TM1, TM2, and TM3) or at 45° C., 50° C., and 55° C. (TM4) on a Veriti 96-well thermal cycle. At different time points, samples were taken out and measured for their remaining activity using PASC as the substrate.
FIG. 82: Amino acid sequence alignment of the CBM3c of Cb1952 with those from other family 9 glycoside hydrolases. The amino acid residues proposed to be involved in cellulose ligand binding based on the works of Jindou et al. (2006) and Li et al. (2010) are indicated with a filled triangle. The sources of the enzymes used for comparison are as follows. Cb1952 (SEQ ID NO: 153): bifunctional cellulase/mannanase of Caldicellulosiruptor bescii (this study); ADQ45731(SEQ ID NO: 154): putative cellulase of Caldicellulosiruptor kronotskyensis; ABP66693 (SEQ ID NO: 155): putative cellulase of Caldicellulosiruptor saccharolyticus; ADL42950 (SEQ ID NO: 156): putative Caldicellulosiruptor obsidiansis cellulase/mannan endo-1,4-beta-mannosidase; AAK06394 (SEQ ID NO: 157): CelE of Caldicellulosiruptor sp. Tok7B.1 (11); AAA73868 (SEQ ID NO: 158): Cel9G of Clostridium cellulolyticum (26); AAC38572 (SEQ ID NO: 159): EngH of Clostridium cellulovorans (38); CAA39010 (SEQ ID NO: 160): Cel9Z of Clostridium stercorarium (18); ABX43720 (SEQ ID NO: 161): Cel9 of Clostridium phytofermentans (39, 48); ABN51860 (SEQ ID NO: 162): Cel9I of Clostridium thermocellum DSM 1313 (50); CAB38941 (SEQ ID NO: 163): Cel9B of Paenibacillus barcinonensis (32); BAB33148 (SEQ ID NO: 164): CelQ of Clostridium thermocellum F1 (2); AAA23086 (SEQ ID NO: 165): CenB of Cellulomonas fimi (27); AAW62376 (SEQ ID NO: 166): CBP105 of Cellulomonas flavigena (28); AAB42155 (SEQ ID NO: 167): Cel9A of Thermobifida fusca (16, 34).
FIG. 83: SDS-PAGE of purified Cb1581.
FIGS. 84A and 84B: Shows the enhancing effect of Cb1581 on enzymatic hydrolysis of microwave pretreated miscanthus at 70° C. (FIG. 84A) or 80° C. (FIG. 84B). Enzymatic hydrolysis of pretreated miscanthus was carried out at pH 6.0 using 0.5 μM each of the cellulase/hemicellulase enzyme mixture in a total volume of 500 μl with 10% miscanthus as the substrate. The enzymes in the mixture include Cb1946TM2, Cb1952TM1, Cb1953TM2, Cb1954TM3, Cb629TM1, Cb486, Cb193, Cb195, Cb2487, Cb1172, Cb909, and Cb162, and variable amounts of recombinant Cb1581, as indicated. The concentration of glucose equivalents was determined following enzymatic hydrolysis of microwave pretreated miscanthus, according to the methods of Lever, M. The releasing of sugars is enhanced with the increasing amount of Cb1581 in the reaction mixture at both 70° C. and 80° C.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present disclosure relates to thermostable cellulose and hemicellulose-degrading enzymes and to methods of using these enzymes for the degradation of cellulose, hemicellulose, and cellulose and hemicellulose-containing materials. The present disclosure also relates to nucleic acids encoding the enzymes disclosed herein, and enzyme cocktails containing various enzymes disclosed herein.
In one aspect, the disclosure provides enzymes having cellulase activity. Provided herein are truncated enzymes that have improved cellulase activity over wild-type cellulase proteins. Also provided herein are truncated enzymes that have similar cellulase activity to wild-type cellulase proteins. Truncated proteins may be advantageous over wild-type proteins, for example, due to lower cost or improved ease of production of truncated proteins.
In another aspect, the disclosure provides enzymes having hemicellulase activity. The hemicellulose-degrading enzymes of the present disclosure can be used alone, or in combination to degrade hemicellulose, i.e., convert hemicellulose into its structural components by cleavage of bonds, or linkages, between the component subunits present in hemicellulose. Bonds or linkages may include bonds between xylose subunits, or bonds between xylose and functional groups, or bonds between functional groups.
In another aspect, the disclosure provides enzymes that enhance the activity of enzymes having cellulase or hemicellulase activity, and/or mixtures thereof. Enzymes that enhance the activity of cellulases and/or hemicellulases may be provided alone, with cellulases, with hemicellulases, or with mixtures of cellulases and hemicellulases.
Cellulose or hemicellulose treated with the methods of the present disclosure may be at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% degraded. Degradation products may include glucose, cellobiose, cellodextrins, xylose, arabinose, glucuronyl groups, and acetyl groups, in addition to other functional groups and hydrocarbons. The degradation products may find use as biofuels or other value-added compounds. For example, sugars released from the cellulose or hemicellulose may be fermented for the production of ethanol.
The cellulose and hemicellulose-degrading enzymes of the present disclosure are thermostable, and are optimally able to degrade cellulose and/or hemicellulose into sugars such as glucose, xylose, or arabinose at temperatures above 50° C. In addition, the enzymes retain substantial activity when maintained at various temperatures above 50° C.
Without wishing to be bound by theory, another important feature of the enzyme cocktails described herein are that they are derived from the same organism, ensuring that the enzymes will function together to degrade cellulose and/or hemicellulose. Caldicellulosiruptor bescii contains a complete set of enzymes for degrading cellulose, and hemicelluloses such as xylan. Xylan is the main hemicellulose in perennial grasses, such as switchgrass, and is most likely the main hemicellulose in the giant grass Miscanthus. 
In one aspect, the present disclosure provides nucleotide and amino acid sequences for thermostable enzymes that degrade hemicellulose, including Cb193, Cb195, Cb1172, Cb909, Cb2487, and Cb162. Cb193 and Cb195 function as endoxylanases. Cb1172 functions as an α-arabinofuranosidase. Cb909 functions as a glucuronidase. Cb2487 functions as a β-xylosidase. Cb162 functions as an acetyl xylan esterase. Variants of the enzymes that retain partial or complete functional activity are also encompassed by the present disclosure. The enzymes disclosed herein can be used in various combinations.
In one aspect, the disclosure provides improved enzyme mixtures for the degradation of cellulose-containing materials. Improved enzyme mixtures for the degradation of cellulose-containing materials may contain, for example, improved mixtures of cellulases and/or truncated cellulase enzymes.
In another aspect, the disclosure provides improved enzyme mixtures for the degradation of materials containing both cellulose and hemicellulose. Enzyme mixtures disclosed herein containing both cellulases and hemicellulases provide the surprising result of synergistic activity on plant material containing both cellulose and hemicellulose. For example, as shown in Example 15 below, an enzyme cocktail provided herein containing a mixture of cellulases and a mixture of hemicellulases has greater cellulase activity on plant material than the same mixture of cellulases alone. Additionally, the enzyme cocktail containing a mixture of cellulases and a mixture of hemicellulases has greater hemicellulase activity on plant material than the same mixture of hemicellulases alone.
Combinations of enzymes, i.e., an enzyme cocktail, can be tailored to the cellulose and/or hemicellulose structure of a specific feedstock to increase the level of degradation. Initial analysis of the enzyme cocktails described herein suggests that the components have a long shelf life, an important characteristic in an industrial enzyme mix.
Abbreviations/Definitions
The following abbreviations are used in the present disclosure: TLC (thin layer chromatography); SWAX (soluble wheat arabinoxylan); OSX (oat-spelt xylan); BWX (birchwood xylan); CMC (carboxymethyl cellulose); RAX (rye arabinoxylan); MeGlcA (4-O-methyl-D-glucuronosyl); pNP-X (para-nitrophenyl-beta-D-xylopyranoside); GH (glycoside hydrolase); CBM (carbohydrate binding module); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); PASC (phosphoric acid swollen cellulose); CMC-Na (sodium carboxymethyl cellulose); LBG (locust bean gum); KGM (konjac glucomannan); WAX (wheat arabinoxylan); HPAEC (high performance anion-exchange chromatography); HPLC (high performance liquid chromatography)
As used herein, a “polypeptide” is a chain of consecutive polymerized amino acid residues (e.g., at least about 5 consecutive polymerized amino acid residues). As used herein, the terms “polypeptide”, “protein”, and “amino acid sequence” are used interchangeably.
As used herein, “cellulase” activity refers to enzymatic activity which cleaves 1-4 β-D-glycosidic linkages between glucose molecules in cellulose and/or cellooligosaccharides. Cellulase activity includes endoglucanase, exoglucanase, and beta-glucosidase activity.
As used herein, “hemicellulase” activity refers to enzymatic activity which cleaves a bond in a molecule that is a component of hemicellulose, including endoxylanase, α-arabinofuranosidase, glucuronidase, β-xylosidase, and acetyl xylan esterase activity.
Polypeptides of the Disclosure
In some aspects, polypeptides of the disclosure relate to recombinant polypeptides of the thermophilic bacterium Caldicellulosiruptor bescii (formerly Anaerocellum thermophilum DSMZ 6725), truncations, and variations thereof.
In one aspect, the present disclosure provides recombinant polypeptides related to the degradation of cellulose. In some aspects, the disclosure provides recombinant Cb1952, Cb1953, Cb1954, Cb1946, Cb629, and Cb486 polypeptides which have cellulase activity.
In one aspect, the present disclosure provides recombinant polypeptides related to the degradation of hemicellulose. In some aspects, the disclosure provides recombinant Cb193, Cb195, Cb1172, Cb909, Cb2487, and Cb162 polypeptides which have hemicellulase activity.
In one aspect, the present disclosure provides recombinant polypeptides that enhance the hydrolysis of cellulose and/or hemicellulose during treatment of cellulose and/or hemicellulose with cellulase and/or hemicellulases. In one aspect, the disclosure provides recombinant Cb1581 polypeptide, which is a heat shock protein that enhances the hydrolysis of cellulose and/or hemicellulose during treatment of cellulose and/or hemicellulose with cellulase and/or hemicellulases.
Cellulases
Cb1952 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb1952 polypeptides. As used herein, a “Cb1952 polypeptide” refers to the polypeptide of SEQ ID NO: 44, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have cellulase activity. “Cb1952 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46. As used herein, “Cb1952 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46.
The polypeptide of SEQ ID NO: 44 is the product of the Cb1952 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb1952 polypeptide of SEQ ID NO: 44 is an endocellulase that has a glycoside hydrolase (GH) family 9 catalytic domain (cellulase domain), three family 3 carbohydrate binding modules (CBMs) and one GH5 catalytic domain (mannanase domain) (FIG. 18)
The present disclosure also includes the Cb1952 polypeptide of SEQ ID NO: 114, which is the Cb1952 polypeptide of SEQ ID NO: 44 without the signal peptide sequence. The signal peptide is produced as part of the initially translated Cb1952 protein to target the protein for secretion from the cell, and it may be cleaved from the protein during the secretion process. The disclosure also includes the Cb1952 polypeptide of SEQ ID NO: 114 with a methionine residue at the start of the polypeptide chain.
The disclosure further includes the Cb1952 polypeptide of SEQ ID NO: 46 (“Cb1952TM1”), which is a truncational mutant (“TM”) of wild-type Cb1952. The Cb1952TM1 polypeptide includes the cellulase domain and CBMs of wt Cb1952, but does not include the mannase domain (FIG. 18).
The disclosure also includes the Cb1952 polypeptide of SEQ ID NO: 124 (“Cb1952TM2”), which is a truncational mutant of wild-type Cb1952 that does not include the mannase domain or the C-terminal CBM (FIG. 18).
The disclosure also includes the Cb1952 polypeptide of SEQ ID NO: 126 (“Cb1952TM3”), which is a truncational mutant of wild-type Cb1952 that does not include the mannase domain or the 2 most C-terminal CBMs (FIG. 18).
The disclosure also includes the Cb1952 polypeptide of SEQ ID NO: 128 (“Cb1952TM4”), which is a truncational mutant of wild-type Cb1952 that includes the GH9 cellulase domain, but that does not contain any of the CBMs or the mannose domain (FIG. 18).
Cb1952 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb1952 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb1952 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb1953 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb1953 polypeptides. As used herein, a “Cb1953 polypeptide” refers to the polypeptide of SEQ ID NO: 60, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have cellulase activity. “Cb1953 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 60, 61, and 111. As used herein, “Cb1953 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 60, 61, and 111.
The Cb1953 polypeptide of SEQ ID NO: 60 is the product of the Cb1953 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb1953 polypeptide of SEQ ID NO: 60 is an endoglucanase that cleaves mostly cellobiose from cellulose, and it has two glycoside hydrolase (GH) family 5 catalytic domains and 3 carbohydrate binding modules (CBM) (FIG. 29).
The present disclosure also includes the Cb1953 polypeptide of SEQ ID NO: 61, which is the Cb1953 polypeptide of SEQ ID NO: 60 without the signal peptide sequence. The signal peptide is produced as part of the initially translated Cb1953 protein to target the protein for secretion from the cell, and it may be cleaved from the protein during the secretion process. The disclosure also includes the Cb1953 polypeptide of SEQ ID NO: 61 with a methionine residue at the start of the polypeptide chain.
The disclosure further includes the Cb1953 polypeptide of SEQ ID NO: 111 (“Cb1953TM2”), which is a truncational mutant (“TM”) of wild-type Cb1953. The Cb1953TM2 polypeptide includes the C-terminal GH5 domain and the 3 CBMs of wt Cb1953, but does not include the N-terminal GH5 domain. (FIG. 29).
Cb1953 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb1953 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb1953 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb1954 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb1954 polypeptides. As used herein, a “Cb1954 polypeptide” refers to the polypeptide of SEQ ID NO: 74, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have cellulase activity. “Cb1954 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 74, 121, and 76. As used herein, “Cb1954 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 74, 121, and 76.
The Cb1954 polypeptide of SEQ ID NO: 74 is the product of the Cb1954 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb1954 polypeptide of SEQ ID NO: 74 is an endoglucanase that has a glycoside hydrolase (GH) family 9 catalytic domain (a cellulase domain), 3 carbohydrate binding modules (CBM), and one GH48 catalytic domain (FIG. 38).
The present disclosure also includes the Cb1954 polypeptide of SEQ ID NO: 121, which is the Cb1954 polypeptide of SEQ ID NO: 74 without the signal peptide sequence. The signal peptide is produced as part of the initially translated Cb1954 protein to target the protein for secretion from the cell, and it may be cleaved from the protein during the secretion process. The disclosure also includes the Cb1954 polypeptide of SEQ ID NO: 121 with a methionine residue at the start of the polypeptide chain.
The disclosure further includes the Cb1954 polypeptide of SEQ ID NO: 76 (“Cb1954TM3”), which is a truncational mutant (“TM”) of wild-type Cb1954. The Cb1954TM3 polypeptide includes the GH9 domain and the N-terminal-most CBM of wt Cb1954, but does not include the middle or C-terminal CBM, or the GH48 domain. (FIG. 38).
Cb1954 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb1954 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb1954 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb1946 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb1946 polypeptides. As used herein, a “Cb1946 polypeptide” refers to the polypeptide of SEQ ID NO: 86, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have cellulase activity. “Cb1946 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 86, 87, and 113. As used herein, “Cb1946 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 86, 87, and 113.
The Cb1946 polypeptide of SEQ ID NO: 86 is the product of the Cb1946 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb1946 polypeptide of SEQ ID NO: 86 is an endoglucanase that has a glycoside hydrolase (GH) family 5 catalytic domain at the N-terminal region, a GH family 44 catalytic domain at the C-terminal region and 2 carbohydrate binding modules (CBMs) between the two GH catalytic domains (FIG. 42).
The present disclosure also includes the Cb1946 polypeptide of SEQ ID NO: 87, which is the Cb1946 polypeptide of SEQ ID NO: 86 without the signal peptide sequence. The signal peptide is produced as part of the initially translated Cb1946 protein to target the protein for secretion from the cell, and it may be cleaved from the protein during the secretion process. The disclosure also includes the Cb1946 polypeptide of SEQ ID NO: 87 with a methionine residue at the start of the polypeptide chain.
The disclosure further includes the Cb1946 polypeptide of SEQ ID NO: 113 (“Cb1946TM2”), which is a truncational mutant (“TM”) of wild-type Cb1946. The Cb1946TM2 polypeptide includes the C-terminal GH44 domain and the 2 CBMs of wt Cb1946, but does not include the N-terminal GH5 domain. (FIG. 42).
Cb1946 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb1946 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb1946 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb629 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb629 polypeptides. As used herein, a “Cb629 polypeptide” refers to the polypeptide of SEQ ID NO: 98, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have cellulase activity. “Cb629 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 98, 119, and 100. As used herein, “Cb629 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 98, 119, and 100.
The Cb629 polypeptide of SEQ ID NO: 98 is the product of the Cb629 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb629 polypeptide of SEQ ID NO: 98 is an endocellulase that initially cleaves glucose, cellobiose, and cellotriose from cellulose, and it has a glycoside hydrolase (GH) family 5 catalytic domain, a Carbohydrate Binding Module (CBM) family 17_28 domain, and three surface layer homology (SLH) modules likely used in anchoring the enzyme to the cell surface (FIG. 47).
The present disclosure also includes the Cb629 polypeptide of SEQ ID NO: 119, which is the Cb629 polypeptide of SEQ ID NO: 98 without the signal peptide sequence. The signal peptide is produced as part of the initially translated Cb629 protein to target the protein for secretion from the cell, and it may be cleaved from the protein during the secretion process. The disclosure also includes the Cb629 polypeptide of SEQ ID NO: 119 with a methionine residue at the start of the polypeptide chain.
The disclosure further includes the Cb629 polypeptide of SEQ ID NO: 100 (“Cb629TM1”), which is a truncational mutant (“TM”) of wild-type Cb629. The Cb629TM1 polypeptide includes the N-terminal GH5 domain and the CBM17_28 domain of wt Cb629, but does not include the C-terminal SLH modules (FIG. 47).
Cb629 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb629 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb629 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb486 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb486 polypeptides.
As used herein, a “Cb486 polypeptide” refers to the polypeptide of SEQ ID NO: 106, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have cellulase activity. “Cb486 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 106. As used herein, “Cb486 polypeptide” also refers to a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 106.
The Cb486 polypeptide of SEQ ID NO: 106 is the product of the Cb486 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb486 polypeptide of SEQ ID NO: 106 is a β-glucosidase that catalyzes the hydrolysis of cellobiose (a disaccharide of glucose) into two units of glucose, and it has a glycoside hydrolase (GH) family 1 catalytic domain (FIG. 53A).
Cb486 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb486 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb486 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Hemicellulases
The disclosure also provides for polypeptides of thermostable hemicellulose-degrading enzymes Cb193 (SEQ ID NO: 3), Cb195 (SEQ ID NO: 7), Cb1172 (SEQ ID NO: 13), Cb909 (SEQ ID NO: 19), Cb2487 (SEQ ID NO: 27), and Cb162 (SEQ ID NO: 33), or subsequences thereof. The disclosure further provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to Cb193, Cb195, Cb1172, Cb2487, Cb909 or Cb162.
Hemicellulases of the present disclosure may contain one or more glycoside hydrolase (GH) domains. Hemicellulases may also contain one or more carbohydrate binding modules (CBM). The CBM modules may interrupt a GH domain or be located in between two GH domains. Hemicellulases may also contain an acetyl xylan esterase domain. In certain embodiments, the GH, CBM and/or acetyl xylan esterase domain sequence is conserved in polypeptide variants.
Cb193 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb193 polypeptides. As used herein, a “Cb193 polypeptide” refers to the polypeptide of SEQ ID NO: 3, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have hemicellulase activity. “Cb193 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 3 and/or 37. As used herein, “Cb193 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 3 and/or 37.
The Cb193 polypeptide of SEQ ID NO: 3 is the product of the Cb193 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb193 polypeptide of SEQ ID NO: 3 or 37 is an endoxylanase cleaves the xylose backbone of hemicellulose at random to generate shorter chains of xylose in β-1,4-linkages. These xylo-oligosaccharides can range from two or more sugar subunits. Cb193 has a signal peptide (corresponding to amino acids 1-41 of SEQ ID NO: 3), which may be removed. The amino acid sequence of the Cb193 protein without the signal peptide is disclosed in SEQ ID NO: 37. The protein has two putative carbohydrate binding modules (CBM) inserted within the glycoside hydrolase (GH) family 10 catalytic domain (FIG. 2A).
The present disclosure also includes the Cb193 polypeptide of SEQ ID NO: 37, which is the Cb193 polypeptide of SEQ ID NO: 3 without the signal peptide sequence. The signal peptide is produced as part of the initially translated Cb193 protein to target the protein for secretion from the cell, and it may be cleaved from the protein during the secretion process. The disclosure also includes the Cb193 polypeptide of SEQ ID NO: 37 with a methionine residue at the start of the polypeptide chain.
Cb193 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb193 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb193 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb195 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb195 polypeptides. As used herein, a “Cb195 polypeptide” refers to the polypeptide of SEQ ID NO: 7, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have hemicellulase activity. “Cb195 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 7. As used herein, “Cb195 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 7.
The Cb195 polypeptide of SEQ ID NO: 7 is the product of the Cb195 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb195 polypeptide of SEQ ID NO: 7 is an endoxylanase that cleaves the xylose backbone of hemicellulose at random to generate shorter chains of xylose in β-1,4-linkages. These xylo-oligosaccharides can range from containing two or more sugar subunits.
Cb195 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb195 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb195 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb1172 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb1172 polypeptides. As used herein, a “Cb1172 polypeptide” refers to the polypeptide of SEQ ID NO: 13, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have hemicellulase activity. “Cb1172 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 13. As used herein, “Cb1172 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 13.
The Cb1172 polypeptide of SEQ ID NO: 13 is the product of the Cb1172 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb1172 polypeptide of SEQ ID NO: 13 is an α-L-arabinofuranosidase that cleaves arabinose moiety from the xylose backbone or from branched or debranched arabinan of hemicellulose to generate exclusively arabinose. The protein has a glycoside hydrolase (GH) family 51 catalytic domain (FIG. 6D).
Cb1172 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb1172 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb1172 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb909 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb909 polypeptides. As used herein, a “Cb909 polypeptide” refers to the polypeptide of SEQ ID NO: 19, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have hemicellulase activity. “Cb909 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 19. As used herein, “Cb909 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 19.
The Cb909 polypeptide of SEQ ID NO: 19 is the product of the Cb909 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb909 polypeptide of SEQ ID NO: 19 is an α-glucuronidase that cleaves the α-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and the β-1,4-xylosidic linkage backbone of xylan.
Cb909 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb909 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb909 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb2487 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb2487 polypeptides. As used herein, a “Cb2487 polypeptide” refers to the polypeptide of SEQ ID NO: 27, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have hemicellulase activity. “Cb2487 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 27. As used herein, “Cb2487 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 27.
The Cb2487 polypeptide of SEQ ID NO: 27 is the product of the Cb2487 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb2487 polypeptide of SEQ ID NO: 27 is a β-xylosidase.
Cb2487 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb2487 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb2487 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Cb162 Polypeptides
In some aspects, the present disclosure relates to recombinant Cb162 polypeptides. As used herein, a “Cb162 polypeptide” refers to the polypeptide of SEQ ID NO: 33, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have hemicellulase activity. “Cb162 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 33. As used herein, “Cb162 polypeptide” also refers to a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 33.
The Cb162 polypeptide of SEQ ID NO: 33 is the product of the Cb162 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb162 polypeptide of SEQ ID NO: 33 is an acetyl xylan esterase that cleaves the linkages between xylose and the side chain of acetyl groups in hemicellulose to provide more accessibility to other hemicellulases such as xylanase and beta-xylosidase to the backbone of xylan. The protein has a single domain of acetyl xylan esterase (FIG. 10A).
Cb162 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb162 polypeptide of the present disclosure has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb162 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Polypeptides that Enhance Enzymatic Hydrolysis of Cellulose and/or Hemicellulose
In some aspects, the disclosure provides for recombinant polypeptides that enhance the enzymatic hydrolysis of cellulose and/or hemicellulose.
In one aspect, a recombinant polypeptide that enhances the enzymatic hydrolysis of cellulose and/or hemicellulose is a recombinant Cb1581 polypeptide.
As used herein, a “Cb1581 polypeptide” refers to the polypeptide of SEQ ID NO: 146, and truncational mutants thereof, homologs thereof, and truncational mutants of homologs thereof, which have enzymatic hydrolysis of cellulose and/or hemicellulose-enhancing activity. “Cb1581 polypeptide” also refers to a polypeptide that has enzymatic hydrolysis of cellulose and/or hemicellulose-enhancing activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 146. As used herein, “Cb1581 polypeptide” also refers to a polypeptide that has enzymatic hydrolysis of cellulose and/or hemicellulose-enhancing activity, and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 146.
The Cb1581 polypeptide of SEQ ID NO: 146 is the product of the Cb1581 gene in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The Cb1581 polypeptide is a small heat shock protein.
Cb1581 polypeptides of the present disclosure are thermophilic and thermostable. In some aspects, a Cb1581 polypeptide of the present disclosure has peak enzymatic hydrolysis of cellulose and/or hemicellulose-enhancing activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, a Cb1581 polypeptide of the present disclosure retains at least 60% of its initial rate of enzymatic hydrolysis of cellulose and/or hemicellulose-enhancing activity for a period of at least 24 hours when incubated at a temperature of about 55, 60, 65, 70, 75, 80, 85, or 90° C.
Polypeptides with Protein “Tags”
Polypeptides of the disclosure further include any of the recombinant polypeptides disclosed herein with a polypeptide “tag.” Polypeptide tags are polypeptides that may be attached to a protein of interest through gene cloning, and may be used to facilitate the purification, increase the solubility, and/or increase the stability of the “tagged” protein. Protein tags are well known in the art and include, without limitation, poly-histidine (e.g. 6 consecutive His-residues), glutathione S-transferase (GST), T7, FLAG, hemagglutinin (HA), MYC and maltose-binding protein (MBP) tags.
Production of Polypeptides
The polypeptides can be expressed in and purified from their native host, Caldicellulosiruptor bescii. Polypeptides may also be expressed in and purified from transgenic expression systems. Transgenic expression systems can be prokaryotic or eukaryotic. Transgenic host cells may include yeast and E. coli. Transgenic host cells may secrete the polypeptide out of the host cell. In certain embodiments, the isolated or recombinant polypeptide lacks a signal sequence. Methods for the production of recombinant polypeptides are further discussed infra.
Nucleic Acids of the Disclosure
The present disclosure further provides recombinant nucleic acids that encode any of the polypeptides disclosed herein. Nucleic acids that encode a polypeptide are also referred to herein as “genes”. Methods for determining the relationship between a polypeptide and a nucleic acid that encodes the polypeptide are well known to one of skill in the art. Similarly, methods of determining the polypeptide sequence encoded by a polynucleotide sequence are well known to one of skill in the art. Due to codon degeneracy, multiple different nucleic acid sequences may encode the same polypeptide sequence.
As used herein, the terms, “nucleic acid” “polynucleotide”, and variations thereof are generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing non-nucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog, and inter-nucleotide modifications. As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature.
As used herein, more than one “nucleic acid” or “polynucleotide” may be present in a single contiguous polydeoxyribonucleotide chain/strand of DNA. Thus, a single strand of DNA (such as in a plasmid) may contain more than one “nucleic acid” or “polynucleotide”, and thus, may contain sequences encoding more than one different polypeptide.
The nucleic acids may be synthesized, isolated, or manipulated using standard molecular biology techniques such as those described in Sambrook, J. et al. 2000. Molecular Cloning: A Laboratory Manual (Third Edition). Techniques may include cloning, expression of cDNA libraries, and amplification of mRNA or genomic DNA.
The nucleic acids of the present disclosure, or subsequences thereof, may be incorporated into a cloning vehicle comprising an expression cassette or vector. The cloning vehicle can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, or an artificial chromosome. The viral vector can comprise an adenovirus vector, a retroviral vector, or an adeno-associated viral vector. The cloning vehicle can comprise a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
The nucleic acids may be operably linked to a promoter. The promoter can be a viral, bacterial, mammalian or plant promoter. The promoter can be a constitutive promoter, an inducible promoter, a tissue-specific promoter, or an environmentally regulated or a developmentally regulated promoter.
Nucleic Acids that Encode Cellulases
Cb1952 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb1952 polypeptide of the disclosure. In some aspects, the disclosure includes recombinant polynucleotides that encode a polypeptide of SEQ ID NOs: 44, 114, 124, 126, 128, or 46.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46.
In some aspects, the disclosure includes the recombinant polynucleotides of SEQ ID NOs: 45, 115, 125, 127, 129, and 47. The polynucleotide of SEQ ID NO: 45 encodes the polypeptide of SEQ ID NO: 44. The polynucleotide of SEQ ID NO: 115 encodes the polypeptide of SEQ ID NO: 45. The polynucleotide of SEQ ID NO: 47 encodes the polypeptide of SEQ ID NO: 46. The polynucleotide of SEQ ID NO: 125 encodes the polypeptide of SEQ ID NO: 124. The polynucleotide of SEQ ID NO: 127 encodes the polypeptide of SEQ ID NO: 126. The polynucleotide of SEQ ID NO: 129 encodes the polypeptide of SEQ ID NO: 128.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the sequences of SEQ ID NOs: 45, 115, 125, 127, 129, and 47, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of any of the sequences of SEQ ID NOs: 45, 115, 125, 127, 129, and 47, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb1952 polypeptides disclosed herein.
Cb1953 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb1953 polypeptide of the disclosure. In some aspects, the disclosure includes recombinant polynucleotides that encode a polypeptide of SEQ ID NOs: 60, 61, or 111.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 60, 61, and 111. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 60, 61, and 111.
In some aspects, the disclosure includes the recombinant polynucleotides of SEQ ID NOs: 62, 63, or 110. The polynucleotide of SEQ ID NO: 62 encodes the polypeptide of SEQ ID NO: 60. The polynucleotide of SEQ ID NO: 63 encodes the polypeptide of SEQ ID NO: 61. The polynucleotide of SEQ ID NO: 110 encodes the polypeptide of SEQ ID NO: 111.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the sequences of SEQ ID NOs: 62, 63, and 110, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 60, 61, and 111. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of any of the sequences of SEQ ID NOs: 62, 63, or 110, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 60, 61, and 111.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb1953 polypeptides disclosed herein.
Cb1954 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb1954 polypeptide of the disclosure. In some aspects, the disclosure includes recombinant polynucleotides that encode a polypeptide of SEQ ID NOs: 74, 121, or 76.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 74, 121, and 76. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 74, 121, and 76.
In some aspects, the disclosure includes the recombinant polynucleotides of SEQ ID NOs: 116, 75, or 77. The polynucleotide of SEQ ID NO: 116 encodes the polypeptide of SEQ ID NO: 74. The polynucleotide of SEQ ID NO: 75 encodes the polypeptide of SEQ ID NO: 121. The polynucleotide of SEQ ID NO: 77 encodes the polypeptide of SEQ ID NO: 76.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the sequences of SEQ ID NOs: 116, 75, and 77, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 74, 121, and 76. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of any of the sequences of SEQ ID NOs: 116, 75, and 77, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 74, 121, and 76.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb1954 polypeptides disclosed herein.
Cb1946 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb1946 polypeptide of the disclosure. In some aspects, the disclosure includes recombinant polynucleotides that encode a polypeptide of SEQ ID NOs: 86, 87, or 113.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 86, 87, and 113. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 86, 87, and 113.
In some aspects, the disclosure includes the recombinant polynucleotides of SEQ ID NOs: 88, 89, or 112. The polynucleotide of SEQ ID NO: 88 encodes the polypeptide of SEQ ID NO: 86. The polynucleotide of SEQ ID NO: 89 encodes the polypeptide of SEQ ID NO: 87. The polynucleotide of SEQ ID NO: 112 encodes the polypeptide of SEQ ID NO: 113.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the sequences of SEQ ID NOs: 88, 89, and 112, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 86, 87, and 113. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of any of the sequences of SEQ ID NOs: 88, 89, or 112, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 86, 87, and 113.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb1946 polypeptides disclosed herein.
Cb629 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb629 polypeptide of the disclosure. In some aspects, the disclosure includes recombinant polynucleotides that encode a polypeptide of SEQ ID NOs: 98, 119, or 100.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 98, 119, and 100. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 98, 119, and 100.
In some aspects, the disclosure includes the recombinant polynucleotides of SEQ ID NOs: 99, 120, or 101. The polynucleotide of SEQ ID NO: 99 encodes the polypeptide of SEQ ID NO: 98. The polynucleotide of SEQ ID NO: 120 encodes the polypeptide of SEQ ID NO: 119. The polynucleotide of SEQ ID NO: 101 encodes the polypeptide of SEQ ID NO: 100.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the sequences of SEQ ID NOs: 99, 120, or 101, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 98, 119, and 100. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of any of the sequences of SEQ ID NOs: 99, 120, or 101, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 98, 119, and 100.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb629 polypeptides disclosed herein.
Cb486 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb486 polypeptide of the disclosure. In some aspects, the disclosure includes recombinant polynucleotides that encode the polypeptide of SEQ ID NO: 106.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 106. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has cellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 106.
In some aspects, the disclosure includes the recombinant polynucleotide of SEQ ID NO: 107. The polynucleotide of SEQ ID NO: 107 encodes the polypeptide of SEQ ID NO: 106.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 107, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 106. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of the sequence of SEQ ID NO: 107, and that encode a polypeptide that has cellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 106.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb486 polypeptides disclosed herein.
Nucleic Acids that Encode Hemicellulases
The present disclosure provides nucleotide sequences encoding the hemicellulose-degrading enzymes Cb193 (SEQ ID NO: 4), Cb195 (SEQ ID NO: 8), Cb1172 (SEQ ID NO: 14), Cb909 (SEQ ID NO: 20), Cb2487 (SEQ ID NO: 28), and Cb162 (SEQ ID NO: 34), or subsequences thereof. The disclosure also provides for nucleotide sequences having at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to the nucleic acid sequences encoding Cb193, Cb195, Cb1172, Cb909, Cb2487, and Cb162.
Nucleotide sequences of the present disclosure may encode polypeptides with one or more glycoside hydrolase (GH) domains. Nucleotide sequences may also encode polypeptides with one or more carbohydrate binding modules (CBM). The CBM modules may interrupt a GH domain or be located in between two GH domains. Nucleotide sequences may also encode polypeptides with an acetyl xylan esterase domain. In certain embodiments, the GH, CBM and/or acetyl xylan esterase domain sequence is conserved in nucleotide variants.
Cb193 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb193 polypeptide of the disclosure. In some aspects, the disclosure includes recombinant polynucleotides that encode a polypeptide of SEQ ID NOs: 3 or 37.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 3 and/or 37. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of any of the polypeptides of SEQ ID NOs: 3 and/or 37.
In some aspects, the disclosure includes the recombinant polynucleotides of SEQ ID NOs: 4 or 38. The polynucleotide of SEQ ID NO: 4 encodes the polypeptide of SEQ ID NO: 3. The polynucleotide of SEQ ID NO: 38 encodes the polypeptide of SEQ ID NO: 37.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the sequences of SEQ ID NOs: 4 and/or 38, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 3 and/or 37. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of any of the sequences of SEQ ID NOs: 4 or 38, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of any of the polypeptides of SEQ ID NOs: 3 and/or 37.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb193 polypeptides disclosed herein.
Cb195 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb195 polypeptide of the disclosure. In some aspects, the disclosure includes a recombinant polynucleotide that encodes a polypeptide of SEQ ID NO: 7.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 7. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 7.
In some aspects, the disclosure includes the recombinant polynucleotide of SEQ ID NO: 8. The polynucleotide of SEQ ID NO: 8 encodes the polypeptide of SEQ ID NO: 7.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 8, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 7. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of the sequence of SEQ ID NO: 8, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence the polypeptide of SEQ ID NO: 7.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb195 polypeptides disclosed herein.
Cb1172 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb1172 polypeptide of the disclosure. In some aspects, the disclosure includes a recombinant polynucleotide that encodes a polypeptide of SEQ ID NO: 13.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 13. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 13.
In some aspects, the disclosure includes the recombinant polynucleotide of SEQ ID NO: 14. The polynucleotide of SEQ ID NO: 14 encodes the polypeptide of SEQ ID NO: 13.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 14, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 13. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of the sequence of SEQ ID NO: 14, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence the polypeptide of SEQ ID NO: 13.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb1172 polypeptides disclosed herein.
Cb909 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb909 polypeptide of the disclosure. In some aspects, the disclosure includes a recombinant polynucleotide that encodes a polypeptide of SEQ ID NO: 19.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 19. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 19.
In some aspects, the disclosure includes the recombinant polynucleotide of SEQ ID NO: 20. The polynucleotide of SEQ ID NO: 20 encodes the polypeptide of SEQ ID NO: 19.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 20, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 19. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of the sequence of SEQ ID NO: 20, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence the polypeptide of SEQ ID NO: 19.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb909 polypeptides disclosed herein.
Cb2487 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb2487 polypeptide of the disclosure. In some aspects, the disclosure includes a recombinant polynucleotide that encodes a polypeptide of SEQ ID NO: 27.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 27. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 27.
In some aspects, the disclosure includes the recombinant polynucleotide of SEQ ID NO: 28. The polynucleotide of SEQ ID NO: 28 encodes the polypeptide of SEQ ID NO: 27.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 28, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 27. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of the sequence of SEQ ID NO: 28, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence the polypeptide of SEQ ID NO: 27.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb2487 polypeptides disclosed herein.
Cb162 Polynucleotides
The present disclosure includes recombinant polynucleotides that encode a Cb162 polypeptide of the disclosure. In some aspects, the disclosure includes a recombinant polynucleotide that encodes a polypeptide of SEQ ID NO: 33.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 33. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that has hemicellulase activity and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 33.
In some aspects, the disclosure includes the recombinant polynucleotide of SEQ ID NO: 34. The polynucleotide of SEQ ID NO: 34 encodes the polypeptide of SEQ ID NO: 33.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 34, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 33. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of the sequence of SEQ ID NO: 34, and that encode a polypeptide that has hemicellulase activity and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence the polypeptide of SEQ ID NO: 33.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb162 polypeptides disclosed herein.
Nucleic Acids that Encode Polypeptides that Enhance Enzymatic Hydrolysis of Cellulose and/or Hemicellulose
The present disclosure includes recombinant polynucleotides that encode a Cb1581 polypeptide of the disclosure. In some aspects, the disclosure includes recombinant polynucleotides that encode the polypeptide of SEQ ID NO: 146.
Polynucleotides of the disclosure include recombinant polynucleotides that encode a polypeptide that enhances enzymatic hydrolysis of cellulose and/or hemicellulose and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 146. Polynucleotides of the disclosure also include recombinant polynucleotides that encode a polypeptide that enhances enzymatic hydrolysis of cellulose and/or hemicellulose and that has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 consecutive amino acids of the polypeptide of SEQ ID NO: 146.
In some aspects, the disclosure includes the recombinant polynucleotide of SEQ ID NO: 147. The polynucleotide of SEQ ID NO: 147 encodes the polypeptide of SEQ ID NO: 146.
Polynucleotides of the disclosure also include recombinant polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 147, and that encode a polypeptide that enhances enzymatic hydrolysis of cellulose and/or hemicellulose and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 146. Polynucleotides of the disclosure also include recombinant polynucleotides that have at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 consecutive nucleotides of the sequence of SEQ ID NO: 147, and that encode a polypeptide that enhances enzymatic hydrolysis of cellulose and/or hemicellulose and that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of the polypeptide of SEQ ID NO: 146.
Polynucleotides of the disclosure further include recombinant polynucleotides that are complementary to polynucleotides that encode Cb1581 polypeptides disclosed herein.
Recombinant Polynucleotides Encoding Polypeptides with Protein “Tags”
Further disclosed herein are recombinant polynucleotides that encode polypeptides of the disclosure with a polypeptide “tag.” Polynucleotides that encode a polypeptide “tag” may be added to a polynucleotide encoding a polypeptide of the disclosure by standard molecular biology cloning techniques. (See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2001)).
Variants, Sequence Identity, and Sequence Similarity
Methods of alignment of sequences for comparison are well-known in the art. For example, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11 17; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443 453; the search-for-similarity-method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444 2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873 5877.
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237 244 (1988); Higgins et al. (1989) CABIOS 5:151 153; Corpet et al. (1988) Nucleic Acids Res. 16:10881 90; Huang et al. (1992) CABIOS 8:155 65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307 331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. BLAST is available, for example, from the National Center for Biotechnology Information (NCBI). Alignment may also be performed manually by inspection.
As used herein “sequence identity” refers to the percentage of residues that are identical in the same positions in the sequences being analyzed. As used herein “sequence similarity” refers to the percentage of residues that have similar biophysical/biochemical characteristics in the same positions (e.g. charge, size, hydrophobicity) in the sequences being analyzed.
The functional activity of enzyme variants can be evaluated using standard molecular biology techniques including thin layer chromatography or a reducing sugar assay. Enzymatic activity can be determined using cellulose, hemicellulose or an artificial substrate.
Compositions
The present disclosure further includes compositions containing one or more recombinant polypeptides disclosed herein. In some aspects, provided herein are compositions containing two or more recombinant polypeptides disclosed herein. Compositions containing two or more recombinant polypeptides may be referred to as a “cocktail” of polypeptides and/or enzymes.
In some aspects, disclosed herein are compositions that contain one or more recombinant polypeptides disclosed herein, wherein the one or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides.
In some aspects, disclosed herein are compositions that contain two or more recombinant polypeptides disclosed herein, wherein the two or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides.
In some aspects, disclosed herein are compositions that contain three or more recombinant polypeptides disclosed herein, wherein the three or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides.
In some aspects, disclosed herein are compositions that contain four or more recombinant polypeptides disclosed herein, wherein the four or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides.
In some aspects, disclosed herein are compositions that contain five or more recombinant polypeptides disclosed herein, wherein the five or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides.
In some aspects, disclosed herein are compositions that contain six or more recombinant polypeptides disclosed herein, wherein the six or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides.
In some aspects, disclosed herein are compositions that contain one or more recombinant polypeptides disclosed herein, wherein the one or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, disclosed herein are compositions that contain two or more recombinant polypeptides disclosed herein, wherein the two or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, disclosed herein are compositions that contain three or more recombinant polypeptides disclosed herein, wherein the three or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, disclosed herein are compositions that contain four or more recombinant polypeptides disclosed herein, wherein the four or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, disclosed herein are compositions that contain five or more recombinant polypeptides disclosed herein, wherein the five or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, disclosed herein are compositions that contain six or more recombinant polypeptides disclosed herein, wherein the six or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, disclosed herein are compositions that contain two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
In some aspects, disclosed herein are compositions that contain three or more recombinant polypeptides disclosed herein, wherein the three or more polypeptides are selected from the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
In some aspects, disclosed herein are compositions that contain four or more recombinant polypeptides disclosed herein, wherein the four or more polypeptides are selected from the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
In some aspects, disclosed herein are compositions that contain five or more recombinant polypeptides disclosed herein, wherein the five or more polypeptides are selected from the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
In some aspects, disclosed herein are compositions that contain six recombinant polypeptides disclosed herein, wherein the six polypeptides are the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
Any of the compositions disclosed herein containing one or more recombinant cellulases disclosed herein may further contain one or more recombinant hemicellulases. Hemicellulases include, without limitation, endoxylanases, exoxylanases, α-arabinofuranosidases, glucuronidases, β-xylosidases, and acetyl xylan esterases. In some aspects, hemicellulases include the polypeptides that contain the amino acid sequence of any of SEQ ID NOs: 3, 7, 13, 19, 27, 33, or 37. Any of the compositions disclosed herein containing one or more recombinant cellulases disclosed herein may further contain an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose. In one aspect, an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose contains the amino acid sequence of SEQ ID NO: 146.
The present disclosure provides for compositions including the recombinant amino acid sequence of Cb193 alone or in combination with one or more of the recombinant amino acid sequences of Cb195, Cb1172, Cb909, Cb2487 and Cb162. The present disclosure also provides for compositions including the recombinant amino acid sequence of Cb195 alone or in combination with one or more of the recombinant amino acid sequences of Cb193, Cb1172, Cb909, Cb2487 and Cb162. The present disclosure also provides for compositions including the recombinant amino acid sequence of Cb1172 alone or in combination with one or more of the recombinant amino acid sequences of Cb193, Cb195, Cb909, Cb2487 and Cb162. The present disclosure also provides for compositions including the recombinant amino acid sequence of Cb909 alone or in combination with one or more of the recombinant amino acid sequences of Cb193, Cb195, Cb1172, Cb2487 and Cb162. The present disclosure also provides for compositions including the recombinant amino acid sequence of Cb2487 alone or in combination with one or more of the recombinant amino acid sequences of Cb193, Cb195, Cb1172, Cb909 and Cb162. The present disclosure also provides for compositions including the recombinant amino acid sequence of Cb162 alone or in combination with one or more of the recombinant amino acid sequences of Cb193, Cb195, Cb1172, Cb2487 and Cb909.
The present disclosure also provides for compositions including two or more of the recombinant amino acid sequences of Cb193, Cb195, Cb1172, Cb909, Cb2487, and Cb162. One composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb1172, Cb909, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb195, Cb1172, Cb909, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb1172, Cb909, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb909, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb1172, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb1172, Cb909, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb1172, Cb909, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb1172, Cb909, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb195, Cb909, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb195, Cb1172, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb195, Cb1172, Cb909, and Cb162. Another composition includes the recombinant amino acid sequences of Cb195, Cb1172, Cb909, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb193, Cb909, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb1172, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb1172, Cb909, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb1172, Cb909, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb909, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb909, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb1172, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb1172, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, Cb1172, and Cb909. Another composition includes the recombinant amino acid sequences of Cb909, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb1172, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb1172, Cb909, and Cb162. Another composition includes the recombinant amino acid sequences of Cb1172, Cb909, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb195, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb195, Cb909, and Cb162. Another composition includes the recombinant amino acid sequences of Cb195, Cb909, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb195, Cb1172, and Cb162. Another composition includes the recombinant amino acid sequences of Cb195, Cb1172, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb195, Cb1172, and Cb909. Another composition includes the recombinant amino acid sequences of Cb193, Cb2487, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb909, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb909, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb193, Cb1172, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb1172, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb193, Cb1172, and Cb909. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, and Cb162. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, and Cb2487. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, and Cb909. Another composition includes the recombinant amino acid sequences of Cb193, Cb195, and Cb1172. Another composition includes the recombinant amino acid sequences of Cb195 and Cb909. Another composition includes the recombinant amino acid sequences of Cb193 and Cb195. Another composition includes the recombinant amino acid sequences of Cb193 and Cb1172. Another composition includes the recombinant amino acid sequences of Cb193 and Cb909. Another composition includes the recombinant amino acid sequences of Cb193 and Cb2487. Another composition includes the recombinant amino acid sequences of Cb193 and Cb162. Another composition includes the recombinant amino acid sequences of Cb195 and Cb1172. Another composition includes the recombinant amino acid sequences of Cb195 and Cb2487. Another composition includes the recombinant amino acid sequences of Cb195 and Cb162. Another composition includes the recombinant amino acid sequences of Cb1172 and Cb909. Another composition includes the recombinant amino acid sequences of Cb1172 and Cb2487. Another composition includes the recombinant amino acid sequences of Cb1172 and Cb162. Another composition includes the recombinant amino acid sequences of Cb909 and Cb2487. Another composition includes the recombinant amino acid sequences of Cb909 and Cb162. Another composition includes the recombinant amino acid sequences of Cb2487 and Cb162.
Compositions may include a transgenic host cell comprising one or more of the amino acid sequences encoding Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162. The one or more polypeptides may be secreted from the transgenic host cell.
The present disclosure provides for compositions including the recombinant nucleotide sequence encoding Cb193 alone or in combination with one or more of the recombinant nucleotide sequences encoding Cb195, Cb1172, Cb909, Cb2487 and Cb162. The present disclosure also provides for compositions including the recombinant nucleotide sequence encoding Cb195 alone or in combination with one or more of the recombinant nucleotide sequences encoding Cb193, Cb1172, Cb909, Cb2487 and Cb162. The present disclosure also provides for compositions including the recombinant nucleotide sequence encoding Cb1172 alone or in combination with one or more of the recombinant nucleotide sequences encoding Cb193, Cb195, Cb909, Cb2487 and Cb162. The present disclosure also provides for compositions including the recombinant nucleotide sequence encoding Cb909 alone or in combination with one or more of the recombinant nucleotide sequences encoding Cb193, Cb195, Cb1172, Cb2487 and Cb162. The present disclosure also provides for compositions including the recombinant nucleotide sequence encoding Cb2487 alone or in combination with one or more of the recombinant nucleotide sequences encoding Cb193, Cb195, Cb1172, Cb909 and Cb162. The present disclosure also provides for compositions including the recombinant nucleotide sequence encoding Cb162 alone or in combination with one or more of the recombinant nucleotide sequences encoding Cb193, Cb195, Cb1172, Cb2487 and Cb909.
The present disclosure also provides for compositions including two or more of the recombinant nucleotide sequences encoding Cb162, Cb193, Cb195, Cb1172, Cb2487 and Cb909. Compositions may include vectors comprising the nucleotide sequence encoding one or more of Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162.
Any of the compositions disclosed herein containing one or more recombinant hemicellulases disclosed herein may further contain one or more recombinant cellulases. In some aspects, cellulases include the polypeptides that contain the amino acid sequence of any of SEQ ID NOs: 44, 114, 124, 126, 128, 46, 60, 61, 111, 74, 121, 76, 86, 87, 113, 98, 119, 100, and 106. Any of the compositions disclosed herein containing one or more recombinant hemicellulases disclosed herein may further contain an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose. In one aspect, an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose contains the amino acid sequence of SEQ ID NO: 146.
Compositions disclosed herein containing one or more recombinant polypeptides disclosed herein may contain the proteins in any form. In some aspects, the polypeptides are in a liquid solution. In some aspects, the polypeptides are lyophilized. In some aspects, additional material is included in compositions containing one or more recombinant polypeptides disclosed herein to help preserve the stability of the polypeptides. In some aspects, additional material is included in compositions containing one or more recombinant polypeptides disclosed herein to help preserve the stability of the polypeptides, wherein the additional material is additional polypeptides. In some aspects, the compositions are stable for at least six months. In some aspects, the compositions are stable for at least one year.
Host Cells
The present disclosure further provides host cells that contain a recombinant nucleic acid encoding a recombinant polypeptide of the disclosure. In some aspects, the disclosure provides host cells containing two or more recombinant nucleic acids encoding two or more recombinant polypeptides of the disclosure.
“Host cell” and “host microorganism” are used interchangeably herein to refer to a living biological cell that can be transformed via insertion of recombinant DNA or RNA. Such recombinant DNA or RNA can be in an expression vector. A host organism or cell as described herein may be a prokaryotic organism or a eukaryotic cell.
Any prokaryotic or eukaryotic host cell may be used in the present disclosure so long as it remains viable after being transformed with a sequence of nucleic acids. Preferably, the host cell is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of the proteins (e.g., transporters), or the resulting intermediates.
In some aspects, the host cell is a prokaryotic cell. Any prokaryotic cell suitable for expression of a recombinant polypeptide may be used to produce recombinant polypeptides of the present disclosure. Prokaryotic host cells of the disclosure include, without limitation, Escherichia coli, Bacillus subtilis, Corynebacterium spp., Pseudomonas spp., Proteus spp., Ralstonia spp., Streptomyces spp., Staphylococcus spp., Lactococcus spp., Zymomonas mobilis, Clostridium spp., Thermoanaerobacterium spp., Caldicellulosiruptor spp. and Klebsiella spp.
In some aspects, the host cell is a eukaryotic cell. Any eukaryotic cell suitable for expression of a recombinant polypeptide may be used to produce recombinant polypeptides of the present disclosure. Suitable eukaryotic cells include, but are not limited to, fungal, plant, insect or mammalian cells.
In certain aspects, the host cell is a fungal strain. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi.
In certain embodiments, the fungal host is a yeast strain. “Yeast” as used herein refers to any single cell fungus that reproduces asexually by budding or division, and it includes fungi of both Ascomycota and Basidiomycota.
In certain embodiments, the fungal host is of the genus Saccharomyces, Schizosaccharomyces, Leucosporidium, Dekkera/Brettanomyces, Zygosaccharomyces, Yarrowia, Hansenula, Kluyveromyces, Scheffersomyces (Pichia), Neurospora or Candida. 
In some aspects, the host cell is a thermophilic microorganism.
The host cells of the present disclosure may be genetically modified in that recombinant nucleic acids have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing one or more nucleic acid constructs encoding one or more proteins for different functions.
“Recombinant nucleic acid” or “heterologous nucleic acid” or “recombinant polynucleotide”, “recombinant nucleotide” or “recombinant DNA” as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host cell; (b) the sequence may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount; (c) the sequence of nucleic acids contains two or more subsequences that are not found in the same relationship to each other in nature; (d) the polynucleotide is isolated from an organism in which the polynucleotide naturally occurs; or (e) the polynucleotide is synthetically prepared. For example, regarding instance (c), a recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid. Specifically, the present disclosure describes the introduction of an expression vector into a host cell, wherein the expression vector contains a nucleic acid sequence coding for a protein that is not normally found in a host cell or contains a nucleic acid coding for a protein that is normally found in a cell but is under the control of different regulatory sequences. With reference to the host cell's genome, then, the nucleic acid sequence that codes for the protein is recombinant. As used herein, the term “recombinant polypeptide” refers to a polypeptide generated from a “recombinant nucleic acid” or “heterologous nucleic acid” or “recombinant polynucleotide”, “recombinant nucleotide” or “recombinant DNA” as described above.
In some aspects, the host cell naturally produces a protein encoded by a polynucleotide of the disclosure. A gene encoding the desired protein may be heterologous to the host cell or the gene may be endogenous to the host cell but is operatively linked to a heterologous promoters and/or control region which results in the higher expression of the gene in the host cell.
Host Cell Components
In some aspects, host cells disclosed herein contain one or more recombinant nucleic acids, wherein the recombinant nucleic acids encode one or more polypeptides selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629, or Cb486 polypeptides.
In some aspects, host cells disclosed herein contain two or more recombinant nucleic acids, wherein the recombinant nucleic acids encode two or more polypeptides selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629, or Cb486 polypeptides.
In some aspects, host cells disclosed herein contain three or more recombinant nucleic acids, wherein the recombinant nucleic acids encode three or more polypeptides selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629, or Cb486 polypeptides.
In some aspects, host cells disclosed herein contain four or more recombinant nucleic acids, wherein the recombinant nucleic acids encode four or more polypeptides selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629, or Cb486 polypeptides.
In some aspects, host cells disclosed herein contain five or more recombinant nucleic acids, wherein the recombinant nucleic acids encode five or more polypeptides selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629, or Cb486 polypeptides.
In some aspects, host cells disclosed herein contain six or more recombinant nucleic acids, wherein the recombinant nucleic acids encode six or more polypeptides selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629, and Cb486 polypeptides.
In some aspects, host cells disclosed herein contain one or more recombinant nucleic acids, wherein the recombinant nucleic acids encode one or more recombinant polypeptides disclosed herein, wherein the one or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, host cells disclosed herein contain two or more recombinant nucleic acids, wherein the recombinant nucleic acids encode two or more recombinant polypeptides disclosed herein, wherein the two or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, host cells disclosed herein contain three or more recombinant nucleic acids, wherein the recombinant nucleic acids encode three or more recombinant polypeptides disclosed herein, wherein the three or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, host cells disclosed herein contain four or more recombinant nucleic acids, wherein the recombinant nucleic acids encode four or more recombinant polypeptides disclosed herein, wherein the four or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, host cells disclosed herein contain five or more recombinant nucleic acids, wherein the recombinant nucleic acids encode five or more recombinant polypeptides disclosed herein, wherein the five or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, host cells disclosed herein contain six or more recombinant nucleic acids, wherein the recombinant nucleic acids encode six or more recombinant polypeptides disclosed herein, wherein the six or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106.
In some aspects, host cells disclosed herein contain two or more recombinant nucleic acids encoding two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
In some aspects, host cells disclosed herein contain three or more recombinant nucleic acids encoding three or more recombinant polypeptides disclosed herein, wherein the three or more polypeptides are selected from the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
In some aspects, host cells disclosed herein contain four or more recombinant nucleic acids encoding four or more recombinant polypeptides disclosed herein, wherein the four or more polypeptides are selected from the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
In some aspects, host cells disclosed herein contain five or more recombinant nucleic acids encoding five or more recombinant polypeptides disclosed herein, wherein the five or more polypeptides are selected from the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
In some aspects, host cells disclosed herein contain six recombinant nucleic acids encoding six recombinant polypeptides disclosed herein, wherein the six polypeptides are the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113.
In some aspects, host cells disclosed herein contain one or more recombinant nucleic acids, wherein the recombinant nucleic acids encode one or more recombinant polypeptides disclosed herein, wherein the one or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the recombinant nucleic acid encoding a Cb1952 polypeptide is selected from the polynucleotides of SEQ ID NOs: 45, 115, 125, 127, 129, and 47, wherein the recombinant nucleic acid encoding a Cb1953 polypeptide is selected from the polynucleotides of SEQ ID NOs: 62, 63, and 110, wherein the recombinant nucleic acid encoding a Cb1954 polypeptide is selected from the polynucleotides of SEQ ID NOs: 116, 75, and 77, wherein the recombinant nucleic acid encoding a Cb1946 polypeptide is selected from the polynucleotides of SEQ ID NOs: 88, 89, and 112, wherein the recombinant nucleic acid encoding a Cb629 polypeptide is selected from the polynucleotides of SEQ ID NOs: 99, 120, and 101, and wherein the recombinant nucleic acid encoding a Cb486 polypeptide is the polynucleotide of SEQ ID NO: 107.
In some aspects, host cells disclosed herein contain two or more recombinant nucleic acids, wherein the recombinant nucleic acids encode two or more recombinant polypeptides disclosed herein, wherein the two or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the recombinant nucleic acid encoding a Cb1952 polypeptide is selected from the polynucleotides of SEQ ID NOs: 45, 115, 125, 127, 129, and 47, wherein the recombinant nucleic acid encoding a Cb1953 polypeptide is selected from the polynucleotides of SEQ ID NOs: 62, 63, and 110, wherein the recombinant nucleic acid encoding a Cb1954 polypeptide is selected from the polynucleotides of SEQ ID NOs: 116, 75, and 77, wherein the recombinant nucleic acid encoding a Cb1946 polypeptide is selected from the polynucleotides of SEQ ID NOs: 88, 89, and 112, wherein the recombinant nucleic acid encoding a Cb629 polypeptide is selected from the polynucleotides of SEQ ID NOs: 99, 120, and 101, and wherein the recombinant nucleic acid encoding a Cb486 polypeptide is the polynucleotide of SEQ ID NO: 107.
In some aspects, host cells disclosed herein contain three or more recombinant nucleic acids, wherein the recombinant nucleic acids encode three or more recombinant polypeptides disclosed herein, wherein the three or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the recombinant nucleic acid encoding a Cb1952 polypeptide is selected from the polynucleotides of SEQ ID NOs: 45, 115, 125, 127, 129, and 47, wherein the recombinant nucleic acid encoding a Cb1953 polypeptide is selected from the polynucleotides of SEQ ID NOs: 62, 63, and 110, wherein the recombinant nucleic acid encoding a Cb1954 polypeptide is selected from the polynucleotides of SEQ ID NOs: 116, 75, and 77, wherein the recombinant nucleic acid encoding a Cb1946 polypeptide is selected from the polynucleotides of SEQ ID NOs: 88, 89, and 112, wherein the recombinant nucleic acid encoding a Cb629 polypeptide is selected from the polynucleotides of SEQ ID NOs: 99, 120, and 101, and wherein the recombinant nucleic acid encoding a Cb486 polypeptide is the polynucleotide of SEQ ID NO: 107.
In some aspects, host cells disclosed herein contain four or more recombinant nucleic acids, wherein the recombinant nucleic acids encode four or more recombinant polypeptides disclosed herein, wherein the four or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the recombinant nucleic acid encoding a Cb1952 polypeptide is selected from the polynucleotides of SEQ ID NOs: 45, 115, 125, 127, 129, and 47, wherein the recombinant nucleic acid encoding a Cb1953 polypeptide is selected from the polynucleotides of SEQ ID NOs: 62, 63, and 110, wherein the recombinant nucleic acid encoding a Cb1954 polypeptide is selected from the polynucleotides of SEQ ID NOs: 116, 75, and 77, wherein the recombinant nucleic acid encoding a Cb1946 polypeptide is selected from the polynucleotides of SEQ ID NOs: 88, 89, and 112, wherein the recombinant nucleic acid encoding a Cb629 polypeptide is selected from the polynucleotides of SEQ ID NOs: 99, 120, and 101, and wherein the recombinant nucleic acid encoding a Cb486 polypeptide is the polynucleotide of SEQ ID NO: 107.
In some aspects, host cells disclosed herein contain five or more recombinant nucleic acids, wherein the recombinant nucleic acids encode five or more recombinant polypeptides disclosed herein, wherein the five or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 or Cb486 polypeptides, and wherein the recombinant nucleic acid encoding a Cb1952 polypeptide is selected from the polynucleotides of SEQ ID NOs: 45, 115, 125, 127, 129, and 47, wherein the recombinant nucleic acid encoding a Cb1953 polypeptide is selected from the polynucleotides of SEQ ID NOs: 62, 63, and 110, wherein the recombinant nucleic acid encoding a Cb1954 polypeptide is selected from the polynucleotides of SEQ ID NOs: 116, 75, and 77, wherein the recombinant nucleic acid encoding a Cb1946 polypeptide is selected from the polynucleotides of SEQ ID NOs: 88, 89, and 112, wherein the recombinant nucleic acid encoding a Cb629 polypeptide is selected from the polynucleotides of SEQ ID NOs: 99, 120, and 101, and wherein the recombinant nucleic acid encoding a Cb486 polypeptide is the polynucleotide of SEQ ID NO: 107.
In some aspects, host cells disclosed herein contain six or more recombinant nucleic acids, wherein the recombinant nucleic acids encode six or more recombinant polypeptides disclosed herein, wherein the six or more recombinant polypeptides are selected from Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides, and wherein the recombinant nucleic acid encoding a Cb1952 polypeptide is selected from the polynucleotides of SEQ ID NOs: 45, 115, 125, 127, 129, and 47, wherein the recombinant nucleic acid encoding a Cb1953 polypeptide is selected from the polynucleotides of SEQ ID NOs: 62, 63, and 110, wherein the recombinant nucleic acid encoding a Cb1954 polypeptide is selected from the polynucleotides of SEQ ID NOs: 116, 75, and 77, wherein the recombinant nucleic acid encoding a Cb1946 polypeptide is selected from the polynucleotides of SEQ ID NOs: 88, 89, and 112, wherein the recombinant nucleic acid encoding a Cb629 polypeptide is selected from the polynucleotides of SEQ ID NOs: 99, 120, and 101, and wherein the recombinant nucleic acid encoding a Cb486 polypeptide is the polynucleotide of SEQ ID NO: 107.
In some aspects, host cells disclosed herein contain two or more recombinant nucleic acids encoding two or more recombinant polypeptides disclosed herein, wherein the two or more recombinant nucleic acids are selected from the polynucleotides of SEQ ID NOs: 47, 110, 77, 112, 101, and 107.
In some aspects, host cells disclosed herein contain three or more recombinant nucleic acids encoding three or more recombinant polypeptides disclosed herein, wherein the three or more recombinant nucleic acids are selected from the polynucleotides of SEQ ID NOs: 47, 110, 77, 112, 101, and 107.
In some aspects, host cells disclosed herein contain four or more recombinant nucleic acids encoding four or more recombinant polypeptides disclosed herein, wherein the four or more recombinant nucleic acids are selected from the polynucleotides of SEQ ID NOs: 47, 110, 77, 112, 101, and 107.
In some aspects, host cells disclosed herein contain five or more recombinant nucleic acids encoding five or more recombinant polypeptides disclosed herein, wherein the five or more recombinant nucleic acids are selected from the polynucleotides of SEQ ID NOs: 47, 110, 77, 112, 101, and 107.
In some aspects, host cells disclosed herein contain six recombinant nucleic acids encoding six recombinant polypeptides disclosed herein, wherein the six recombinant nucleic acids are the polynucleotides of SEQ ID NOs: 47, 110, 77, 112, 101, and 107.
Any of the host cells disclosed herein containing one or more recombinant nucleic acids encoding one or more recombinant cellulases disclosed herein may further contain one or more recombinant nucleic acids encoding one or more recombinant hemicellulases. In some aspects, polynucleotides that encode hemicellulases include nucleic acids that contain the polynucleotide sequence of any of SEQ ID NOs: 4, 8, 14, 20, 28, 34, or 38. Any of the host cells disclosed herein containing one or more recombinant cellulases disclosed herein may further contain an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose. In one aspect, an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose contains the amino acid sequence of SEQ ID NO: 146.
The disclosure further provides for a transformed transgenic host cell comprising one or more of the nucleic acids encoding Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162. The transformed cell can be, without limitation, a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell, or a plant cell. In certain embodiments, the transformed cell is E. coli. In certain embodiments, the transformed cell is a thermophilic microorganism.
Any of the host cells disclosed herein containing one or more recombinant nucleic acids encoding one or more recombinant hemicellulases disclosed herein may further contain one or more recombinant nucleic acids encoding one or more recombinant cellulases. In some aspects, polynucleotides that encode cellulases include nucleic acids that contain the polynucleotide sequence of any of SEQ ID NOs: 44, 114, 124, 126, 128, 46, 60, 61, 111, 74, 121, 76, 86, 87, 113, 98, 119, 100, and 106. Any of the host cells disclosed herein containing one or more recombinant hemicellulases disclosed herein may further contain an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose. In one aspect, an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose contains the amino acid sequence of SEQ ID NO: 146.
Methods of Producing and Culturing Host Cells of the Disclosure
Methods of producing and culturing host cells of the disclosure may include the introduction or transfer of expression vectors containing the recombinant nucleic acids of the disclosure into the host cell. Such methods for transferring expression vectors into host cells are well known to those of ordinary skill in the art. For example, one method for transforming cells with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, e.g., calcium phosphate, may also be used following a similar procedure. In addition, electroporation (i.e., the application of current to increase the permeability of cells to nucleic acid sequences) may be used to transfect the host cell. Cells also may be transformed through the use of spheroplasts (Schweizer, M, Proc. Natl. Acad. Sci., 78: 5086-5090 (1981)). Also, microinjection of the nucleic acid sequences provides the ability to transfect host cells. Other means, such as lipid complexes, liposomes, and dendrimers, may also be employed. Those of ordinary skill in the art can transfect a host cell with a desired sequence using these or other methods.
In some cases, cells are prepared as protoplasts or spheroplasts prior to transformation. Protoplasts or spheroplasts may be prepared, for example, by treating a cell having a cell wall with enzymes to degrade the cell wall. Fungal cells may be treated, for example, with zymolyase or chitinase.
The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host, or a transposon may be used.
The vectors preferably contain one or more selectable markers which permit easy selection of transformed hosts. A selectable marker is a gene the product of which provides, for example, biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selection of bacterial cells may be based upon antimicrobial resistance that has been conferred by genes such as the amp, gpt, neo, tet, camR and hyg genes.
Selectable markers for use in fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Suitable markers for S. cerevisiae hosts include, for example, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
The vectors may contain an element(s) that permits integration of the vector into the host's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host genome, the vector may rely on the gene's sequence or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host. The additional nucleotide sequences enable the vector to be integrated into the host genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, or 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host by non-homologous recombination.
For autonomous replication, the vector may further contain an origin of replication enabling the vector to replicate autonomously in the host in question. The origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a sequence that enables a plasmid or vector to replicate in vivo.
The vector may further contain a promoter for regulation of expression of a recombinant nucleic acid in the vector. Promoters for the regulation of expression of a gene are well-known in the art, and include constitutive promoters, and inducible promoters. Promoters are described, for example, in Sambrook, et al. Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, (2001). In some aspects, vectors for use in Saccharomyces spp. may include the TDH1 or PGK1 promoter, which are strong and constitutive promoters.
More than one copy of a gene may be inserted into the host to increase production of the gene product. An increase in the copy number of the gene can be obtained by integrating at least one additional copy of the gene into the host genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the gene, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 2001, supra).
Once the host cell has been transformed with the expression vector, the host cell is allowed to grow. Growth of a host cell in a medium may involve the process of fermentation. Methods of the disclosure may include culturing the host cell such that recombinant nucleic acids in the cell are expressed. Media, temperature ranges and other conditions suitable for growth are known in the art.
Expression of Recombinant Polypeptides of the Disclosure
The disclosure further provides for the expression of polypeptides of the disclosure. Polypeptides of the disclosure may be prepared by standard molecular biology techniques such as those described herein and in Sambrook, et al. Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, (2001). Recombinant polypeptides may be expressed in and purified from transgenic expression systems. Transgenic expression systems can be prokaryotic or eukaryotic. In some aspects, transgenic host cells may secrete the polypeptide out of the host cell. In some aspects, transgenic host cells may retain the expressed polypeptide in the host cell.
In certain aspects, recombinant polypeptides of the disclosure are partially or substantially isolated from a host cell, or from the growth media of the host cell. In certain aspects, a recombinant polypeptide of the disclosure is prepared with a protein “tag” to facilitate protein purification, such as a GST-tag or poly-His tag. In some aspects, a recombinant polypeptide of the disclosure is prepared with a signal sequence to direct the export of the polypeptide out of the cell. In some aspects, recombinant polypeptides may be only partially purified (e.g. <80% pure, <70% pure, <60% pure, <50% pure, <40% pure, <30% pure, <20% pure, <10% pure, <5% pure). In some aspects, recombinant polypeptides of the present disclosure may be purified to a high degree of purity (e.g. >99% pure, >98% pure, >95% pure, >90% pure, etc.). Recombinant polypeptides may be purified through a variety of techniques known to those of skill in the art, including for example, ion-exchange chromatography, size exclusion chromatography, and affinity chromatography.
In one aspect, a method for producing any of the recombinant polypeptides disclosed herein (including cellulases, hemicellulases, and enzymes that enhances enzymatic hydrolysis of cellulose and/or hemicellulose) includes the steps of: A) culturing a host cell containing one or more recombinant nucleic acids encoding the one or more recombinant polypeptides disclosed herein in media under conditions necessary to support the expression of the recombinant nucleic acid(s), and collecting the one or more polypeptides from the media and/or host cell.
In one aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing one or more recombinant nucleic acids encoding one or more recombinant polypeptides disclosed herein, wherein the one or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acid(s), and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing two or more recombinant nucleic acids encoding two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing three or more recombinant nucleic acids encoding three or more recombinant polypeptides disclosed herein, wherein the three or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing four or more recombinant nucleic acids encoding four or more recombinant polypeptides disclosed herein, wherein the four or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing five or more recombinant nucleic acids encoding five or more recombinant polypeptides disclosed herein, wherein the five or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing six or more recombinant nucleic acids encoding six or more recombinant polypeptides disclosed herein, wherein the six or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell.
In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing one or more recombinant nucleic acids encoding one or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acid(s), and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing two or more recombinant nucleic acids encoding two or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing three or more recombinant nucleic acids encoding three or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing four or more recombinant nucleic acids encoding four or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing five or more recombinant nucleic acids encoding five or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell. In another aspect, a method for producing cellulases includes the steps of: A) culturing a host cell containing six or more recombinant nucleic acids encoding six of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and collecting the one or more polypeptides from the media and/or host cell.
Thermostability of Enzymes
The enzymes of the present disclosure are thermophilic and thermostable. As used herein, “thermophilic” refers to the characteristic of an enzyme to have peak activity at a high temperature (e.g. above 50° C.). As used herein, “thermostable” refers to the characteristic of an enzyme to retain activity at high temperatures (e.g. above 50° C.) for a significant period of time. For Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides of the disclosure, “enzymatic” activity refers to cellulase activity (including β-glucosidase activity). For Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 polypeptides of the disclosure, “enzymatic” activity refers to hemicellulase activity. For Cb1581 polypeptides of the disclosure, “enzymatic” activity refers to activity that enhances enzymatic hydrolysis of cellulose and/or hemicellulose.
Cellulases
In certain aspects, one or more of the Cb1952, Cb1953, Cb1954, Cb1946, Cb629 and Cb486 polypeptides of the disclosure has a peak rate of enzymatic activity on cellulose or cellulose-containing material at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail has a peak rate of enzymatic activity on cellulose or a cellulose-containing material at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 has a peak rate of enzymatic activity on cellulose or a cellulose-containing material at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C.
In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the cocktail retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In another aspect, an enzyme cocktail is provided herein, wherein the cocktail contains the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
Hemicellulases
In certain embodiments, one or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 has peak rate of enzymatic activity at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In other embodiments, an enzyme ‘cocktail’ that contains two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 has peak rate of enzymatic activity on hemicellulose or a hemicellulose-derived substrate at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In one embodiment, an enzyme ‘cocktail’ that contains all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 has peak rate of enzymatic activity on hemicellulose or a hemicellulose-derived substrate at a temperature of about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C.
Enzymes of the present disclosure retain significant enzymatic activity for significant periods of time at high temperatures. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In one embodiment, an enzyme cocktail containing two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 90% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 75% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 50% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 55° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 60° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 65° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 70° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 75° C. In one embodiment, an enzyme cocktail containing all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 retains at least 25% of its initial rate of enzymatic activity for a period of at least 24 hours when incubated at a temperature of about 80° C.
Any of the cellulase cocktails disclosed herein having the thermophilic and thermostable characteristics disclosed herein may further include any of the hemicellulases disclosed herein and/or an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose disclosed herein. Additionally, any of the hemicellulase cocktails disclosed herein having the thermophilic and thermostable characteristics disclosed herein may further include any of the cellulases disclosed herein and/or an enzyme that enhances enzymatic hydrolysis of cellulose and/or hemicellulose disclosed herein.
Applications
Methods of Degrading Cellulose-Containing Material
In one aspect, provided herein are methods for degrading cellulose-containing material.
In one aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing one or more recombinant polypeptides disclosed herein, wherein the one or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the polypeptide(s) and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing three or more recombinant polypeptides disclosed herein, wherein the three or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing four or more recombinant polypeptides disclosed herein, wherein the four or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing five or more recombinant polypeptides disclosed herein, wherein the five or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing six or more recombinant polypeptides disclosed herein, wherein the six or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation.
In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing one or more recombinant polypeptides disclosed herein, wherein the one or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106, and incubating the polypeptide(s) and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing three or more recombinant polypeptides disclosed herein, wherein the three or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing four or more recombinant polypeptides disclosed herein, wherein the four or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing five or more recombinant polypeptides disclosed herein, wherein the five or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing six or more recombinant polypeptides disclosed herein, wherein the six or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and wherein the Cb1952 polypeptide is selected from the polypeptides of SEQ ID NOs: 44, 114, 124, 126, 128, and 46, wherein the Cb1953 polypeptide is selected from the polypeptides of SEQ ID NOs: 60, 61, and 111, wherein the Cb1954 polypeptide is selected from the polypeptides of SEQ ID NOs: 74, 121, and 76, wherein the Cb1946 polypeptide is selected from the polypeptides of SEQ ID NOs: 86, 87, and 113, wherein the Cb629 polypeptide is selected from the polypeptides of SEQ ID NOs: 98, 119, and 100, and wherein the Cb486 polypeptide is the polypeptide of SEQ ID NO: 106, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation.
In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing one or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the polypeptide(s) and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing two or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing three or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing four or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing five or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing six of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation.
In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing one or more recombinant nucleic acids encoding one or more recombinant polypeptides disclosed herein, wherein the one or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acid(s), and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing two or more recombinant nucleic acids encoding two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing three or more recombinant nucleic acids encoding three or more recombinant polypeptides disclosed herein, wherein the three or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing four or more recombinant nucleic acids encoding four or more recombinant polypeptides disclosed herein, wherein the four or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing five or more recombinant nucleic acids encoding five or more recombinant polypeptides disclosed herein, wherein the five or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing six or more recombinant nucleic acids encoding six or more recombinant polypeptides disclosed herein, wherein the six or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation.
In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing one or more recombinant nucleic acids encoding one or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acid(s), and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing two or more recombinant nucleic acids encoding two or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing three or more recombinant nucleic acids encoding three or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing four or more recombinant nucleic acids encoding four or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing five or more recombinant nucleic acids encoding five or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing six or more recombinant nucleic acids encoding six of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, in media under conditions necessary to support the expression of the recombinant nucleic acids, and incubating the cell and cellulose-containing material under conditions that support cellulose degradation.
In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing one or more recombinant polypeptides disclosed herein, wherein the one or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, or Cb629 polypeptides, and wherein the composition does not contain a Cb486 polypeptide, and incubating the polypeptides and cellulose-containing material under conditions that support cellulose degradation. In another aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing one or more recombinant nucleic acids encoding one or more recombinant polypeptides disclosed herein, wherein the one or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, or Cb629 polypeptides, and wherein the host cell does not contain a recombinant nucleic acid encoding a Cb486 polypeptide, in media under conditions necessary to support the expression of the recombinant nucleic acid(s), and incubating the cell and cellulose-containing material under conditions that support cellulose degradation. Contacting a cellulose-containing material with one or more cellulases disclosed herein, but not Cb486, may lead to the accumulation of cellobiose and/or other oligosaccharides during the degradation of the cellulose-containing material. Products containing cellobiose and/or oligosaccharides may be useful as feedstocks for organisms or processes that effectively utilize cellobiose and/or oligosaccharides to generate desired end products, such as biofuels.
As used herein, a “cellulose-containing material” is any material that contains cellulose, including biomass. Biomass suitable for use with the currently disclosed methods include any cellulose-containing material, and includes, without limitation, Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, rye hulls, wheat hulls, sugarcane bagasse, copra meal, copra pellets, palm kernel meal, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine wood, birch wood, willow wood, aspen wood, poplar wood, energy cane, waste paper, sawdust, forestry wastes, municipal solid waste, waste paper, crop residues, other grasses, and other woods. In some aspects, biomass is lignocellulosic material.
Commonly, cellulose-containing materials also contain hemicellulose. For example, unprocessed or partially processed plant materials generally contain hemicellulose. In some aspects, any of the methods for degrading a cellulose-containing material disclosed herein may further include contacting a cellulose-containing material with one or more hemicellulases.
Any of the methods disclosed herein for degrading a cellulose-containing material that includes contacting a cellulose-containing material with a composition containing one, two, three, four, five, six or more polypeptides selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, may further include contacting the cellulose-containing material with one or more, two or more, three or more, four or more, five or more, or the six recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37. In one aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a composition containing the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 and the recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37. In one aspect, provided herein is a method for degrading a biomass-containing material, including contacting a cellulose-containing material with a composition containing the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 and the recombinant polypeptides of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37, and incubating the polypeptides and biomass-containing material under conditions that support cellulose degradation.
Any of the methods disclosed herein for degrading a cellulose-containing material that includes contacting a cellulose-containing material with a host cell containing one, two, three, four, five, or six recombinant nucleic acids encoding one, two, three, four, five, or six recombinant polypeptides disclosed herein, wherein the one, two, three, four, five, six or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, in media under conditions necessary to support the expression of the recombinant nucleic acids, may further include contacting the cellulose-containing material with one or more, two or more, three or more, four or more, five or more, or six or more recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37. In one aspect, a method for degrading a cellulose-containing material includes contacting a cellulose-containing material with a host cell containing recombinant nucleic acids encoding the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 and the recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37. In one aspect, provided herein is a method for degrading a biomass-containing material, including contacting a cellulose-containing material with a host cell containing recombinant nucleic acids encoding the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 and the recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37, and incubating the host cell and biomass-containing material under conditions that support cellulose degradation.
In some aspects, any of the methods disclosed herein for degrading a cellulose-containing material may be carried out at a high temperature. In some aspects, any of the methods disclosed herein for degrading a cellulose-containing material may be carried out at about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, any of the methods disclosed herein for degrading a cellulose-containing material may be carried out for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, or 24 hours at about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. If a method disclosed herein for degrading a cellulose containing material is carried out at a high temperature and it uses host cells expressing recombinant polypeptides disclosed herein, in some aspects, the host cell is a thermophilic organism.
Methods of Reducing Viscosity of Pretreated Biomass Mixtures
Further provided herein are methods for reducing the viscosity of pre-treated biomass.
Biomass that that is used for degradation into component sugars or oligosaccharides may contains high levels of lignin, which can block hydrolysis of the cellulosic component of the biomass. Typically, biomass is pretreated with, for example, high temperature and/or high pressure to increase the accessibility of the cellulosic component to hydrolysis. Other pretreatments include, without limitation, ammonia fiber expansion (AFEX), steam explosion, and treatment with alkaline aqueous solutions, acidic solutions, organic solvents, ionic liquids (IL), electrolyzed water, phosphoric acid, or combinations thereof. However, pretreatment generally results in a biomass mixture that is highly viscous. The high viscosity of the pretreated biomass mixture can increase the difficulty in handling the pretreated biomass, and it can also interfere with effective hydrolysis of the pretreated biomass. Advantageously, recombinant polypeptides disclosed herein can be used to reduce the viscosity of pretreated biomass mixtures prior to further degradation of the biomass.
Accordingly, certain aspects of the present disclosure relate to methods of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing one or more recombinant polypeptides disclosed herein, wherein the one or more polypeptides are selected from one or more of the cellulases, hemicellulases, and polypeptides that enhance enzymatic hydrolysis of cellulose and/or hemicellulose, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture.
Certain aspects of the present disclosure relate to methods of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing one or more recombinant polypeptides disclosed herein, wherein the one or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing two or more recombinant polypeptides disclosed herein, wherein the two or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing three or more recombinant polypeptides disclosed herein, wherein the three or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing four or more recombinant polypeptides disclosed herein, wherein the four or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing five or more recombinant polypeptides disclosed herein, wherein the five or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing six or more recombinant polypeptides disclosed herein, wherein the six or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture.
In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing one or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing two or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing three or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing four or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing five or more of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture. In another aspect, the disclosure includes a method of reducing the viscosity of a pretreated biomass mixture, by contacting a pretreated biomass mixture having an initial viscosity with a composition containing six of the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of the pretreated biomass mixture.
In some aspects, the disclosed methods are carried out as part of a pretreatment process. The pretreatment process may include the additional step of adding a composition containing one, two, three, four, five, six or more recombinant polypeptides disclosed herein, wherein the one, two, three, four, five, six or more polypeptides are selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, to pretreated biomass mixtures after a step of pretreating the biomass, and incubating the pretreated biomass with the polypeptides under conditions sufficient to reduce the viscosity of the mixture. The polypeptides or compositions may be added to pretreated biomass mixture while the temperature of the mixture is high, or after the temperature of the mixture has decreased. In some aspects, the methods are carried out in the same vessel or container where the pretreatment was performed. In other aspects, the methods are carried out in a separate vessel or container where the pretreatment was performed.
In some aspects, the methods are carried out in the presence of high salt, such as solutions containing saturating concentrations of salts, solutions containing sodium chloride (NaCl) at a concentration of at least at or about 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 1 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M, or 4 M sodium chloride, or potassium chloride (KCl), at a concentration at or about 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 1 M, 1.5 M, 2 M, 2.5 M 3.0 M or 3.2 M KCl and/or ionic liquids, such as 1,3-dimethylimidazolium dimethyl phosphate ([DMIM]DMP) or [EMIM]OAc, or in the presence of one or more detergents, such as ionic detergents (e.g., SDS, CHAPS), sulfydryl reagents, such as in saturating ammonium sulfate or ammonium sulfate between at or about 0 and 1 M. In some aspects, the methods are carried out at a temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90° C. In some aspects, the methods are carried out over a broad temperature range, such as between at or about 20° C. and 50° C., 25° C. and 55° C., 30° C. and 60° C., 40° C. and 80° C., 60° C. and 80° C., or 60° C. and 100° C. In some aspects, the methods may be performed over a broad pH range, for example, at a pH of between about 4.5 and 8.75, at a pH of greater than 7 or at a pH of 8.5, or at a pH of at least 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5.
Any of the methods disclosed herein for reducing the viscosity of a pretreated biomass mixture that includes contacting a pretreated biomass mixture with a composition containing one, two, three, four, five, six or more polypeptides selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, may further include contacting the pretreated biomass mixture with one or more, two or more, three or more, four or more, five or more, or six recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37. In one aspect, a method for reducing the viscosity of a pretreated biomass mixture includes contacting a pre-treated biomass mixture with a composition containing the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 and the recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37. In one aspect, provided herein is a method for reducing the viscosity of a pretreated biomass mixture, including contacting a pre-treated biomass mixture with a composition containing the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 and the recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37, and incubating the polypeptides and reducing the viscosity of a pretreated biomass mixture to reduce the viscosity of the pretreated biomass mixture.
Methods of Converting Cellulose-Containing Materials to Fermentation Product
Further provided herein are methods for converting cellulose-containing materials to a fermentation production. In one aspect, a method for converting a cellulose-containing material into a fermentation product includes the steps of: A) contacting a cellulose-containing material with a composition containing one, two, three, four, five, six or more polypeptides selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides; B) incubating the cellulose-containing material with the composition containing one, two, three, four, five, six or more polypeptides under conditions that support cellulose degradation, in order to obtain sugars; and C) culturing the sugars with a fermentative microorganism under conditions sufficient to produce a fermentation product.
In another aspect, a method for converting a cellulose-containing material into a fermentation product includes the steps of: A) contacting a cellulose-containing material with a composition containing the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113; B) incubating the cellulose-containing material with the composition containing the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, under conditions that support cellulose degradation, in order to obtain sugars; and C) culturing the sugars with a fermentative microorganism under conditions sufficient to produce a fermentation product.
Any of the methods disclosed herein for converting a cellulose-containing material into a fermentation product may further include contacting the pretreated biomass mixture with one or more, two or more, three or more, four or more, five or more, or six recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37. In one aspect, a method for converting a cellulose-containing material into a fermentation product includes contacting a cellulose-containing material with a composition containing the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and the recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37. In one aspect, provided herein is a method for converting a cellulose-containing material into a fermentation product including the steps of: A) contacting a cellulose-containing material with a composition containing the recombinant polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113 and the recombinant polypeptides of SEQ ID NOs: 7, 13, 19, 27, 33, and 37; B) incubating the cellulose-containing material with the composition containing the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, 113, 7, 13, 19, 27, 33, and 37 under conditions that support cellulose degradation, in order to obtain sugars; and C) culturing the sugars with a fermentative microorganism under conditions sufficient to produce a fermentation product.
Sugars that may be obtained from the degradation of cellulose-containing materials include, without limitation, glucose, cellobiose, xylose, arabinose, galactose, glucuronic acid, and mannose.
Fermentation products that may be produced from sugars obtained from the degradation of cellulose-containing materials include, without limitation, ethanol, n-propanol, n-butanol, iso-butanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and octanol.
Fermentative organisms include, without limitation, Saccharomyces spp.
Methods of Consolidated Bioprocessing
Further provided herein are methods for converting cellulose-containing materials to a fermentation production, by consolidated bioprocessing. Consolidated bioprocessing combines enzyme generation, biomass hydrolysis, and biofuel production into a single stage. In one aspect, a method for converting a cellulose-containing material into a fermentation product by consolidated bioprocessing includes the steps of: A) contacting a cellulose-containing material with a cell having recombinant nucleic acids encoding one, two, three, four, five, six or more polypeptides selected from: Cb1952 polypeptides, Cb1953 polypeptides, Cb1954 polypeptides, Cb1946 polypeptides, Cb629 polypeptides, or Cb486 polypeptides, and one or more recombinant nucleic acids encoding one or more polypeptides involved in a biochemical pathway for the production of a biofuel, under conditions sufficient to support expression of the nucleic acids; B) incubating the cellulose-containing material with the cell expressing recombinant nucleic acids under conditions that support cellulose degradation and fermentation, in order to produce a fermentation product.
In another aspect, a method for converting a cellulose-containing material into a fermentation product by consolidated bioprocessing includes the steps of: A) contacting a cellulose-containing material with a cell having recombinant nucleic acids encoding the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, and 113, and one or more recombinant nucleic acids encoding one or more polypeptides involved in a biochemical pathway for the production of a biofuel, under conditions sufficient to support expression of the nucleic acids; B) incubating the cellulose-containing material with the cell expressing recombinant nucleic acids under conditions that support cellulose degradation and fermentation, in order to produce a fermentation product.
In another aspect, a method for converting a cellulose-containing material into a fermentation product by consolidated bioprocessing includes the steps of: A) contacting a cellulose-containing material with a cell having recombinant nucleic acids encoding the polypeptides of SEQ ID NOs: 46, 76, 100, 106, 111, 113, 7, 13, 19, 27, 33, and 37, and one or more recombinant nucleic acids encoding a polypeptide involved in a biochemical pathway for the production of a biofuel under conditions sufficient to support expression of the nucleic acids; B) incubating the cellulose-containing material with the cell expressing recombinant nucleic acids under conditions that support cellulose degradation and fermentation, in order to produce a fermentation product.
Fermentation products that may be produced from sugars obtained from the degradation of cellulose-containing materials include, without limitation, ethanol, n-propanol, n-butanol, iso-butanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-1-pentanol, and octanol.
Concentration of Polypeptides
In certain aspects, polypeptides of the disclosure are provided with a substrate at a concentration of at least 0.01 nM of each polypeptide. In certain aspects, the polypeptides are provided with a substrate at a concentration of at least 0.1 nM of each polypeptide. In certain aspects, the polypeptides are provided with a substrate at a concentration of at least 1 nM of each polypeptide. In certain aspects, the polypeptides are provided with a substrate at a concentration of at least 10 nM of each polypeptide. In certain aspects, the polypeptides are provided with a substrate at a concentration of at least 0.1 μM of each polypeptide. In certain aspects, the polypeptides are provided with a substrate at a concentration of at least 10 μM of each polypeptide. In certain aspects, the polypeptides are provided with a substrate at a concentration of at least 100 μM of each polypeptide.
Combination of Thermostable Cellulases with Thermostable Hemicellulose-Degrading Enzymes
In some aspects, thermostable cellulose-degrading enzymes of the present disclosure are provided with thermostable hemicellulases. Thermostable hemicellulases may be provided with the thermostable cellulose-degrading enzymes of the present disclosure in order to increase the degradation of materials containing both cellulose and hemicellulose, such as biomass from terrestrial plants.
In some aspects disclosed herein, mixtures of cellulases of the present disclosure exhibit surprising synergistic effects when combined with mixtures of hemicellulases. In such examples, mixtures containing multiple cellulases have greater cellulase activity when they are combined in a cocktail with a mixture containing multiple hemicellulases, as compared to when the mixture of cellulases is not combined with a mixture containing multiple hemicellulases. Also, in some examples, mixtures containing multiple hemicellulases have greater hemicellulase activity when they are combined in a cocktail with a mixture containing multiple cellulases, as compared to the activity of the mixture of hemicellulases when it is not combined with a mixture containing multiple cellulases. Thus, cellulase and hemicellulase mixtures provided herein may have surprising synergistic effects together, wherein each enzyme mixture has greater activity when combined with the other than when either enzyme mixture is provided with a substrate alone.
Thermostable hemicellulases may be obtained from organisms capable of degrading hemicellulose. In one aspect, thermostable hemicellulases may be isolated directly from organisms capable of degrading cellulose. In another aspect, thermostable hemicellulases are produced recombinantly, through the use of host cells and expression vectors containing genes encoding thermostable hemicellulases. Thermostable hemicellulases and/or genes encoding thermostable hemicellulases may be isolated from various organisms capable of degrading hemicellulose including, for example and without limitation, archaeal, bacterial, fungal, and protozoan organisms.
In some aspects, thermostable hemicellulases are recombinant polypeptides related to thermostable hemicellulases of C. bescii. In some aspects, thermostable hemicellulases contain the amino acid sequence of any of SEQ ID NOs: 3, 7, 13, 19, 27, 33, and 37. In some aspects, polynucleotides encoding thermostable hemicellulases contain the nucleic acid sequence of any of SEQ ID NOs: 4, 8, 14, 20, 28, 34, and 38.
Synergy of Hemicellulase Enzymatic Activity
In certain embodiments, the enzymes of the present disclosure are provided as an enzyme ‘cocktail’ wherein two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are provided together to degrade hemicellulose or a hemicellulose-derived substrate. In certain embodiments, the enzymes function synergistically and the combination of two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 is more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than the activity of a single enzyme. Similarly, in certain embodiments, enzyme cocktails with three or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than enzyme cocktails with one or two of the enzymes. In certain embodiments, enzyme cocktails with four or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than enzyme cocktails with one, two, or three of the enzymes. In certain embodiments, enzyme cocktails with five or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than enzyme cocktails with one, two, three, or four of the enzymes. In certain embodiments, enzyme cocktails with all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than enzyme cocktails with one, two, three, four, or five of the enzymes.
In other embodiments, enzyme cocktails with two or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than enzyme cocktails with the same total amount of enzyme units but with only one of the species of enzymes. In other embodiments, enzyme cocktails with three or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than enzyme cocktails with the same total amount of enzyme units but with only one or two of the species of enzymes. In other embodiments, enzyme cocktails with four or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than enzyme cocktails with the same total amount of enzyme units but with only one, two, or three of the species of enzymes. In other embodiments, enzyme cocktails with five or more of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than enzyme cocktails with the same total amount of enzyme units but with only one, two, three, or four of the species of enzymes. In other embodiments, enzyme cocktails with all six of the enzymes Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162 are more effective at degrading hemicellulose and releasing monosaccharides from hemicellulose than enzyme cocktails with the same total amount of enzyme units but with only one, two, three, four, or five of the species of enzymes.
Treatment Methods of Hemicellulose and Hemicellulose-Containing Materials
The above-described hemicellulase enzymes and variants can be used alone or in combination to degrade hemicellulose by cleaving one or more functional groups from the xylose backbone to form cleaved hemicellulose.
Hemicellulose treated with the methods of the present disclosure may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% degraded. The hemicellulose substrate is degraded when the enzymes cleave the bonds or linkages present between the subunits present in the hemicellulose. Degradation products may comprise xylose, arabinose, glucuronyl groups, acetyl groups, in addition to other functional groups and hydrocarbons.
In one aspect, plant material containing hemicellulose, or isolated hemicellulose, is treated with one or more of the above-described enzymes, such as Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162. In one embodiment, hemicellulose is treated with Cb193 in combination with one or more enzymes including Cb195, Cb1172, Cb2487, Cb909, and Cb162. In one embodiment, hemicellulose is treated with Cb195 in combination with one or more enzymes including Cb193, Cb1172, Cb909, Cb2487, and Cb162.
Without wishing to be bound by theory, Applicants believe that the methods of the present disclosure degrade hemicellulose via the following mechanisms. Treatment of hemicellulose with endoxylanases Cb193, Cb195, or a variant cleaves β-1,4-xylose linkages in the xylose backbone to generate shorter chains of xylose in β-1,4-linkages. Treatment of hemicellulose with the α-L-arabinofuranosidase Cb1172 or a variant cleaves arabinose moiety from the xylose backbone or from branched or debranched arabinan of hemicelluloses to generate exclusively arabinose. Treatment of hemicellulose with the α-glucuronidase Cb909 or a variant cleaves the alpha-1,2,-glycosidic bond between 4-O-methyl-D-glucuronic acid and the beta-1,4-xylosidic linkage backbone of xylan. Treatment of hemicellulose with the β-xylosidase Cb2487 or a variant cleaves beta-1,4-xylosidic linkages in the xylose backbone. Treatment of hemicellulose with Cb162 or a variant cleaves the linkages between xylose and the side chain of acetyl groups in hemicellulose to provide more accessibility to other hemicellulases such as xylanase and β-xylosidase to the backbone of xylan. Using a combination or two or more enzymes is believed to provide synergistic hemicellulose degradation activity.
In certain embodiments, plant material containing hemicellulose, or isolated hemicellulose, may be treated with one or more isolated or recombinant polypeptides comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity/sequence similarity to Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162.
The polypeptides may be administered directly, either alone, or as a composition.
In other methods of the present disclosure, hemicellulose is degraded by contact with a transgenic host cell secreting one or more polypeptides including Cb193, Cb195, Cb1172, Cb2487, Cb909, and Cb162. In some embodiments, the transgenic host cell may be Escherichia, Pseudomonas, Proteus, Ralstonia, Streptomyces, Staphylococcus, Lactococcus, Bacillus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Aspergillus, Chrysosporium lucknowense, or Trichoderma reesei. In some embodiments, the transgenic host cell may be a thermophilic microorganism. In one embodiment, the thermophilic host cell is Caldicellulosiruptor bescii. 
The transgenic host cell may contain a vector encoding Cb193, Cb195, Cb1172, Cb909, Cb2487, Cb162 or variants thereof. In some embodiments, the hemicellulose is degraded by treating with Cb193 or a variant alone, or in combination with one or more of Cb195, Cb1172, Cb909, Cb2487, Cb162, and variants thereof. In some embodiments, the hemicellulose is degraded by treating with Cb195 or a variant alone, or in combination with one or more of Cb193, Cb1172, Cb909, Cb2487, Cb162, and variants thereof.
The methods of the present disclosure can be practiced with any plant material that contains hemicellulose. Plant material suitable for use with the currently disclosed methods include Miscanthus, switchgrass, cord grass, rye grass, reed canary grass, elephant grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, sorghum, rice hulls, rye hulls, wheat hulls, sugarcane bagasse, corn fiber, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine, birch, willow, aspen, poplar wood, and energy cane. The methods may also be practiced on isolated hemicellulose.
In certain embodiments, thermophilic enzymes of the present disclosure are provided with a substrate at a concentration of at least 0.01 nM enzyme of each enzyme. In certain embodiments, the enzymes are provided with a substrate at a concentration of at least 0.1 nM enzyme of each enzyme. In certain embodiments, the enzymes are provided with a substrate at a concentration of at least 1 nM enzyme of each enzyme. In certain embodiments, the enzymes are provided with a substrate at a concentration of at least 10 nM enzyme of each enzyme. In certain embodiments, the enzymes are provided with a substrate at a concentration of at least 0.1 μM enzyme of each enzyme. In certain embodiments, the enzymes are provided with a substrate at a concentration of at least 10 μM enzyme of each enzyme. In certain embodiments, the enzymes are provided with a substrate at a concentration of at least 100 μM enzyme of each enzyme.
The methods of the present disclosure can be practiced at any pH and temperature at which hemicellulose can be degraded; however, in certain embodiments, the methods of the present disclosure are practiced in a pH range of about 5 to about 7 and at or between a temperature between about 60 and about 80° C.
Combination of Thermostable Hemicellulose-Degrading Enzymes with Thermostable Cellulases
In some embodiments, thermostable hemicellulose-degrading enzymes of the present disclosure are provided with thermostable cellulases. Cellulases are enzymes that can hydrolyze cellulose, and they include, but are not limited to, exoglucanases, endoglucanases, and β-glucosidases. In some aspects, thermostable cellulases have optimal enzymatic activity at temperatures above 55° C. Thermostable cellulases may be provided with the thermostable hemicellulose-degrading enzymes of the present disclosure in order to increase the degradation of materials containing both cellulose and hemicellulose, such as biomass from terrestrial plants. For example and without limitation, in one aspect, microorganisms can be provided that express hemicellulose-degrading enzymes of the present disclosure and thermostable cellulases. In one aspect, compositions containing hemicellulose-degrading enzymes of the present disclosure may also contain thermostable cellulases. In other aspects, methods of degrading biomass, of converting biomass into fermentation product, and of converting biomass to fuel are provided, in which biomass is contacted with hemicellulose-degrading enzymes of the present disclosure and with thermostable cellulases
Thermostable cellulases may be obtained from organisms capable of degrading cellulose. In one aspect, thermostable cellulases are obtained directly from organisms capable of degrading cellulose. In another aspect, thermostable cellulases are produced recombinantly, through the use of host cells and expression vectors containing genes encoding thermostable cellulases. Thermostable cellulases and/or genes encoding thermostable cellulases may be isolated from various organisms capable of degrading cellulose including, for example and without limitation, archaeal, bacterial, fungal, and protozoan organisms.
Organisms capable of degrading cellulose include for example and without limitation, those belonging to the genera Aquifex, Bacillus, Rhodothermus, Thermobifida, Thermotoga, Anaerocellum, Sulfolobus, Pyrococcus and Caldicellulosiruptor. A recombinant thermostable endoglucanase of Aquifex aeolicus produced in E. coli showed maximal activity at 80° C. and pH 7.0 with a half-life of 2 h at 100° C. (Kim J S, Lee Y Y, Torget, R W. (2001). Cellulose hydrolysis under extremely low sulfuric acid and high-temperature conditions. Appl. Biochem. Biotechnol. 91-93. 331-340)). The endoglucanases produced by Anaerocellum thermophilum and Caldicellulosiruptor saccharolyticus are multidomain enzymes composed of two catalytic domains, linked to carbohydrate binding domains by proline-threonine-rich regions (Zverlov V, Mahr S, Riedel K, Bronnenmeier K (1998a), “Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile ‘Anaerocellum thermophilum’ with separate glycosyl hydrolase family 9 and 48 catalytic domains,” Microbiology 144 (Pt 2): 457-465; Te'o V S, Saul D J, Bergquist P L (1995), “celA, another gene coding for a multidomain cellulase from the extreme thermophile Caldocellum saccharolyticum,” Appl Microbiol Biotechnol 43: 291-296; Saul et al. 1990. The recombinant endoglucanase of Rhodothermus marinus has a pH optimum of 6.0-7.0 and a temperature optimum at 100° C. (Halldórsdóttir S, Thórólfsdóttir ET, Spilliaert R, Johansson M, Thorbjarnardóttir S H, Palsdottir A, Hreggvidsson G O, Kristjánsson J K, Holst O, Eggertsson G. (1998), “Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12,” Appl Microbiol Biotechnol 49: 277-284). The aerobic thermophilic bacterium Thermus caldophilus also produces an endoglucanase which exhibits high activity on CMC with cellobiose and cellotriose as products (Kim D, Park B H, Jung B-W, Kim M-K, Hong S I, Lee, D S (2006) Identification and molecular modeling of a family 5 endocellulase from Thermus caldophilus GK24, a cellulolytic strain of Thermus thermophilus. Int J Mol Sci 7: 571-589). Thermostable cellulases have also been described from Bacillus subtilis (Mawadza, C, Hatti-Kaul, R., Zvauya, R. and Mattiasson, B., 2000. Purification and characterization of cellulases produced by two Bacillus strains. J. Biotechnol. 83, pp. 177-187), from Pyrococcus furiosus (Kengen, S., Luesink, E., Stams, A. and Zehnder, A., 1993. Purification and characterization of an extremely thermostable β-glucosidase from the hyperthermophilic archaeon Pyrococccus furiosus. Eur. J. Biochem. 213, pp. 305-312.), from Pyrococcus horikoshi (Ando, S., Ishida, H., Kosugi, Y. and Ishikawa, K., 2002. Hyperthermostable endoglucanase from Pyrococcus horikoshi. Appl. Environ. Microbiol. 68, pp. 430-433.), from Rhodothermus marinus (Hreggvidsson, G O., Kaiste, E., Hoist, O., Eggertsson, G., Palsdottir, A. and Kristjansson, J. K., 1996. An extremely thermostable cellulase from the thermophilic eubacterium Rhodothermus marinus. Appl. Environ. Microbiol. 62, pp. 3047-3049.), from Thermatoga maritema (Bronnenmeier, K., Kern, A., Libel, W. and Staudenbauer, W., 1995. Purification of Thermatoga maritema enzymes for the degradation of cellulose materials. Appl. Environ. Microbiol. 61, pp. 1399-1407.), and from Thermatoga neapolitana (Bok, J., Goers, S. and Eveleigh, D., 1994. Cellulase and xylanase systems of Thermatoga neapolitana. ACS Symp. Ser. 566, pp. 54-65; Bok, J., Dienesh, A., Yernool, D. and Eveleigh, D., 1998. Purification, characterization and molecular analysis of thermostable cellulases CeIA and CeIB from Thermatoga neapolitana. Appl. Environ. Microbiol. 64, pp. 4774-4781.).
In some aspects, the thermostable cellulases are any of Cb1952, Cb1953, Cb1954, Cb1946, Cb629, or Cb486 polypeptides.
In some aspects, any mixture of hemicellulases or hemicellulase with cellulases provided herein may further be provided with Cb1581 polypeptides.
Additional Applications
The methods described herein can be practiced in combination with other methods useful for converting lignocellulosic materials into biofuels.
For example, plant material may be subjected to pretreatment including ammonia fiber expansion (AFEX), steam explosion, treatment with alkaline aqueous solutions, acidic solutions, organic solvents, ionic liquids (IL), electrolyzed water, phosphoric acid, and combinations thereof. Pretreatments that remove lignin from the plant material may increase the overall amount of sugar released from the hemicellulose.
In certain embodiments, where a cellulase mixture is being used to release glucose from plant cell walls, a hemicellulase enzyme cocktail of the present disclosure may be used to hydrolyze the hemicellulosic component of the plant material and increase accessibility of the cellulase cocktail to the cellulose fraction of the plant material.
Typically, the compositions and methods of the present disclosure are used to generate biofuels or specialty chemicals. In one aspect, the compositions and methods of the present disclosure are used to degrade hemicellulose into fermentable sugars. The fermentable sugars are then converted into biofuel components, such as ethanol, propanol, and butanol, or specialty chemicals, such as ketones and aldehydes. The fermentable sugars may be converted by a microorganism, such as yeast, or by isolated enzymes.
The hemicellulose-related methods described herein can be practiced in combination with cellulases. Additional methods are provided for the use of the polypeptides and compositions as feed additives for monogastric animal agriculture, including pigs and poultry production.
EXAMPLES
The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.
Example 1: Endoxylanase Cb193 (SEQ ID NOs: 3 and 4)
An endoxylanase, Cb193, was identified in Caldicellulosiruptor bescii. The enzyme is the gene product of Cb193, where Cb stands for C. bescii. The endoxylanase cleaves the xylose backbone of hemicellulose at random to generate shorter chains of xylose in β-1,4-linkages. These xylo-oligosaccharides can range from two or more sugar subunits. The Cb193 protein is 671 amino acids long and has a molecular mass of 77.7 kDa (His-tag+truncated Cb193 protein). The protein has two putative carbohydrate binding modules (CBM) inserted within the glycoside hydrolase (GH) family 10 catalytic domain (FIG. 2A).
Cloning of Cb193
The gene for Cb193 was amplified from Caldicellulosiruptor bescii genomic DNA by PCR using iProof HF DNA polymerase (BIO-RAD). The Cb193 gene was amplified using the following primer set:









Cb193For



(SEQ ID NO: 134)



5′-GACGACGACAAGATGAACTTTGAAGGAAGAGAC-3′ 



 



Cb193Rev



(SEQ ID NO: 135)



5′-GAGGAGAAGCCCGGTTATTTT TTAGCCTTTAC-3′ 






The polymerase chain reaction mixture contained the following:










PCR reaction


















2 U/μL iProof HF DNA polymerase
0.5



13.7 ng/μL C. bescii gDNA
1



50 μM Fw Primer
0.5



50 μM Rv Primer
0.5



10 mM dNTP Mixture
1



5x iProof HF Buffer
10



dH2O
36.5



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol




















Denaturing
98° C.
10 sec




Annealing
62° C.
30 sec
35 cycles



Elongation
72° C.
120 sec 




Last
 4° C.
∞









After the PCR amplification described above, the amplification of Cb193 gene was confirmed by 1% agarose gel electrophoresis. T4 DNA polymerase (Novagen) was then added to the purified PCR product to generate compatible overhangs.












T4 DNA polymerase treatment
Incubation



















2.5 U/mL T4 DNA Polymerase
0.2
22° C.
30 min


Purified PCR Product
2.1
75° C.
20 min


25 mM dATP
1
 4° C.
∞


100 mM DTT
0.5




10x T4 DNA Polymerase Buffer
1




dH2O
5.2




Total
10 μL











After the reaction, the following annealing reaction was prepared with pET46 Ek/LIC vector.














Annealing
Incubation





















pET46 Ek/LIC vector
0.5
22° C.
5 min



Reaction Mixture
1





Total
1.5 μL











After the incubation, EDTA was added to the reaction.














Annealing
Incubation





















25 mM EDTA
0.5
22° C.
5 min



pET46 Ek/LIC vector
0.5





Reaction Mixture
1





Total
2 μL











The annealing mixture for Cb193-pET46 Ek/LIC was introduced into E. coli JM109 by electroporation and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., three colonies were selected and used to inoculate 10 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours and minipreps were made of the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to confirm the size of plasmid/insert DNA. Next, the integrity of the gene was confirming by nucleotide sequencing.
For gene expression, one of the plasmids was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol and ampicillin at 100 μg/ml and 50 μg/ml and incubated at 37° C. overnight. Five to six colonies were inoculated into 3 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 4 hours. One mL of the culture was added to 500 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.25. The inducer, IPTG, was then added at 0.5 mM final concentration, and the culturing continued at 16° C. overnight.
Protein Purification
Cultures were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (50 mM Tris-HCL pH 7.5, 300 mM of NaCl). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed several times to remove the unbound proteins. The bound protein (6-Histidine-tagged Cb193) was then eluted from the resin with an elution buffer composed of the lysis buffer supplemented with 150 mM imidazole.
The gene product of Cb193 was expressed in a truncated form. The first 41 amino acids, which represent a signal peptide, were removed. In the native organism, C. bescii, the signal peptide facilitates transport of the Cb193 out of the cell so that it can act on its target substrate (xylan or plant cell wall) in the medium. Usually after transportation outside the cell, the signal peptide is processed (cleaved) off the protein. Signal peptides can often become a problem during production of recombinant proteins. To circumvent this potential problem, i.e., to prevent secretion of the protein into the periplasm, the PCR primers were designed to remove the signal peptide. The signal peptide does not influence catalytic activity. The design of the PCR primers also ensured that the protein was fused to 6-histidines encoded in the plasmid. The six histidines will bind to either a nickel-charged resin or a cobalt-charged resin. The bound protein can be displaced from the resin with a buffer containing imidazole. This method facilitates quick purification of the protein of interest.
Cb193 (Amino Acid Sequence)
The Cb193 [ENDO-1,4-BETA-XYLANASE A PRECURSOR (EC 3.2.1.8)] amino acid sequence is disclosed in SEQ ID NO: 3. The signal peptide of Cb193, corresponding to amino acid numbers 1-41 of SEQ ID NO: 3 was removed to create the Cb193 protein expression vector. Thus, the expressed Cb193 protein did not contain amino acids 1-41 of SEQ ID NO: 3. The amino acid sequence of the Cb193 protein without the signal peptide is disclosed in SEQ ID NO: 37.
Cb193 (Nucleotide Sequence)
The Cb193 nucleotide sequence is disclosed in SEQ ID NO: 4. Nucleotide numbers 1-123 of SEQ ID NO: 4 correspond to the signal peptide of Cb193, and were not present in the gene cloned to make Cb193. The Cb193 gene without the first 123 nucleotides is disclosed in SEQ ID NO: 38, which encodes the amino acid sequence of SEQ ID NO: 37.
The procedure of cloning the gene for Cb193 into the plasmid pET46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The short peptide comprises the first 15 amino acids of SEQ ID NO: 6. The nucleotide sequence encoding SEQ ID NO: 6 is SEQ ID NO: 5.
The Cb193 gene was expressed in E. coli cells, and the protein was purified in three steps (TALON affinity chromatography, ion exchange chromatography, and gel filtration). FIG. 2B shows an SDS-PAGE of purified Cb193. The molecular markers are in the lane marked M.
Enzyme Activity
The enzymatic activity of Cb193 was measured according to the methods of Morag, E., Bayer, E. A., and Lamed, R. (Relationship of cellulosomal and non-cellulosomal xylanases of Clostridium thermocellum to cellulose degrading enzymes. J. Bacteriol. 1990: 172; 6098-6105). 1 μL of sample supernatant (substrate reacted with enzyme) was spotted on TLC plate. A marker mixture was made by combining each 0.2 μL of 1% xylose/xylobiose/xylotriose/xylotetraose/xylopentaose. All sugars were purchased from Megazyme. The spots were dried and the TLC plate was developed in a developing tank for 1 hour. The plate was dried in a chamber for 30 min. The plate was sprayed with visualizing reagent and incubated for 5 to 10 min at 75° C. to visualize the results.
FIG. 2C shows the enzymatic activity of Cb193 on natural substrates using TLC analysis. Various substrates were tested: soluble wheat arabinoxylan (SWAX), oat-spelt xylan (OSX), birchwood xylan (BWX), carboxymethyl cellulose (CMC), lichenan, glucomannan, 1,4 β-mannan, arabinan. In the case of SWAX, OSX, and BWX, in the presence of Cb193 (+), short xylose chains were released. In the minus (−) lanes, no enzyme was added and therefore no products of hydrolysis were released. X1 (xylose monomer), X2 (xylose dimer or a disaccharide), X3 (trisaccharide), X4 (tetrasaccharide), and pentasaccharide (X5) were loaded in the first lane (M) as markers. The results showed that this enzyme releases shorter chains or oligosaccharides from the complex substrates (SWAX, OSX, and BWX).
The concentration of glucose equivalents was determined following enzymatic hydrolysis of SWAX and OSX according to the methods of Lever, M. (A new reaction for colorimetric determination carbohydrates. Anal. Biochem. 1972: 47; 273-279). 1.5 mL microcentrifuge tubes were “zeroed” in an analytical balance. Next, 5±0.1 mg SWAX or OSX were added to each tube, and the mass measured and recorded. The volumes needed to be added to each tube were calculated based on the mass. Sodium phosphate reaction buffer and enzymes were added to each tube beginning with the reaction buffer. The tubes were incubated with constant mixing in a Rotisserie-style tube mixer at 37° C. for 15 h. The tubes were centrifuged at 10,000 rpm for 5 min at 4° C. 100 μL of sample supernatant was transferred to a clean 1.5 mL centrifuge tube for the pHBAH assay, and 150 μL of sodium citrate reaction buffer was added for a final volume of 250 μL. 1 mL of a stock solution of glucose was made at a concentration of 20 mM in sodium citrate buffer, and then serial dilutions were made in sodium citrate buffer to the following concentrations (20 mM, 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM). 50 mg of pHBAH was dissolved in 50 mL of ice-cold citrate/NaOH solution for a final concentration of 0.1% (w/v), and the solution kept on ice. 112.5 μL of pHBAH solution was added to 37.5 μL of the sample and glucose standard solutions, and the tubes were incubated at 100° C. for 10 min. The tubes were incubated at room temperature for 5 min. The wavelength at 410 nm was measured for the standards and samples. The A410nm and glucose concentrations were plotted against each other, and linear regression was used to fit a line to the data. The correlation coefficient (R2) value was between 0.98 and 1.0. The equation from the standard curve was used to calculate the concentrations of reducing ends in the samples based upon their absorbances.
FIG. 2E shows the enzymatic activity of Cb193 on natural substrates from a reducing sugar assay. In this experiment, a different assay for reducing sugars was used to determine the release of products from the substrates. A standard was made based on known glucose concentrations and their absorbance (color development) in the presence of para-hydroxy-benzoic acid hydrazide (Cann et al. 1999. J. Bacterial. 181:1643-1651 and other reference above—Laver, M. 1972.). Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents.
FIG. 3A shows the thermostability of Cb193. Final 5 nM of Cb193 was incubated at different temperatures from 70 ̃90° C. The Cb193 enzymes were incubated at 70° C., 75° C., 80° C., 85° C., 90° C. The incubated enzymes were taken out at certain time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h, 16 h, and 24 h) and immediately incubated with wheat arabinoxylan (final 1%, w/v) to measure the enzyme activity. The initial velocity of reaction was calculated. The residue activity (%) was calculated by dividing the activity of each sample by the initial activity at zero time. Bars are shown with standard errors for three independent experiments.
FIG. 4 shows the kinetic data of Cb193 on hydrolysis of wheat arabinoxylan, oat spelt xylan, and birchwood xylan. The Km, kcat, and kcat/Km are indicated as well. In FIGS. 4AA, AB, and AC, the experiment was conducted at 75° C. with 50 mM citrate buffer (pH 6.0). In FIGS. BA, BB, and BC, the experiment was conducted at 85° C. with 50 mM citrate buffer (pH 6.0). Xylan substrates (final 2.5-50 mg/mL) were incubated with CB193 (final 5 nM for wheat arabinoxylan and final 50 nM for oat spelt xylan and birchwood xylan). The initial velocity of reaction was calculated. The initial velocities were then plotted against the concentrations of xylan substrates. The Km and kcat were calculated by non-linear fit using the Graphpad software. Bars are shown with standard errors for three independent experiments.
Example 2: Endoxylanase Cb195 (SEQ ID NOs: 7 and 8)
An endoxylanase, Cb195, was identified in Caldicellulosiruptor bescii. The enzyme is the gene product of Cb195, where Cb stands for C. bescii. The endoxylanase cleaves the xylose backbone of hemicellulose at random to generate shorter chains of xylose in β-1,4-linkages. These xylo-oligosaccharides can range from containing two or more sugar subunits. The Cb195 protein is 351 amino acids long and has a molecular weight of 41.9 kDa (His-tag+Cb195 protein) (FIG. 2A).
Cloning of Cb195
The gene for Cb195 was amplified from Caldicellulosiruptor bescii genomic DNA by PCR using iProof HF DNA polymerase (BIO-RAD).
The polymerase chain reaction mixture contained the following:










PCR reaction


















2 U/μL iProof HF DNA polymerase
0.5



13.7 ng/μL C. bescii gDNA
1



50 μM Fw Primer
0.5



50 μM Rv Primer
0.5



10 mM dNTP Mixture
1



5x iProof HF Buffer
10



dH2O
36.5



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol




















Denaturing
98° C.
10 sec




Annealing
62° C.
30 sec
35 cycles



Elongation
72° C.
120 sec 




Last
 4° C.
∞









After the PCR amplification described above, the amplification of Cb195 gene was confirmed by 1% agarose gel electrophoresis. T4 DNA polymerase (Novagen) was then added to the purified PCR product to generate compatible overhangs.












T4 DNA polymerase treatment
Incubation



















2.5 U/mL T4 DNA Polymerase
0.2
22° C.
30 min


Purified PCR Product
2.1
75° C.
20 min


25 mM dATP
1
 4° C.
∞


100 mM DTT
0.5




10x T4 DNA Polymerase Buffer
1




dH2O
5.2




Total
10 μL











After the reaction, the following annealing reaction was prepared with pET46 Ek/LIC vector.














Annealing
Incubation





















pET46 Ek/LIC vector
0.5
22° C.
5 min



Reaction Mixture
1





Total
1.5 μL











After the incubation, EDTA was added to the reaction.














Annealing
Incubation





















25 mM EDTA
0.5
22° C.
5 min



pET46 Ek/LIC vector
0.5





Reaction Mixture
1





Total
2 μL











The annealing mixtures for Cb195-pET46 Ek/LIC was introduced into E. coli JM109 by electroporation and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., three colonies were selected and used to inoculate 10 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours and minipreps were made of the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to confirm the size of plasmid/insert DNA. Next, the integrity of the gene was confirming by nucleotide sequencing.
For gene expression, one of the plasmids was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol and ampicillin at 100 μg/ml and 50 μg/ml and incubated at 37° C. overnight. Five to six colonies were inoculated into 3 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 4 hours. One mL of the culture was added to 500 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.25. The inducer, IPTG, was then added at 0.5 mM final concentration, and the culturing continued at 16° C. overnight.
Protein Purification
Cultures were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (50 mM Tris-HCL pH 7.5, 300 mM of NaCl). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed several times to remove the unbound proteins. The bound protein (6-Histidine-tagged Cb195) was then eluted from the resin with an elution buffer composed of the lysis buffer supplemented with 150 mM imidazole. The protein was purified in three steps (TALON affinity chromatography, ion exchange chromatography, and gel filtration). FIG. 2B shows an SDS-PAGE of purified Cb195. The molecular mass markers are in the lane marked M.
The Cb195 [ENDO-1,4-BETA-XYLANASE A PRECURSOR (EC 3.2.1.8)] amino acid sequence is disclosed in SEQ ID NO: 7. The nucleotide sequence encoding Cb195 is disclosed in SEQ ID NO: 8.
For protein expression, Cb195 was cloned into the plasmid pET46 Ek/LIC. The amino acid sequence of Cb195-pET46 Ek/LIC is SEQ ID NO: 10. Amino acid numbers 1-15 of SEQ ID NO: 10 are from the pET46 Ek/LIC plasmid, and include a sequence of six histidines to facilitate protein purification. The nucleotide sequence encoding SEQ ID NO: 10 is disclosed in SEQ ID NO: 9. Nucleotide numbers 1-45 of SEQ ID NO: 9 are from the pET46 Ek/LIC plasmid.
Enzyme Activity
The enzymatic activity of Cb195 was measured according to the methods of Morag, E., Bayer, E. A., and Lamed, R. (Relationship of cellulosomal and non-cellulosomal xylanases of Clostridium thermocellum to cellulose degrading enzymes. J. Bacteriol. 1990: 172; 6098-6105). 1 μL of sample supernatant (substrate reacted with enzyme) was spotted on TLC plate. A marker mixture was made by combining each 0.2 μL of 1% xylose/xylobiose/xylotriose/xylotetraose/xylopentaose. All sugars were purchased from Megazyme. The spots were dried and the TLC plate was developed in a developing tank for 1 hour. The plate was dried in a chamber for 30 min. The plate was sprayed with visualizing reagent and incubated for 5 to 10 min at 75° C. to visualize the results.
FIG. 2D shows the enzymatic activity of Cb195 on natural substrates using TLC analysis. Various substrates were tested: soluble wheat arabinoxylan (SWAX), oat-spelt xylan (OSX), birchwood xylan (BWX), carboxymethyl cellulose (CMC), lichenan, glucomannan, 1,4 β-mannan, arabinan. In the case of SWAX, OSX, and BWX, in the presence of Cb195 (+), short xylose chains were released. In the minus (−) lanes, no enzyme was added and therefore no products of hydrolysis were released. X1 (xylose monomer), X2 (xylose dimer or a disaccharide), X3 (trisaccharide), X4 (tetrasaccharide), and pentasaccharide (X5) were loaded in the first lane (M) as markers. The results showed that this enzyme releases shorter chains or oligosaccharides from the complex substrates (SWAX, OSX, and BWX).
The concentration of glucose equivalents was determined following enzymatic hydrolysis of soluble wheat arabinoxylan (SWAX) and oat-spelt xylan (OSX) according to the methods of Lever, M. (A new reaction for colorimetric determination carbohydrates. Anal. Biochem. 1972: 47; 273-279). 1.5 mL microcentrifuge tubes were “zeroed” in an analytical balance. Next, 5±0.1 mg SWAX or OSX were added to each tube, and the mass measured and recorded. The volumes needed to be added to each tube were calculated based on the mass. Sodium phosphate reaction buffer and enzymes were added to each tube beginning with the reaction buffer. The tubes were incubated with constant mixing in a Rotisserie-style tube mixer at 37° C. for 15 h. The tubes were centrifuged at 10,000 rpm for 5 min at 4° C. 100 μL of sample supernatant was transferred to a clean 1.5 mL centrifuge tube for the pHBAH assay, and 150 μL of sodium citrate reaction buffer was added for a final volume of 250 μL. 1 mL of a stock solution of glucose was made at a concentration of 20 mM in sodium citrate buffer, and then serial dilutions were made in sodium citrate buffer to the following concentrations (20 mM, 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM). 50 mg of pHBAH was dissolved in 50 mL of ice-cold citrate/NaOH solution for a final concentration of 0.1% (w/v), and the solution kept on ice. 112.5 μL of pHBAH solution was added to 37.5 μL of the sample and glucose standard solutions, and the tubes were incubated at 100° C. for 10 min. The tubes were incubated at room temperature for 5 min. The wavelength at 410 nm was measured for the standards and samples. The A410nm and glucose concentrations were plotted against each other, and linear regression was used to fit a line to the data. The correlation coefficient (R2) value was between 0.98 and 1.0. The equation from the standard curve was used to calculate the concentrations of reducing ends in the samples based upon their absorbances.
FIG. 2E shows the enzymatic activity of Cb195 on natural substrates from a reducing sugar assay. In this experiment, a different assay for reducing sugars was used to determine the release of products from the substrates. A standard was made based on known glucose concentrations and their absorbance (color development) in the presence of para-hydroxy-benzoic acid hydrazide (Cann et al. 1999. J. Bacterial. 181:1643-1651 and other reference above-Laver, M. 1972.). Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents.
FIG. 3B shows the thermostability of Cb195. Final 5 nM of Cb195 were incubated at different temperatures ranging from 65 ̃80° C. The Cb195 enzymes were incubated at 65° C., 70° C., 75° C., and 80° C. The incubated enzymes were taken out at certain time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h, 16 h, and 24 h) and immediately incubated with wheat arabinoxylan (final 1%, w/v) to measure the enzyme activity. The initial velocity of reaction was calculated. The residue activity (%) was calculated by dividing the activity of each sample by the initial activity at zero time. Bars are shown with standard errors for three independent experiments.
FIG. 5 shows the kinetic data of Cb195 on hydrolysis of wheat arabinoxylan, oat spelt xylan, and birchwood xylan. The Km, kcat, and kcat/Km are indicated as well. In FIGS. 5AA, 5AB, and 5AC, the experiment was conducted at 75° C. with 50 mM citrate buffer (pH 6.0). In FIGS. 5BA, 5BB, and 5BC, the experiment was conducted at 75° C. with 50 mM sodium phosphate buffer (pH 6.5). Xylan substrates (final 2.5-50 mg/mL) were incubated with Cb195 (final 5 nM for wheat arabinoxylan and final 50 nM for oat spelt xylan and birchwood xylan). The initial velocity of reaction was calculated. The initial velocities were then plotted against the concentrations of xylan substrates. The Km and kcat were calculated by non-linear fit using the Graphpad software. Bars are shown with standard errors for three independent experiments.
Example 3: α-L-Arabinofuranosidase Cb1172 (SEQ ID NOs: 13 and 14)
An α-L-arabinofuranosidase, Cb1172, was identified in Caldicellulosiruptor bescii. The enzyme is the gene product of Cb1172. The α-L-arabinofuranosidase cleaves arabinose moiety from the xylose backbone or from branched or debranched arabinan of hemicellulose to generate exclusively arabinose. The Cb1172 protein is 505 amino acids long and has a molecular mass of 59.6 kDa (His-tag+Cb1172 protein). The protein has a glycoside hydrolase (GH) family 51 catalytic domain (FIG. 6D).
Cloning of Cb1172
The gene for Cb1172 was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using iProofTM High-Fidelity DNA Polymerase (BIO-RAD). The Cb1172 gene was amplified using the following primer set:









Cb1172Forward



(SEQ ID NO: 136)



5′-GAC GAC GAC AAG ATG AAA AAA GCA AAA GTC  



ATC TAC-3′



 



Cb1172Reverse



(SEQ ID NO: 137)



5′-GAG GAG AAG CCC GGT TAA TTT TCT TTC TTC  



TTT AAC CTG-3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2 U/μL iProoF TM High-Fidelity DNA Polymerase
0.5



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
0.5



50 μM Rv Primer
0.5



10 mM dNTP Mixture
1



5 × iProof HF Buffer
10



dH2O
36.5



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
98° C.
30
sec
1 cycle



Denaturing
98° C.
10
sec
35 cycles



Annealing
62° C.
30
sec




Elongation
72° C.
2
min




Elongation
72° C.
10
min
1 cycle












Last
 4° C.
∞










After the PCR reaction described above, the amplification of Cb1172 gene was confirmed by 1% agarose gel electrophoresis. The DNA corresponding to the expected band on the gel was cut out and applied to a Qiagen Gel Extraction kit to extract the DNA out of the gel.
The Novagen pET-46 Ek/LIC kit was used to treat the purified DNA and ligate it into the pET-46 Ek/LIC vector. The treatment of the purified DNA was as follows:














Unit




Reaction
(μl)
Incubation







0.1 pmol purified PCR product
X




10X T4 DNA Polymerase buffer
1




25 mM dATP
1




100 mM DTT
  0.5




Nuclease-free water
7.3-X




2.5 U/μl T4 DNA Polymerase
  0.2




Total
10 
22° C.
30 min









After the reaction, the enzyme was deactivated by incubating at 75° C. for 20 min.
The following protocol was used to anneal the insert into the pET-46 Ek/LIC vector.














Unit




Reaction
(μl)
Incubation


















pET-46 Ek/LIC vector
0.5




T4 DNA Polymerase treated EK/LIC insert
1




Total
1.5
22° C.
5 min









Then add 0.5 μl 25 mM EDTA. Mix by stirring with pipet tip. Incubate at 22° C. for 5 min.

The ligation mixture for Cb1172-pET-46 Ek/LIC was introduced into E. coli JM109 by electroporation method, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., four colonies were selected and used individually to inoculate 6 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours, and plasmid minipreps (QIAGEN) were made from the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to confirm the size of the plasmid DNA. The plasmid inserts (genes) were sequenced to confirm their identity.
For gene expression, one of the correct plasmids was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol (100 μg/ml) and ampicillin (50 μg/ml) and incubated at 37° C. overnight. Five to six colonies were inoculated into 3 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 4 hours. One mL of the culture was added to 500 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.25. The inducer, IPTG, was then added at 0.01 mM final concentration, and the culturing continued at 16° C. overnight.
Protein Purification
Cultures were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (25 mM Tris-HCL pH 7.8, 750 mM of NaCl, 5% glycerol, 20 mM imidazole, 1.25% Tween-20). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed three times to remove the unbound proteins. The bound protein (6-Histidine-tagged Cb1172) was then eluted from the resin with an elution buffer (50 mM Tris-HCL, pH7.5, 250 mM imidazole).
The gene product of Cb1172 was expressed in its full length form. The design of the PCR primers ensured that the protein was fused to 6-histidines encoded in the plasmid. The six histidines will bind to either a nickel-charged resin or a cobalt-charged resin. The bound protein can then be displaced from the resin with a buffer containing imidazole. This method facilitated quick purification of the protein.
The Cb1172 [α-L-arabinofuranosidase (EC 3.2.1.55)] amino acid sequence is disclosed in SEQ ID NO: 13. The nucleotide sequence encoding Cb1172 is disclosed in SEQ ID NO: 14.
For protein expression, Cb1172 was cloned into the plasmid pET46 Ek/LIC. The amino acid sequence of Cb195-pET46 Ek/LIC is SEQ ID NO: 16. Amino acid numbers 1-15 of SEQ ID NO: 16 are from the pET46 Ek/LIC plasmid, and include a sequence of six histidines to facilitate protein purification. The nucleotide sequence encoding SEQ ID NO: 16 is disclosed in SEQ ID NO: 15. Nucleotide numbers 1-45 of SEQ ID NO: 15 are from the pET46 Ek/LIC plasmid.
The Cb1172 gene was expressed in E. coli cells, and the protein was purified in two steps, including a talon resin purification (immobilized metal affinity chromatography) step making use of the 6-histidines encoded by the plasmid and an anion exchange step using Hitrap Q column. FIG. 6A shows an SDS-PAGE of purified Cb1172.
Enzyme Activity
FIG. 6B shows the enzymatic activity of Cb1172 on natural substrates from a reducing sugar assay. Five different hemicellulosic substrates were tested: arabinan (sugar beet), soluble wheat arabinoxylan (SWAX), rye arabinoxylan (RAX), oat spelt xylan (OSX) and debranched arabinan. Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of arabinose equivalents. Hydrolysis of arabinan (from sugar beet) was higher than hydrolysis of other natural substrates.
The concentration of arabinose equivalents was determined following enzymatic hydrolysis of arabinan (sugar beet), soluble wheat arabinoxylan (SWAX), rye arabinoxylan (RAX), oat spelt xylan (OSX) and debranched arabinan, according to the methods of Lever, M. (A new reaction for colorimetric determination carbohydrates. Anal. Biochem. 1972: 47; 273-279). 1.5 mL microcentrifuge tubes were “zeroed” in an analytical balance. Next, 2±0.1 mg arabinan (sugar beet), SWAX, RAX, OSX and debranched arabinan were added to each tube, and the mass measured and recorded. The volumes needed to be added to each tube were calculated based on the mass. Sodium citrate reaction buffer and enzymes were added to each tube beginning with the reaction buffer. The tubes were incubated with constant mixing in a Thermomixer R (Eppendorf) at 75° C. for 16 h. The tubes were centrifuged at 10,000 rpm for 5 min at 4° C. 50 μL of sample supernatant was transferred to a clean 1.5 mL centrifuge tube for the pHBAH assay. 1 mL of a stock solution of arabinose was made at a concentration of 100 mM in sodium citrate buffer, and then serial dilutions were made in sodium citrate buffer to the following concentrations (50 mM, 25 mM, 12.5 mM and 6.25 mM). 50 mg of pHBAH was dissolved in 50 mL of ice-cold citrate/NaOH solution for a final concentration of 0.1% (w/v), and the solution was kept on ice. 150 μL of pHBAH solution was added to 50 μL of the sample and arabinose standard solutions, and the tubes were incubated at 100° C. for 10 min. The tubes were incubated at room temperature for 5 min. The wavelength at 410 nm was measured for the standards and samples. The A410nm and arabinose concentrations were plotted against each other, and linear regression was used to fit a line to the data. The correlation coefficient (R2) value was between 0.98 and 1.0. The equation from the standard curve was used to calculate the concentrations of reducing ends in the samples based upon their absorbances.
FIG. 6C shows the enzymatic activity of Cb1172 on natural substrates using HPLC analysis. Five different hemicellulosic substrates were tested: arabinan (sugar beet), soluble wheat arabinoxylan (SWAX), rye arabinoxylan (RAX), oat spelt xylan (OSX) and debranched arabinan. In each case, in the presence of Cb1172, arabinose was released. In the absence of Cb1172, only minor amount of arabinose was observed for debranched arabinan; no products of hydrolysis were released for other natural polysaccharides. The results showed that this enzyme releases arabinose from complex substrates (arabinan, SWAX, RAX, OSX and debranched arabinan).
FIG. 6E shows the thermostability of Cb1172. Cb1172 has 57%, 45%, 35% and 22% activity after incubation at 70° C., 75° C., 80° C. and 85° C. for 24 h, respectively. Fifty nM Cb1172 was kept at different temperatures (70° C., 75° C., 80° C., 85° C. and 90° C.). The samples were taken out at the following time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately applied to enzyme activity measurement. The enzyme activity was measured at 85° C. using Cary 300 UV-Vis spectrophotometer (Varian). One hundred μl 1.25 mM pNP-α-L-arabinofuranoside substrate was kept at 85° C. for three minutes to equilibrate. Then twenty five μl of the protein sample was added to the substrate and mixed by pipetting up and down for several times. The optical density at 400 nm was recorded by the spectrophotometer for 2.5 minutes. And the initial velocity of reaction in the first minute was calculated. The initial velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the initial velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the initial velocity of reaction at time 0, then multiplied by 100, respectively.
FIG. 7 shows the kinetic data of Cb1172 on hydrolysis of pNP-α-L-arabinofuranoside. The Km, kcat, and kcat/Km are indicated as well. In FIG. 7A, the experiment was conducted at 90° C.; in FIG. 7B, the experiment was conducted at 75° C. One hundred μl pNP-α-L-arabinofuranoside substrate of different concentrations was kept at 85° C. for three minutes to equilibrate. Then twenty five μl of the protein sample (fifty nM) was added to the substrate and mixed by pipetting up and down for several times. The optical density at 400 nm was recorded by a Cary 300 UV-Visible spectrophotometer for 2.5 minutes. The initial velocity of reaction in the first minute was calculated. The initial velocities were then plotted against the concentrations of pNP-α-L-arabinofuranoside. The Km and kcat were calculated by non-linear fit using the Graphpad software.
Example 4: α-Glucuronidase Cb909 (SEQ ID NOs: 19 and 20)
An α-glucuronidase, Cb909, was identified in Caldicellulosiruptor bescii. The α-glucuronidase cleaves the α-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and the β-1,4-xylosidic linkage backbone of xylan.
The Cb909 gene was amplified by PCR using iProofTM High-Fidelity DNA Polymerase (Bio-Rad) and subcloned into pET46 Ek/LIC vector using Ek/LIC Cloning Kits (Novagen). The forward (For) and reverse (Rev) primer sequences are below:







CB909For


(SEQ ID NO: 138)


5′-GAC GAC GAC AAG ATG ATT TTA TCA AGG AGC AGT


AAC-3′


 


CB909Rev


(SEQ ID NO: 139)


5′-GAG GAG AAG CCC GGT TAC GGA TAT ATT AGT CTT


C-3′






The PCR mixture and the amplification procedure appear below:










PCR mixture









μL













2 U/μL iProoF TM High-Fidelity DNA
0.5



Polymerase




Genomic DNA
1



50 μM Fw Primer
0.5



50 μM Rv Primer
0.5



10 mM dNTP Mixture
1



5 x iProof HF Buffer
10



dH2O
36.5



Total
50



















PCR Protocol





















Denature
98° C.
30
sec




Denature
98° C.
10
sec
35 Cycles



Anneal
62° C.
30
sec




Elongate
72° C.
2
min




Elongate
72° C.
10
min













Final
 4° C.
∞










After the PCR amplification described above, the amplification of Cb909 gene was confirmed by 1% agarose gel electrophoresis. T4 DNA polymerase (Novagen) was then added to the purified PCR product to generate compatible overhangs.












T4 DNA polymerase treatment
Incubation



















2.5 U/μL T4 DNA Polymerase
0.2
22° C.
30 min


Purified PCR Product
0.5
75° C.
20 min


25 mM dATP
1
 4° C.
∞


100 mM DTT
0.5




10x T4 DNA Polymerase Buffer
1




dH2O
6.8




Total
10 μL











After the reaction, the following annealing reaction was prepared with pET46 Ek/LIC vector.














Annealing
Incubation





















pET46 Ek/LIC vector
0.5
22° C.
5 min



Reaction Mixture
1





Total
1.5 μL











After the incubation, EDTA was added to terminate the annealing reaction.














Termination reaction
Incubation





















25 mM EDTA
0.5
22° C.
5 min



pET46 Ek/LIC vector
0.5





Reaction Mixture
1





Total
2 μL











The annealing mixture for Cb909-pET46 Ek/LIC was used to transform E. coli JM109 by electroporation and the cells were plated on LB-ampicillin plates. After overnight incubation at 37° C., three colonies were selected and each was used to inoculate 10 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours and plasmid minipreps were made of each cell culture. The individual plasmid preparations were then electrophoresed on a 1% agarose gel to confirm the size of plasmid/insert DNA. Next, the integrity of the gene was confirmed by nucleotide sequencing.
The Cb909 (α-glucuronidase) amino acid sequence is disclosed in SEQ ID NO: 19.
The nucleotide sequence encoding Cb909 is disclosed in SEQ ID NO: 20.
For protein expression, Cb909 was cloned into the plasmid pET46Ek/LIC. The amino acid sequence of Cb909-pET46 Ek/LIC is SEQ ID NO: 24. Amino acid numbers 1-15 of SEQ ID NO: 24 are from the pET46 Ek/LIC plasmid, and include a sequence of six histidines to facilitate protein purification. The nucleotide sequence encoding SEQ ID NO: 24 is disclosed in SEQ ID NO: 23. Nucleotide numbers 1-45 of SEQ ID NO: 23 are from the pET46 Ek/LIC plasmid.
FIG. 8A shows putative domain architecture of Cb909. FIG. 8B show SDS-PAGE of purified Cb909.
FIG. 8C shows the activity of Cb909. The substrate is aldouronic acids, that is a mixture of xylo-oligosaccharides decorated with 4-O-methyl-D-glucuronosyl (MeGlcA). After incubation with Cb909 at 75° C. for 60 minutes, MeGlcA group was cleaved by Cb909 from aldouronic acids to release undecorated xylose, xylobiose, xylotriose and xylotetraose as products. The condition of the reaction was as follows: 6 nM Cb909, 50 mM Phosphate buffer pH 6.0, 150 mM NaCl, 1 mg/ml aldouronic acids.
FIG. 8D shows the results of pH optimization assay for Cb909. The maximum activity was detected at pH 5.5. This assay was carried out as follows: 1 mg/ml aldouronic acids solution was incubated with 6 nM Cb909 for 10 minutes at 75° C. at each pH. 50 mM citrate buffer containing 150 mM NaCl was used in the range from pH 5 to pH 6.50 mM phosphate buffer containing 150 mM NaCl was used in the range of pH 6 to pH 7. After the reaction, the temperature was quickly increased to 100° C. to terminate the reaction. The amounts of products were detected by HPLC.
FIG. 8E shows the results of optimum temperature assay. The maximum activity of Cb909 was detected at 75° C. (xylobiose and xylotriose). Xylose was produced most efficiently at 70° C. but the amounts of produced xylose at 70° C. and 75° C. were almost the same. This assay was carried out as follows: 1 mg/ml aldouronic acids solution was incubated with 6 nM Cb909 for 10 minutes in 50 mM citrate buffer pH 5.5 that contained 150 mM NaCl. After the reaction the temperature was quickly increased to 100° C. to terminate the reaction. The amounts of products were detected by HPLC.
Example 5: β-Xylosidase Cb2487 (SEQ ID NOs: 27 and 28)
Another enzyme in the enzyme cocktail is a β-xylosidase that was amplified from a Caldicellulosiruptor bescii, Cb2487.
The Cb2487 gene was amplified by PCR using iProofTM High-Fidelity DNA Polymerase (Bio-Rad) and subcloned into pET46 Ek/LIC vector using Ek/LIC Cloning Kits (Novagen). The forward (For) and reverse (Rev) primer sequences are below:









CB2487For



(SEQ ID NO: 140)



5′-GACGACGACAAGATGTCAATTGAAAAAAGGGTAAAC-3′ 



 



CB2487Rev



(SEQ ID NO: 141)



5′-GAGGAGAAGCCCGGTTATTCACACCATGCA-3′ 






The PCR mixture and the amplification procedure appear below:










PCR mixture









μL













2 U/μL iProoF TM High-Fidelity DNA
0.5



Polymerase




Genomic DNA
1



50 μM Fw Primer
0.5



50 μM Rv Primer
0.5



10 mM dNTP Mixture
1



5 x iProof HF Buffer
10



dH2O
36.5



Total
50



















PCR Protocol





















Denature
98° C.
30
sec




Denature
98° C.
10
sec
35 Cycles



Anneal
62° C.
30
sec




Elongate
72° C.
2
min




Elongate
72° C.
10
min













Final
 4° C.
∞










After the PCR amplification described above, the amplification of Cb2487 gene was confirmed by 1% agarose gel electrophoresis. T4 DNA polymerase (Novagen) was then added to the purified PCR product to generate compatible overhangs.












T4 DNA polymerase treatment
Incubation



















2.5 U/μL T4 DNA Polymerase
0.2
22° C.
30 min


Purified PCR Product
0.5
75° C.
20 min


25 mM dATP
1
 4° C.
∞


100 mM DTT
0.5




10x T4 DNA Polymerase Buffer
1




dH2O
6.8




Total
10 μL











After the reaction, the following annealing reaction was prepared with pET46 Ek/LIC vector.














Annealing
Incubation





















pET46 Ek/LIC vector
0.5
22° C.
5 min



Reaction Mixture
1





Total
1.5 μL











After the incubation, EDTA was added to terminate the reaction.














Termination reaction
Incubation





















25 mM EDTA
0.5
22° C.
5 min



pET46 Ek/LIC vector
0.5





Reaction Mixture
1





Total
2 μL











The annealing mixtures for Cb2487-pET46 Ek/LIC was transformed into E. coli JM109 by electroporation and the cells were plated on LB-ampicillin plates. After overnight incubation at 37° C., three colonies were selected and each was used to inoculate 10 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours and plasmid minipreps were made from each cell culture. The individual plasmid preparations were then electrophoresed on a 1% agarose gel to confirm the size of plasmid/insert DNA. Next, the integrity of the gene was confirmed by nucleotide sequencing.
The Cb2487 (β-xylosidase) amino acid sequence is disclosed in SEQ ID NO: 27. The nucleotide sequence encoding Cb2487 is disclosed in SEQ ID NO: 28.
For protein expression, Cb2487 was cloned into the plasmid pET46 Ek/LIC. The amino acid sequence of Cb2487-pET46 Ek/LIC is SEQ ID NO: 30. Amino acid numbers 1-15 of SEQ ID NO: 30 are from the pET46 Ek/LIC plasmid, and include a sequence of six histidines to facilitate protein purification. The nucleotide sequence encoding SEQ ID NO: 30 is disclosed in SEQ ID NO: 29. Nucleotide numbers 1-45 of SEQ ID NO: 29 are from the pET46 Ek/LIC plasmid.
FIG. 9A shows putative domain architecture of Cb2487. FIG. 9B shows SDS-PAGE of purified Cb2487. FIG. 9C shows biochemical assay to determine the optimum pH of Cb2487. FIG. 9D shows biochemical assay to determine the optimum temperature of Cb2487. FIG. 9E shows the kinetic parameter of Cb2487 with pNP-β-D-xylopyranoside as substrate. FIG. 9F shows xylo-oligosaccharides hydrolysis products analysis through thin layer chromatography (TLC). FIG. 9G shows thermostability assay for Cb2487. FIG. 9H shows synergism of β-xylosidase (Cb2487) and α-glucuronidase (Cb909).
FIG. 9A shows putative domain architecture of Cb2487. The putative conserved domains of Cb2487 were analyzed through the NCBI Conserved Domains Database search tool.
FIG. 9B shows SDS-PAGE of purified Cb2487. The lane next to MW shows the protein molecular mass marker. The lane Cb2487 shows the purified protein.
Purification of Cb2487
For Cb2487 purification, the cell pellet was re-suspended in binding buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5), then lysed by passing through an EmulsiFlex C-3 cell homogenizer. The lysate was centrifuged at 20,000×g for 20 min at 4° C. to remove cell debris. The supernatant was incubated at 75° C. for 30 min and centrifuged at 20,000×g for 15 min at 4° C. to remove heat labile proteins. The supernatant after heating was purified by Talon Metal Affinity Resin pre-equilibrated with binding buffer and incubated for 1 h at 4° C. The resin was washed with 50 column volumes of binding buffer, then eluted with 10 column volumes of elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM Imidazole, pH 7.5). The elution fractions were pooled and concentrated with Amicon Ultra-15 centrifugal filter units (50,000 MMCO), and exchanged into Tris-HCl buffer (20 mM, pH 7.5) by three successive concentration and dilution cycles, then purified with Hitrap Q HP column. The elution fractions were pooled and concentrated with Amicon Ultra-15 centrifugal filter units (50,000 MMCO), and exchanged into Tris-HCl buffer (50 mM, pH 7.5, 300 mM NaCl). The proteins were then purified with a SuperdexTM 200 HiloadTM 16/60 size exclusion column using an AKTAxpress system equipped with a UV detector.
FIG. 9C shows a biochemical assay to determine the optimum pH of Cb2487. For the pH optimum assay, para-nitrophenyl-beta-D-xylopyranoside (pNP-X, 0.8 mM) was incubated with Cb2487 concentration (10 nM) at 75° C. in different buffer: pH 4.0-6.0 (citrate buffer, 50 mM, 150 mM NaCl), pH6.0-8.0 (phosphate buffer, 50 mM, 150 mM NaCl), pH 8.5-9.0 (Tris-HCl, 50 mM, 150 mM NaCl).
FIG. 9D shows a biochemical assay to determine the optimum temperature of Cb2487. For temperature optimum assay, pNP-X (0.8 mM) was incubated with Cb2487 (10 nM) in citrate buffer (50 mM, pH 6.0, 150 mM NaCl) at different temperatures (40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100° C.).
FIGS. 9EA and 9EB show a determination of kinetic parameters for Cb2487 with pNP-β-D-xylopyranose as substrate. For the left side panel (FIG. 9EA), the kinetic parameters were determined at 90° C., pH 6.0. For the right side panel (FIG. 9EB), the kinetic parameters were determined at 75° C., pH 6.0. For these assays, different concentrations of pNP-X (0.08-24 mM) were incubated with Cb2487 (10 nM) in citrate buffer (50 mM, pH 6.0, 150 mM NaCl) at 75 and 90° C., respectively.
FIG. 9F shows hydrolytic activity of Cb2487 on xylo-oligosaccharides. Cb2487 (0.5 μM) was incubated with different xylo-oligosaccharides (X2-6) at 75° C. for 15 hr and then the products were separated by TLC.
FIG. 9G shows a thermostability assay for Cb2487. Cb2487 was incubated in citrate buffer (pH 6.0, 50 mM) at different temperatures (70, 75, 80, 85, 90, and 95° C.) without substrate addition, the protein was taken at different times (0, 10 min, 30 min, 1 h, 3 h, 4, 8 h, 12 h, 24 h) and the residual activity was assayed with pNP-X as substrate.
FIG. 9H shows synergism of β-xylosidase (Cb2487) & α-glucuronidase (Cb909). Aldouronic acids were incubated with Cb2487 (0.5 μM), Cb909 (0.5 μM) in citrate buffer (pH 6.0), 75° C. overnight, then assayed with HPLC. Adding Cb909 cleaved off the methylglucuronic acid decorations in aldouronic acids to release xylose and xylo-oligosaccharides. Adding Cb2487 cleaved available beta-1,4-xylosidic linkages to release more xylose. Mixing the two enzymes led to the conversion of the xylo-oligosaccharides released by Cb909 to xylose by Cb2487.
Example 6: Acetyl Xylan Esterase Cb162 (SEQ ID NOs: 33 and 34)
An acetyl xylan esterase, Cb162, was identified in Caldicellulosiruptor bescii. The enzyme is the gene product of Cb162, where Cb stands for C. bescii. The acetyl xylan esterase cleaves the linkages between xylose and the side chain of acetyl groups in hemicellulose to provide more accessibility to other hemicellulases such as xylanase and beta-xylosidase to the backbone of xylan. The Cb162 protein is 321 amino acids long and has a predicted molecular mass of 38.7 kDa (His-tag+Cb162 protein). The protein has a single domain of acetyl xylan esterase (FIG. 10A).
Cloning of Cb162
The gene for Cb162 was amplified from Caldicellulosiruptor bescii genomic DNA by PCR using PrimeSTAR HS DNA polymerase (TaKaRa). The Cb162 gene was amplified using the following primer set:







Cb162-Fw


(SEQ ID NO: 142)


5′-GACGACGACAAGATGGTTTTTGAAATGCCACTTGAAAAG-3′ 


 


Cb162-Rv


(SEQ ID NO: 143)


5′-GAGGAGAAGCCCGGTTATTTTATCATCTCCATAAGATACATAAA


TATCTTGTC-3′ 






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/mL PrimeSTAR DNA polymerase
0.5



19 ng/mL C. bescii gDNA
1



10 mM Fw Primer
1



10 mM Rv Primer
1



2.5 mM dNTP Mixture
4



5x PrimeSTAR Buffer
10



dH2O
32.5



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol




















Denaturing
98° C.
10 sec
30 cycles



Annealing
55° C.
 5 sec




Elongation
72° C.
60 sec




Last
 4° C.
∞









The Ek/LIC cloning kit was utilized (Novagen). Both ends of the amplified gene fragment were digested, in the presence of dATP, with the 3′ to 5′ exonuclease activity of T4 DNA polymerase. The resultant fragment was annealed to the pET-46 Ek/LIC vector.
The ligation mixtures for Cb162-pET46 were introduced into E. coli JM109 by heat shock method and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., four colonies were selected and used to inoculate, individually, 10 mL of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours, and minipreps were made of the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to check the size of the plasmid DNA. For gene expression, one of the plasmids was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol and ampicillin at 100 μg/ml and 50 μg/ml and incubated at 37° C. overnight. Five to six colonies were inoculated into 3 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 4 hours. One mL of the culture was added to 500 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.25. The inducer, IPTG, was then added at 0.1 mM final concentration, and the culturing continued at 16° C. overnight.
Protein Purification
Cultures were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (50 mM Tris-HCL pH 7.5, 20 mM imidazole and 300 mM of NaCl). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a nickel-charged resin (GE Healthcare) and washed several times to remove the unbound proteins. The bound protein (6-Histidine-tagged Cb162) was then eluted from the resin with an elution buffer composed of the lysis buffer supplemented with 250 mM imidazole. The eluted protein was further purified by passing through Hiload 16/20 prepgrade gel-filtration column (GE Healthcare) under the 50 mM Na2HPO4—HCl pH 6.5 and 100 mM NaCl buffer.
The Cb162 (acetyl xylan esterase) amino acid sequence is disclosed in SEQ ID NO: 33. The nucleotide sequence encoding Cb162 is disclosed in SEQ ID NO: 34.
For protein expression, Cb162 was cloned into the plasmid pET46 Ek/LIC. The amino acid sequence of Cb162-pET46 Ek/LIC is SEQ ID NO: 36. Amino acid numbers 1-15 of SEQ ID NO: 36 are from the pET46 Ek/LIC plasmid, and include a sequence of six histidines to facilitate protein purification. The nucleotide sequence encoding SEQ ID NO: 36 is disclosed in SEQ ID NO: 35. Nucleotide numbers 1-45 of SEQ ID NO: 35 are from the pET46 Ek/LIC plasmid.
FIG. 10A shows the domain structure of Cb162; the protein has an acetyl xylan esterase domain.
The Cb162 gene was expressed in E. coli cells, and the protein was purified in two steps, making use of the 6-histidines encoded by the plasmid. FIG. 10B shows an SDS-PAGE of purified Cb162. The molecular markers are in the lane next to the purified Cb162.
FIG. 10C shows the enzymatic activity of Cb162 at different pHs using para-nitrophenol adducted acetate (pNP-acetate) as a substrate. The released pNP was monitored continuously at an absorbance of 400 nm using Synergy 2 Microplate reader (BioTek). The initial rate of hydrolysis was adopted as an enzyme activity. The figure shows the pH profile of Cb162 on pNP-acetate. The pH effect on the Cb162 was examined at 50° C. in the presence of 50 mM citrate-NaOH (pH 4.0 to 6.0), 50 mM Na2HPO4—HCl (pH 6.0 to 8.0), with 150 mM NaCl, respectively. 0.1 μM of purified Cb162 and 2 mM pNP-acetate were used for this assay.
FIG. 10D shows the temperature profile of Cb162 on pNP-acetate. The temperature profile was performed in 50 mM Na2HPO4—HCl, pH 7.0, and 150 mM NaCl, at temperatures between 40° C. and 75° C. with 5° C. increments. 0.04 μM of purified Cb162 and 2 mM pNP-acetate were used for this assay.
FIG. 10E shows the thermostability profile of Cb162 on pNP-acetate. 0.02 μM of purified Cb162 in 50 mM Na2HPO4—HCl, pH 7.0, and 150 mM NaCl was incubated for 0 to 24 hours at temperatures between 60° C. and 80° C. with 5° C. intervals, and the residual activities were measured.
FIG. 10F shows the kinetic study of Cb162. 0.04 μM of purified Cb162 in 50 mM Na2HPO4—HCl, pH 6.0, and 150 mM NaCl was incubated with a various concentration of pNP-acetate, and the initial rate of hydrolysis was plotted on the graph. The kinetic parameters were determined by Michaelis-Menten equation utilizing Graph Pad Prism v5.01 (GraphPad Software).
Example 7: Hydrolysis of Polysaccharides with Enzyme Cocktails of Caldicellulosiruptor bescii Hemicellulases Containing a Single Type of Endoxylanase
Mixtures of one or more of the enzymes endoxylanase (Cb193), α-arabinofuranosidase (Cb1172), β-xylosidase (Cb2487), α-glucuronidase (Cb909), and acetyl xylan esterase (Cb162) were incubated with the polysaccharides soluble wheat arabinoxylan, birch wood xylan, and oat spelt xylan. For each substrate, incubation of the substrate with a cocktail containing all of the enzymes endoxylanase (Cb193), α-arabinofuranosidase (Cb1172), 3-xylosidase (Cb2487), α-glucuronidase (Cb909), and acetyl xylan esterase (Cb162) yielded a greater release of monosaccharides from xylan than incubating the substrate with an enzyme cocktail containing less than all of the enzymes.
FIG. 11 shows synergy of C. bescii hemicellulolytic enzymes on soluble wheat arabinoxylan (SWAX) hydrolysis. SWAX (8.0%, w/v) was incubated with different hemicellulase mixes at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar [FIG. 11A] and HPLC [FIG. 11B] analysis. The hemicellulases applied include Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM).
FIG. 12 shows synergy of C. bescii hemicellulolytic enzymes on oatspelt xylan (OSX) hydrolysis. OSX (8.0%, w/v) was incubated with different hemicellulase at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar [FIG. 12A] and HPLC [FIG. 12B] analysis. The hemicellulases applied include Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM).
FIG. 13A shows SWAX hydrolysis with a hemicellulase cocktail at different temperatures. SWAX (8.0%, w/v) was incubated with Cb193 (0.5 μM), Cb2487 (4 μM), Cb1172 (0.5 μM), Cb162 (0.5 μM), and Cb909 (0.5 μM) at 65° C., 70° C., 75° C., 80° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay.
FIG. 13B shows BWX hydrolysis with a hemicellulase cocktail at different temperatures. BWX (8.0%, w/v) was incubated with Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM) at 65° C., 70° C., 75° C., 80° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay.
FIG. 13C shows OSX hydrolysis with a hemicellulase cocktail at different temperatures. OSX (8.0%, w/v) was incubated with Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM) at 65° C., 70° C., 75° C., 80° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay.
Example 8: Hydrolysis of Polysaccharides with Enzyme Cocktails of Caldicellulosiruptor bescii Hemicellulases Containing Two Types of Endoxylanase
Mixtures containing the enzymes α-arabinofuranosidase (Cb1172), β-xylosidase (Cb2487), α-glucuronidase (Cb909), acetyl xylan esterase (Cb162), and one or both of the endoxylanases (Cb193 and Cb195) were incubated with the polysaccharides soluble wheat arabinoxylan, birch wood xylan, and oat spelt xylan. For each substrate, incubation of the substrate with a cocktail containing both of the endoxylanases (Cb193 and Cb195) yielded a greater release of monosaccharides from xylan than incubating the substrate with an enzyme cocktail containing only one of the endoxylanases.
FIG. 14A shows SWAX hydrolysis was improved by adding two xylanases (Cb195 and Cb193) in the hemicellulase mixture. SWAX (8.0%, w/v) was incubated with different hemicellulase mixes at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar analysis. Different hemicellulase mixtures were applied in the hydrolysis: Mix I) Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); Mix II) Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); Mix III) Cb195 (0.25 μM), Cb193 (0.25 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM).
FIG. 14B shows BWX hydrolysis was improved by adding two xylanases (Cb195 and Cb193) in the hemicellulase mixture. BWX (8.0%, w/v) was incubated with different hemicellulase mixes at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar analysis. Different hemicellulase mixtures were applied in the hydrolysis: Mix I) Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); Mix II) Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); Mix III) Cb195 (0.25 μM), Cb193 (0.25 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM).
FIG. 14C shows OSX hydrolysis was improved by adding two xylanases (Cb195 and Cb193) in the hemicellulase mixture. OSX (8.0%, w/v) was incubated with different hemicellulase mixes at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar analysis. Different hemicellulase mixtures were applied in the hydrolysis: Mix I) Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); Mix II) Cb193 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM); Mix III) Cb195 (0.25 μM), Cb193 (0.25 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb909 (0.5 μM), and Cb162 (0.5 μM).
FIG. 15 shows soluble wheat arabinoxylan hydrolysis with hemicellulase cocktail of Caldicellulosiruptor bescii. Different concentrations of SWAX (1.0, 2.0, 4.0, 6.0, 8.0%, w/v) were incubated with Cb193 (0.5 μM), Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb162 (0.5 μM), and Cb909 (0.5 μM) for 15 hr at 75° C. in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay. FIG. 15A shows reducing sugar in the control and hydrolysis mixtures, and FIG. 15B shows comparison of calculated and average of actual reducing sugar in hydrolysis mixtures with different substrate concentrations.
FIG. 16 shows birch wood xylan hydrolysis with hemicellulase cocktails of Caldicellulosiruptor bescii. Different concentrations of BWX (1.0, 2.0, 4.0, 6.0, 8.0%, w/v) were incubated with Cb193 (0.5 μM), Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb162 (0.5 μM), and Cb909 (0.5 μM) at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay. FIG. 16A shows reducing sugar in the control and hydrolysis mixtures, and FIG. 16B shows comparison of calculated and average of actual reducing sugar in hydrolysis mixtures with different substrate concentrations.
FIG. 17 shows oat spelt xylan hydrolysis with hemicellulase cocktail of Caldicellulosiruptor bescii. Different concentrations of OSX (1.0, 2.0, 4.0, 6.0, 8.0%, w/v) were incubated with Cb193 (0.5 μM), Cb195 (0.5 μM), Cb1172 (0.5 μM), Cb2487 (4 μM), Cb162 (0.5 μM), and Cb909 (0.5 μM) at 75° C. for 15 hr in citrate buffer (50 mM, pH 6.0, 150 mM NaCl), and subjected to reducing sugar assay. FIG. 17A shows reducing sugar in the control and hydrolysis mixtures, and FIG. 17B shows comparison of calculated and average of actual reducing sugar in hydrolysis mixtures with different substrate concentrations.
Example 9: Endocellulase/Mannanase Cb1952
An endocellulase/mannanase, Cb1952, was identified in Caldicellulosiruptor bescii. The enzyme is the gene product of Cb1952, wherein Cb stands for Caldicellulosiruptor bescii. The protein has a Glycoside Hydrolase (GH) family 9 catalytic domain (cellulase domain), three family 3 carbohydrate binding modules (CBMs) (one CBM3c and two CBM3b modules) and one GH5 catalytic domain (mannanase domain) (FIG. 18).
A wild-type Cb1952 protein, lacking the signal peptide, and several truncational mutations (TM1, TM2, TM3, TM4, TM5, TM6, and TM7) were systematically constructed for functional analysis (FIG. 18).
As shown in FIG. 18, TM1 contained the GH9 module and the three CBMs, TM2 contained the GH9 module and two CBMs, TM3 contained the GH9 module and one CBM (CBM3c), and TM4 was made up of only the GH9 module. The truncated mutant TM5 was composed of the three CBMs linked to the GH5 module, whereas TM6 and TM7 were composed of the CBM3c and CBM3b, respectively. The SDS-PAGE results in FIG. 19 show that all protein constructs were successfully expressed as soluble proteins and highly purified.
Cloning of Cb1952 Wild-Type
The gene for Cb1952 wild-type was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using PrimeSTAR DNA Polymerase (TAKARA). The Cb1952 wild-type gene was amplified using the following primer set:









Cb1952 wild-type Forward:



(SEQ ID NO: 39)



5′- GAC GAC GAC AAG ATG GCA ACA ACC TTT



AACTAT GGT GAA GCT C -3′


 



Cb1952 wild-type Reverse:



(SEQ ID NO: 40)



5′- GA GGA GAA GCC CGG TTA TTC AGC ACC



AAT CGC ATT AGT TTT ATA CC -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
5
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










Cloning of Cb1952TM1

The gene for Cb1952TM1 was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using PrimeSTAR DNA Polymerase (TAKARA). The Cb1952TM1 gene was amplified using the following primer set:









Cb1952TM1Forward:



(SEQ ID NO: 41)



5′- GAC GAC GAC AAG ATG GCA ACA ACC TTT AAC



TAT GGT GAA GCT C -3′


 



Cb1952TM1Reverse:



(SEQ ID NO: 42)



5′- GAG GAG AAG CCC GGT TAG CTA GTA TCT ATC



TTC ACT ATT CCA CTG -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
4
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










Cloning of Cb1952TM5

The gene for Cb1952TM5 was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using PrimeSTAR DNA Polymerase (TAKARA). The Cb1952TM5 gene was amplified using the following primer set:









Cb1952TM5Forward:



(SEQ ID NO: 43)



5′- GAC GAC GAC AAG ATG A AT TTC AAA GCT



ATC GAA AAG CCA AC -3′


 



Cb1952TM5Reverse:



(SEQ ID NO: 40)



5′- GA GGA GAA GCC CGG TTA TTC AGC ACC AAT



CGC ATT AGT TTT ATA CC -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
4
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










After the PCR reactions described above, the amplification of Cb1952 wild-type, Cb1952TM1 and Cb1952TM5 gene was confirmed by 1% agarose gel electrophoresis. The DNA corresponding to the expected band on the gel was cut out and applied to a Qiagen Gel Extraction kit to extract the DNA out of the gel.
A Novagen pET-46 Ek/LIC kit was used to treat the purified DNA and ligate it into the pET-46 Ek/LIC vector. The treatment of the purified DNA was as follows:














Unit




Reaction
(μl)
Incubation







0.1 pmol purified PCR product
X




10X T4 DNA Polymerase buffer
1




25 mM dATP
1




100 mM DTT
  0.5




Nuclease-free water
7.3-X




2.5 U/μl T4 DNA Polymerase
  0.2




Total
10 
22° C.
30 min









After the reaction, the enzyme was inactivated by incubating at 75° C. for 20 min.
The following protocol was used to anneal the insert into the pET-46 Ek/LIC vector.














Unit




Reaction
(μl)
Incubation


















pET-46 Ek/LIC vector
0.5




T4 DNA Polymerase treated EK/LIC insert
1




Total
1.5
22° C.
5 min









Then add 0.5 μl 25 mM EDTA. Mix by stirring with pipet tip. Incubate at 22° C. for 5 min.

The ligation mixtures for Cb1952 wild-type, Cb1952TM1- or Cb1952TM5-pET-46 Ek/LIC were introduced into E. coli NovaBlue competent cells by chemical transformation method, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., four colonies were selected and each was used to inoculate 6 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours, and minipreps (QIAGEN) were made of the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to check the size of the plasmid DNA. After confirmation that the gene has been inserted into plasmids, the genes were sequenced to confirm their identity. The plasmids with the right insertion sequences were selected for recombinant protein production.
Cb1952TM2, Cb1952TM3, Cb1952TM4, Cb1952TM6, and Cb19527 were prepared through similar steps as above, with different steps as appropriate (e.g. primer sequences).
For expression of each enzyme, plasmid containing the wild type, TM1, TM2, TM3, TM4, TM5, TM6, or TM7 was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol (50 μg/ml) and ampicillin (100 μg/ml) and incubated at 37° C. overnight. Five to six colonies were inoculated into 10 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 6 hours. Ten mL of the culture was added to 1000 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.3. The inducer, IPTG, was then added at 0.1 mM final concentration, and the culturing continued at 16° C. overnight.
Protein Purification
Cultures were centrifuged to collect the cell pellet. For Cb1952 wild-type, the pellet was then suspended in a lysis buffer (25 mM Tris-HCL pH 7.8, 750 mM of NaCl, 5% glycerol, 20 mM imidazole, 1.25% Tween-20). For Cb1952TM1, the pellet was then suspended in a lysis buffer (25 mM Tris-HCL pH 7.8, 100 mM of NaCl, 10% glycerol, 10 mM imidazole, 1.25% Tween-20). For the other Cb1952 TM mutants, the pellet was then suspended in a lysis buffer without imidazole (50 mM Tris-HCL pH 7.5, 300 mM of NaCl). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed three times to remove the unbound proteins. The bound protein was then eluted from the resin with an elution buffer (50 mM Tris-HCL, pH7.5, 250 mM imidazole).
The design of the PCR primers ensured that each of the proteins was fused to 6-histidines (N-terminal tag) encoded in the plasmid. The six histidines will bind to either a nickel-charged resin or a cobalt-charged resin. The bound protein can be displaced from the resin with a buffer containing imidazole. This method facilitates quick purification of the protein of interest. All recombinant proteins were purified by immobilized metal ion affinity chromatography (IMAC) using talon resin (Clontech, Mountain View, Calif.) according to the manufacturer's instructions. For Cb1952 wild-type, the eluted protein was dialyzed against a protein storage buffer (50 mM Tris-HCl, 150 mM NaCl, pH7.5). The protein was heated at 75° C. for 10 min and centrifuged at 16,400 rpm for 20 min to precipitate any co-eluting thermo-labile host proteins. The recombinant protein was further purified by gel filtration using an ÄKTAxpress TWIN fast protein liquid chromatograph (FPLC) system equipped with a Hiload 16/60 Superdex 200 column (GE Healthcare, Piscataway, N.J.). For Cb1952TM1, the eluted protein was dialyzed against the protein storage buffer. The protein was then heated at 75° C. for 20 min and centrifuged at 16,400 rpm for 20 min. The supernatant was further purified by gel filtration as described above. For the other mutants, the recombinant proteins eluted from Talon resin were directly applied to gel filtration for purification close to homogeneity. FIG. 19 shows an SDS-PAGE of purified Cb1952 proteins.
Gene and Protein Sequences of Cb1952WT, Cb1952TM1, and Cb1952TM5
Cb1952 Full-Length Amino Acid Sequence
The full-length Cb1952 endocellulase/mannanase (EC 3.2.1.4/EC 3.2.1.78) amino acid sequence is disclosed in SEQ ID NO: 44. The signal peptide of Cb1952, corresponding to amino acid numbers 1-28 of SEQ ID NO: 44 was removed during all PCR amplifications. Thus, the expressed wild-type Cb1952 protein did not contain amino acid numbers 1-28 of SEQ ID NO: 44. The amino acid sequence of the wild-type Cb1952 protein without the signal peptide is disclosed in SEQ ID NO: 114.
The procedure of cloning the gene for wild-type Cb1952 (without the signal peptide) into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The wild-type Cb1952 amino acid sequence (without the signal peptide) with the short peptide is disclosed in SEQ ID NO: 51. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 51.
Cb1952 Full-Length Nucleotide Sequence
The full-length Cb1952 nucleotide sequence is disclosed in SEQ ID NO: 45. The signal peptide of Cb1952, corresponding to nucleotide numbers 1-84 of SEQ ID NO: 45 was removed during all PCR amplifications. Thus, the nucleotide sequence used to express wild-type Cb1952 protein did not contain nucleotide numbers 1-84 of SEQ ID NO: 45. The nucleotide sequence encoding the wild-type Cb1952 protein without the signal peptide is disclosed in SEQ ID NO: 115.
The wild-type Cb1952 nucleotide sequence (without the signal peptide) with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 50. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 50.
Cb1952TM1 Amino Acid Sequence
The Cb1952TM1 endocellulase (EC 3.2.1.4) amino acid sequence is disclosed in SEQ ID NO: 46. The procedure of cloning the gene for Cb1952TM1 into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The Cb1952TM1 amino acid sequence with the short peptide is disclosed in SEQ ID NO: 53. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 53.
Cb1952TM1 Nucleotide Sequence
The Cb1952TM1 nucleotide sequence is disclosed in SEQ ID NO: 47. The Cb1952TM1 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 52. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 52.
Cb1952TM5 Amino Acid Sequence
The Cb1952TM5 amino acid sequence is disclosed in SEQ ID NO: 48. The procedure of cloning the gene for Cb1952TM5 into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The Cb1952TM5 amino acid sequence with the short peptide is disclosed in SEQ ID NO: 55. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 55.
Cb1952TM5 Nucleotide Sequence
The Cb1952TM5 nucleotide sequence is disclosed in SEQ ID NO: 49. The Cb1952TM5 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 54. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 54.
Enzyme Activity
Determination of Optimal pH, Optimal Temperature, and Thermostability
The optimal pH for Cb1952 WT, TM1, TM2, and TM3 with PASC, as substrate, were in the range of pH5.0-5.5 and the optimal temperature for each of these proteins was 85° C. In the case of TM4, the optimal pH and temperature with PASC were 6.5 and 55° C., respectively. The thermostability assays were carried out on the wild type and truncation mutants harboring cellulase activities. At 80° C. and 85° C., the residual activities of WT, TM1, and TM2 after 24 h incubation were less than 20% except TM3, which retained 61.8% activity. At 75° C., the residual activities of WT, TM1, TM2, and TM3 after 24 h incubation were 43.1%, 75.7%, 53.6%, and 101.7%, respectively. Deletion of CBM3c dramatically reduced the thermostability of the enzyme. The truncated mutant TM4 remained stable at 45° C. and 50° C., but the enzyme rapidly lost its activity at temperatures above 55° C. (FIG. 81C). The pH and temperature optima were also determined for hydrolysis of mannan substrates. For the wild-type enzyme the optimal pH and temperature for mannan hydrolysis were 5.5-6.5 and 90° C., respectively, and for TM5 the values were 6.5 and 90° C., respectively (data not shown).
Hydrolysis of Phosphoric Acid Swollen Cellulose, Cello- and Manno-Oligosaccharides by Cb1952 and its Mutants
The capacity of the wild-type Cb1952 and its TM1 and TM5 mutants, representing the mutants that harbored the GH9 module with the 3 CBMs and the GH5 module together with 3 CBMs (FIG. 18) were investigated in a time course approach for hydrolysis of PASC. As shown in the chromatograph in FIG. 78, release of products, mostly cellobiose and glucose, was observed for the wild-type (A) (FIG. 78A) and the TM1 (B) (FIG. 78B) mutant which contains the GH9 module. Very little to no hydrolysis of PASC was detected from TM5 (C) (FIG. 78C) (the construct with the GH5 module). By further testing hydrolysis of cello-oligosaccharides, it was confirmed that the β-1,4-glucose cleaving activity was present in the GH9 domain (FIG. 23). On manno-oligosaccharides hydrolysis, the wild-type and TM5 showed cleavage activity of oligosaccharides with degree of polymerization (DP) of 3 and above (FIG. 24). Interestingly, TM1 also showed activity on substrates of DP of 5 or higher, albeit the activity was lower than the wild-type enzyme and the TM5 mutant (FIG. 24). No transglycosylation activities were found for the wild-type, TM1, and TM5 on glucose, cello-oligosaccharides, mannose, and manno-oligosaccharides.
Activities and Kinetic Parameters of Cb1952 and its Mutants on Cellulosic Substrates
Specific activities were determined for the wild-type protein and each of the mutants with Avicel, a model crystalline cellulose, and filter paper, as substrates. On Avicel, deletion of the individual CBMs led to a decrease in specific activity of the truncated mutant (TM1, TM2, and TM3) (Table 1). The truncated mutant with either two or one of the CBM3b (TM1 and TM2, respectively) only showed a slight decrease in specific activity compared with the WT enzyme. In contrast, deleting the two CBM3b's led to a protein with less than half the specific activity of the WT protein on Avicel. A similar trend was observed for specific activity on filter paper as substrate, although the decreases in activity were less pronounced (Table 1). On both substrates, a construct made up of the GH9 catalytic module alone had only 3.8% and 16.2% of the activities observed for the WT protein on Avicel and filter paper, respectively.







TABLE 1







Specific activities and kinetic parameters of Cb1952 wild-type, its


truncation mutants, and the mutants of TM3 on cellulose substratesa











Avicel
Filter paper




(μmol sugar/
(μmol sugar/
PASCb













min/μmol
min/μmol
kcat
Km
kcat/Km


Protein
protein)
protein)
(s−1)
(mg/ml)
(s−1 ml/mg)















WT
10.15 ± 0.51 
16.12 ± 2.86
2.58 ± 0.15
0.36 ± 0.10
7.16


TM1
8.53 ± 1.47
17.27 ± 2.06
2.12 ± 0.13
0.14 ± 0.07
15.14


TM2
8.94 ± 0.89
14.31 ± 3.13
2.16 ± 0.18
0.19 ± 0.10
11.37


TM3
4.47 ± 0.81
12.87 ± 1.44
3.09 ± 0.30
0.65 ± 0.24
4.75


TM3G208WG
3.68 ± 0.69
13.74 ± 1.80
7.92 ± 0.78
1.71 ± 0.45
4.63


TM3G208W
4.86 ± 0.49
14.61 ± 3.41
6.36 ± 0.74
1.35 ± 0.46
4.71


TM3T298F
5.53 ± 0.53
15.14 ± 1.71
8.53 ± 0.67
2.17 ± 0.42
3.93


TM4
0.39 ± 0.02
 2.62 ± 0.56
0.08 ± 0.01
3.73 ± 0.81
0.02





aThe reactions were carried out at 75° C. except that for TM4, which was done at 45° C.


bPASC: phosphoric acid swollen cellulose.






The phosphoric acid swollen cellulose, derived from Avicel, was used to examine the kinetic parameters of the WT protein and its mutants (Table 1). The estimated kcat for the WT (2.58 s−1) and its truncated mutants (2.12-3.09 s−1) was very modest. Interestingly TM1 exhibited a catalytic efficiency twice higher than that of the wild type, suggesting that the catalytic activities of the GH9 and GH5 modules are functionally coupled. Similar functional coupling of different catalytic modules within a single polypeptide was proposed for another plant cell wall degrading enzyme Prevotella ruminicola Xyn10D-Fae1A (9), a two-domain arginine kinase from the deep-sea clam Calyptogena kaikoi (40), and a flagellar creatine kinase from Chaetopterus variopedatus (13). The kinetic parameters of TM4, the protein with only the GH9 catalytic module were very poor compared to the proteins linked to the CBMs, alluding to the importance of these auxiliary modules to the function of Cb1952.
Activities and Kinetic Parameters of Cb1952 and its Mutants on Mannan-Like Substrates
The enzymatic activities of Cb1952 and its mutants on mannan-like substrates were also investigated. The substrates tested were locust bean gum, guar gum, and konjac glucomannan. The wild type enzyme exhibited very high kcat on all tested mannose based substrates. On locust bean gum, konjac glucomannan, and guar gum, the kcat values were 1420 s−1, 1068 s−1, and 696 s−1, respectively (Table 2). Based on the data in Table 2, the catalytic activity for degradation of mannan and mannose-configured substrates is located in the GH5 module. It was observed that cleaving the GH9 module from the polypeptide to create the TM5 mutant increased the kcat of this mutant, compared to the wild-type, by 2.4-, 2.8-, and 1.6-fold for locust bean gum, guar gum, and konjac glucomannan, respectively. Note that the standard error was quite high for the kcat for guar gum. A corresponding increase in the Km of TM5 on each mannose-configured substrate led to catalytic efficiencies that were lower than those determined for the wild-type protein (Table 2). The truncated mutants containing the GH9 catalytic module in addition to either all three CBMs (TM1) or only the CBM3c (TM3) were almost devoid of activity on both locust bean gum and guar gum. These mutants, however, exhibited very high activity on konjac glucomannan.







TABLE 2







Kinetic parameters of Cb1952 wild-type, its truncation mutants, and the mutants of TM3 on


mannan substrates and konjac glucomannana











Locust bean gum
Guar gum
Konjac glucomannan



















kcat/Km


kcat/Km


kcat/Km



kcat
Km
(s−1
kcat
Km
(s−1
kcat
Km
(s−1


Protein
(s−1)
(mg/ml)
ml/mg)
(s−1)
(mg/ml)
ml/mg)
(s−1)
(mg/ml)
ml/mg)





WT
1420 ± 158 
0.62 ± 0.27
2290
 696 ± 56.7
2.26 ± 0.42
308
1068 ± 271 
1.84 ± 1.03
581


TM1
0.23 ± 0.01
3.89 ± 0.41
5.9 × 10−2
n.d
n.d
n.d
 907 ± 50.7
1.85 ± 0.30
490


TM3
0.15 ± 0.06
4.36 ± 2.82
3.5 × 10−2
(1.03 ± 0.17) × 10−2
0.94 ± 0.50
1.10 × 10−2
 611 ± 68.9
1.30 ± 0.43
470


TM3G208WG
2.31 ± 0.15
1.93 ± 0.31
   1.2
1.03 ± 0.35
9.28 ± 4.36
1.11 × 10−1
1614 ± 143 
2.37 ± 0.49
681


TM3G208W
0.12 ± 0.03
3.33 ± 1.49
3.7 × 10−2
(1.01 ± 0.01) × 10−2
0.50 ± 0.20
2.01 × 10−2
1119 ± 160 
1.80 ± 0.68
621


TM3T298F
1.12 ± 0.55
12.58 ± 7.94 
8.9 × 10−2
(8.92 ± 1.98) × 10−2
3.62 ± 1.53
2.47 × 10−2
1102 ± 77.4
2.61 ± 0.43
422


TM5
3446 ± 367 
1.82 ± 0.48
1893
1940 ± 570 
11.98 ± 4.69 
162
1710 ± 119 
3.72 ± 0.48
460





aKonjac glucomannan is a polysaccharide with mixed linkage of glucose and mannose.






Site-Directed Mutagenesis

The architectural diversity of GH9 modules have been assigned to four different groups known as theme A, B, C, and D (19). In Cb1952, the GH9 catalytic module is linked to an accessory CBM3c at its C-terminus, and this is the architecture of the members of theme B1. In theme B1, there are both processive endoglucanases (7, 12, 34) and non-processive endoglucanases (2, 10). The distribution of reducing ends in the soluble and insoluble fractions of cellulase-hydrolyzed filter paper is commonly used to estimate the processivity of a cellulase (17). Our results, based on such an experiment, determined that Cb1952 and its truncation mutants (TM1, TM2, TM3, and TM4) do not harbor a processive GH9 catalytic module since their end products contained 40%-50% insoluble reducing ends (Table 3).







TABLE 3







Distribution of reducing sugars in soluble and insoluble


fractions of filter paper hydrolyzed by Cb1952 wild-type,


its truncation mutants, and the mutants of TM3a









Ratio










Reducing
(Sol./



sugar (%)
Insol.b













Soluble
Insoluble
Sol-
Insol-
Reducing


Protein
(mM)
(mM)
uble
uble
sugar)





WTc
1.32 ± 0.07
1.32 ± 0.03
50.0
50.0
1.00


TM1c
2.02 ± 0.04
1.38 ± 0.08
59.4
40.6
1.46


TM2c
2.13 ± 0.11
1.42 ± 0.06
60.0
40.0
1.50


TM3c
1.98 ± 0.08
1.56 ± 0.12
55.9
44.1
1.27


TM4d
3.18 ± 0.15
2.72 ± 0.40
53.9
46.1
1.17


TM3G208WGc
1.80 ± 0.12
1.44 ± 0.13
55.6
44.4
1.25


TM3G208Wc
1.74 ± 0.08
1.57 ± 0.11
52.6
47.4
1.11


TM3T298Fc
1.87 ± 0.10
1.52 ± 0.12
55.2
44.8
1.23





aThe reactions were carried out at 75° C. for 16 h for all enzymes except TM4, which was carried out at 45° C.


bSol./Insol.: soluble versus insoluble.


cEnzyme concentration was 0.5 μM.


dEnzyme concentration was 10 μM.






An amino acid sequence alignment of the GH9 domain of Cb1952 with those of Clostridium cellulolyticum Cel9G (a non-processive endoglucanase) and Thermobifida fusca Cel9A (a processive endoglucanase) was examined. The C. cellulolyticum and T. fusca proteins represent two types of family 9 theme B1 endoglucanases with enzyme-cello-oligosaccharides co-crystal structures solved (26, 34). The amino acid sequence alignment showed that most of the residues involved in cellulose substrate binding are well conserved in the GH9 module of Cb1952 (FIG. 79). However, neither of two aromatic residues (Trp-209 in T. fusca and Phe-308 in C. cellulolyticum) responsible for hydrophobic stacking at subsite −3, is present in Cb1952 (FIG. 79). As aromatic residues involved in hydrophobic stacking interactions with the substrates contribute to the processivity of the enzyme during hydrolysis of crystalline substrate (15, 47), we mutated the corresponding amino acid residue in Cb1952TM3 to an aromatic residue by changing Gly-208 to Trp-208 or by inserting a tryptophan before Gly-208 to obtain a TM3G208W and a TM3G208WG mutant, respectively. These mutants mimicked the T. fusca enzyme. In addition, T-298 was also changed to Phe-298 to obtain TM3T298F mutant, which mimicked the C. cellulolyticum enzyme.
The secondary structures of the three mutants did not show any gross differences compared to Cb1952TM3 as revealed by circular dichroism (CD) scans (Table 4), suggesting that the mutations did not result in gross changes in the secondary structural elements of the proteins compared to Cb1952TM3. Compared to parental protein (TM3), the specific activities of the three mutants on Avicel and filter paper were not different (Table 1). The mutations also did not aid us in modifying TM3 into a processive endoglucanase, as the ratio of soluble versus insoluble reducing ends remained unchanged (Table 3). The kcat values of the mutants with PASC as substrate increased by about 2-fold. However, the Km values also increased leading to catalytic efficiencies (kcat/Km) that were similar to that of Cb1952TM3 (Table 1).







TABLE 4







CD spectroscopy analysis of CbCelB/Man5ATM3 and its mutants















Unordered


Protein
α-helix (%)
β-sheet (%)
Turn (%)
(%)





TM3
35.0 ± 1.7
24.0 ± 1.0
16.0 ± 1.0
25.0 ± 1.0


TM3G208WG
36.0 ± 1.0
22.7 ± 2.0
16.3 ± 1.1
25.3 ± 0.6


TM3G208W
35.7 ± 1.5
23.3 ± 1.1
16.3 ± 1.1
25.3 ± 0.6


TM3T298F
32.7 ± 0.6
23.7 ± 0.6
17.3 ± 0.6
26.7 ± 0.6









Unexpectedly, the kcat values of TM3G208WG with locust bean gum and guar gum, as substrates, were increased 15- and 100-fold compared with the values determined for TM3 (Table 2). Moreover, the catalytic efficiencies of this mutant for locust bean gum and guar gum also increased by 34-fold and 10-fold, respectively, (Table 2). The site-directed mutagenesis of the TM3 truncated mutant also increased its kcat on konjac glucomannan by two-fold or higher (Table 2).
Binding of Cb1952 to Insoluble Cellulose Substrates
The Cb1952 wild-type, TM1, and TM5, which harbored all three CBMs (one CBM3c and two CBM3b) bound tightly to Avicel (FIG. 80A) and PASC (FIG. 80B). The truncated mutant TM2, which harbored the CBM3c and one CBM3b, also bound tightly to the two cellulosic substrates. The binding of TM3, which was composed of the GH9 module and the CBM3c, to the insoluble cellulose was weaker than those for wild-type, TM1, TM2, and TM5 (FIGS. 80A and 80B). Depletion binding isotherms were used to estimate the dissociation constant and maximal binding capacity of TM3 to Avicel as 0.52±0.20 M−1 and 423.9±50.7 nmol protein/g Avicel, respectively. The two components of TM3, i.e., the GH9 module and CBM3c, were observed to weakly bind to insoluble cellulose (FIGS. 80A and 80B). The binding of the CBM3c of CbCel9AMan5B (TM6) to insoluble cellulose was unexpected since this binding was not observed for other CBM3c characterized by this method (7, 10, 12, 16). Note, however, that the bindings were weak and thus preventing us from obtaining the binding constants of the GH9 and CBM3c modules for Avicel. The CBM3b (TM7) also bound to Avicel and PASC (FIGS. 80A and 80B), although in this case also the binding constants could not be determined.
Methods Used with Cb1952 Polypeptides
Methods used with the experiments above for Cb1952 polypeptides include the following:
Determination of Optimal pH and Temperature:
Two buffers were used for pH profiling of Cb1952: 50 mM sodium citrate, 150 mM NaCl (pH 4.0-pH 6.0) and 50 mM Na2HPO4—NaH2PO4, 150 mM NaCl (pH 6.5-pH 8.0). To measure the optimal pH of the enzymes on cellulose substrate, 0.5 μM Cb1952 wild-type or one of its truncation mutants was incubated with 2.5 mg/ml PASC in each buffer at a given pH at 75° C., and the activities in a 10 min assay were determined. The reducing sugars released were measured using the pHBAH assay. For determination of optimal temperature, 0.5 μM of each enzyme was incubated with 2.5 mg/ml PASC at pH 5.5 at different temperatures ranging from 40° C. to 95° C. with a 5° C. interval. The optimal pH and temperature for mannanase activity were determined as described above, except for the replacement of PASC with mannan as the substrate and change of the enzyme concentration to 12.5 nM.
Enzymatic Assays:
The specific activities of Cb1952 wild-type and its mutants on Avicel and filter paper were determined at 75° C. in the optimal buffer for the enzymes. The enzyme concentrations were 0.3 μM for each protein except for TM4 (5 μM). At different time intervals in a 90 min assay, samples were taken out and the products released determined as the amount of reducing ends present in the reaction mixture. The specific activities were determined in the region where the relation of reducing sugar versus time was linear.
The kinetics of Cb1952 wild-type and its mutants on PASC, locust bean gum, guar gum, and konjac glucomannan were determined in a 30 min assay. Different concentrations of the enzymes were incubated with a range of concentrations of substrates at 75° C. The velocities of release of reducing ends were determined and plotted against the concentrations of the substrates to estimate the kinetic parameters using the software GraphPad Prism 5.01 (GraphPad, San Diego, Calif.).
Time Course Hydrolysis of Phosphoric Acid Swollen Cellulose (PASC):
Two point five mg/ml PASC was incubated with 0.5 μM Cb1952 WT, TM1, and TM5 at 75° C. At different time intervals (0 min, 2 min, 10 min, 60 min, 4 h, and 24 h), samples were taken out and applied to HPAEC-PAD analysis as described earlier (29).
Analyses of Oligosaccharides Hydrolysis and Transglycosylation Activity:
Glucose, cello-oligosaccharides (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose), mannose, and manno-oligosaccharides (mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexaose), each at a final concentration of 1 mg/ml were incubated with 0.1 μM Cb1952 wild-type, Cb1952TM1, and Cb1952TM5 in a citrate buffer (10 mM sodium citrate, 150 mM NaCl, pH5.5) at 75° C. for 14 h. The total reaction volume was 40 μl. The reaction products were dried using a SpeedVac concentrator (Thermo Fisher Scientific, Pittsburgh, Pa.) and dissolved in 3.5 μl of H2O, and 1 μl of the products were analyzed by thin-layer chromatography (TLC) using a 250 μm thick Whatman silica gel 60A (Maidstone, England). The TLC method was the same as described in our earlier report (29).
Thermostability Assay:
The thermostability of Cb1952 and its truncation mutants harboring cellulase activity were determined by incubating the enzymes at 75° C., 80° C., and 85° C. (WT, TM1, TM2, and TM3) or at 45° C., 50° C., and 55° C. (TM4) on a Veriti 96-well thermal cycler (Applied Biosystems, Carlsbad, Calif.). At different time points, aliquots were taken from the reaction mixture and residual enzymatic activity was determined with PASC as the substrate.
Site-Directed Mutagenesis and Circular Dichroism:
For site-directed mutagenesis, the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) was used according to the manufacturer's instructions. One hundred nanograms of the plasmid encoding Cb1952TM3 were used as the template in the PCR amplification. The reaction mixture contained 100 ng of the mutagenic primer, 1 μl dNTP mix, 0.75 μl QuikSolution and 1 μl QuikChange Multi enzyme blend. The nucleotide sequences of the mutagenic primers used for mutagenesis are shown in Supplemental Table 1. The PCR amplification steps were carried out as follows: an initial denaturation at 95° C. for 1 min, followed by 30 cycles of 95° C. for 1 min, 55° C. for 1 min, and 65° C. for 15 min. The PCR product was digested with DpnI (New England Biolabs) at 37° C. for 4 hours to degrade the parental plasmid DNA. The product from the DpnI digestion was used in electrotransforming JM109 competent cells using a Gene Pulser Xcell electroporation system (BioRAD, Hercules, Calif.). The E. coli cells were spread on LB plates containing 100 μg/ml ampicillin and incubated at 37° C. overnight. Single colonies were inoculated in 7 ml LB medium supplemented with 100 μg/ml ampicillin and cultured for 10 h. The plasmids were extracted from the recombinant E. coli cells and the inserts were sequenced (W. M. Keck Center for Comparative and Functional Genomics, UIUC) to confirm the presence of the desired mutation. Circular dichroism scans of mutated proteins were carried out as described in our previous report (37).
Measurement of Reducing Sugar in the Soluble and Insoluble Fraction of Hydrolyzed Filter Paper:
The reducing sugars in the soluble and insoluble fractions of filter paper hydrolysis products were determined as described by Irwin et al. (17). The Cb1952 wild-type and its mutants (0.5 μM each except TM4, which was 10 μM) were incubated with five plates of Whatman No. 1 filter paper (0.6 cm in diameter) in a citrate buffer (pH5.5) at 75° C. (for TM4, the temperature was 45° C., since this construct has lower thermostability) in 200 μl. The mixtures were shaken end-over-end for 16 h. The reaction products were centrifuged, and the supernatants (soluble fractions) were analyzed for the amounts of reducing ends. For reducing sugar determination in the insoluble fraction, the filter papers were initially washed four times each with 1 ml of the citrate buffer. Two hundred microliters of the citrate buffer was then added to the insoluble fraction (precipitated filter paper) followed by assaying for reducing ends through the pHBAH method.
Binding of Cb1952 Wild-Type and its Truncated Mutants to Cellulose:
For qualitative measurements of the capacity of the individual polypeptides to bind to cellulose, thirty micrograms of Cb1952 wild-type and its mutants were incubated with 40 mg/ml Avicel cellulose or 2.5 mg/ml PASC in 50 mM Tris-HCl, 150 mM NaCl (pH 7.5). The mixture was shaken end-over-end at 4° C. for 1 h. Then the bound and unbound proteins were separated by centrifugation of the mixture at 16,400 rpm for 3 min. The cellulose pellet was washed four times with 1 ml buffer (50 mM Tris buffer, 150 mM NaCl, pH 7.5). Seventy microliters of 1×SDS-PAGE loading buffer was added to the pellet and boiled for 5 min to release bound proteins. The protein present in one tenth of the volume of the supernatant (unbound protein) and the cellulose pellet (bound protein) was examined by a 12% SDS-PAGE.
For quantitative binding assay, different concentrations of proteins were mixed with 2 mg/ml Avicel in 50 mM Tris-HCl, 150 mM NaCl, pH7.5 buffer in a 2-ml tube. As a control, proteins with the same concentrations were incubated without Avicel in the tube. After 1.5 h end-over-end incubation at 4° C., the mixtures were centrifuged at 16,400 rpm for 3 min. The protein concentrations in the supernatant were determined using a bicinchoninic acid (BCA) Protein Assay Reagent Kit (Thermo Scientific, Rockford, Ill.). Taking the protein concentration from the tube without cellulose as the total protein, the concentrations of bound protein were obtained by subtracting the protein concentration of the sample with cellulose from the total protein concentration. For determination of the binding parameters, the Michaelis/Langmuir equation (qad/q=Kp×qmax/(1+Kp×q)) as described in our previous report (46) was used. The qad in the equation represents the amount of bound protein (nmol of protein per gram of Avicel), q is the free protein (μM), and qmax is the maximal amount of bound protein to Avicel. The calculation of the binding parameters was carried out with GraphPad Prism 5.01.
Amino Acid Sequence Alignment:
The amino acid sequences of the family 9 glycoside hydrolase catalytic module of the Clostridium cellulolyticum Cel9G (GenBank accession number AAA73868)(26) and that of the Thermobifida fusca Cel9A (GenBank accession number: AAB42155)(34) were retrieved from Carbohydrate Active enZYme database and the Genbank database and aligned with the GH9 sequence of Cb1952 by using ClustalX. Similarly, the amino acid sequences of the CBM3c modules from the characterized cellulases of different bacterial sources in the published literatures were aligned. These include: ADQ45731: putative cellulase of Caldicellulosiruptor kronotskyensis; ABP66693: putative cellulase of Caldicellulosiruptor saccharolyticus; ADL42950: putative Caldicellulosiruptor obsidiansis cellulase/mannan endo-1,4-beta-mannosidase; AAK06394: CelE of Caldicellulosiruptor sp. Tok7B.1 (11); AAA73868: Cel9G of Clostridium cellulolyticum (26); AAC38572: EngH of Clostridium cellulovorans (38); CAA39010: Cel9Z of Clostridium stercorarium (18); ABX43720: Cel9 of Clostridium phytofermentans (39, 48); ABN51860: Cel9I of Clostridium thermocellum DSM 1313 (50); CAB38941: Cel9B of Paenibacillus barcinonensis (32); BAB33148: CelQ of Clostridium thermocellum F1 (2); AAA23086: CenB of Cellulomonas fimi (27); AAW62376: CBP105 of Cellulomonas flavigena (28); AAB42155: Cel9A of Thermobifida fusca (16, 34). The aligned sequences were analyzed using the BOXSHADE 3.21 with a default setting of the fraction of sequences parameter as 0.5.
Additional Assays
FIG. 20 shows the enzymatic activity of Cb1952 wild-type on natural substrates from a reducing sugar assay. Twelve different substrates were tested: Avicel, phosphoric acid swollen cellulose (PASC), sodium carboxymethyl cellulose (CMC-Na), lichenin, mannan, locust bean gum (LBG), guar gum, konjac glucomannan (KGM), wheat arabinoxylan (WAX), birchwood xylan (BWX), oat-spelt xylan (OSX) and xyloglucan were used. Incubation of enzymes with Avicel, PASC, CMC-Na, lichenin, mannan, LBG, guar gum, KGM, WAX and OSX substrates led to release of products that were quantified as a concentration of glucose equivalents. The Cb1952 wild-type mainly hydrolyzes glucose- and mannose-configured substrates, but not xylose-configured substrates.
FIG. 21 shows the enzymatic activity of Cb1952TM1 on natural substrates from a reducing sugar assay. Twelve different substrates were tested: Avicel, phosphoric acid swollen cellulose (PASC), sodium carboxymethyl cellulose (CMC-Na), lichenin, mannan, locust bean gum (LBG), guar gum, konjac glucomannan (KGM), wheat arabinoxylan (WAX), birchwood xylan (BWX), oat-spelt xylan (OSX) and xyloglucan were used. Incubation of enzymes with Avicel, PASC, CMC-Na, lichenin, mannan, LBG, guar gum, KGM, WAX, BWX, OSX and xyloglucan substrates led to release of products that were quantified as a concentration of glucose equivalents. The results show that Cb1952TM1 mainly hydrolyzes glucose-configured substrates. It also has some activities on mannose-configured substrates. Its activities on xylose-configured substrates are low.
FIG. 22 shows the enzymatic activity of Cb1952TM5 on natural substrates from a reducing sugar assay. Twelve different substrates were tested: Avicel, phosphoric acid swollen cellulose (PASC), sodium carboxymethyl cellulose (CMC-Na), lichenin, mannan, locust bean gum (LBG), guar gum, konjac glucomannan (KGM), wheat arabinoxylan (WAX), birchwood xylan (BWX), oat-spelt xylan (OSX) and xyloglucan were used. Incubation of enzymes with CMC-Na, lichenin, mannan, LBG, guar gum and KGM substrates led to release of products that were quantified as a concentration of mannose equivalents. The Cb1952TM5 mainly hydrolyzes mannose-configured substrates, but does not have obvious activity on glucose- or xylose-configured substrates.
FIG. 23 shows the enzymatic activity of Cb1952 wild-type, Cb1952TM1 and Cb1952TM5 on glucose and cellooligosaccharides from a thin-layer chromatography (TLC) assay. Glucose and five different cellooligosaccharides were used: cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose. Cb1952 wild-type and Cb1952TM1 hydrolyze cellotriose, cellotetraose, cellopentaose and cellohexaose into glucose and cellobiose, but have no activity on cellobiose. Cb1952TM5 has no activity on glucose and any of the cellooligosaccharides tested. None of the enzyme has transglycosylation activity on glucose and cellooligosaccharides.
FIG. 24 shows the enzymatic activity of Cb1952 wild-type, Cb1952TM1 and Cb1952TM5 on mannose and mannooligosaccharides from a thin-layer chromatography (TLC) assay. Mannose and five different mannooligosaccharides were used: mannobiose, mannotriose, mannotetraose, mannopentaose and mannohexaose. Cb1952 wild-type and Cb1952TM5 hydrolyze mannotriose, mannootetraose, mannopentaose and mannohexaose into mannose and smaller mannooligosaccharides, but have no hydrolyzing activity on mannobiose. Cb1952TM1 hydrolyzes mannopentaose and mannohexaose into smaller oligosaccharides but has no hydrolyzing activity on mannobiose, mannotriose, mannotriose and mannotetraose. None of the enzyme has transglycosylation activity on mannose and mannooligosaccharides.
The concentration of glucose or mannose equivalents was determined following enzymatic hydrolysis of the natural polysaccharides, according to the methods of Lever, M. (A new reaction for colorimetric determination carbohydrates. Anal. Biochem. 1972: 47; 273-279). 1.5 mL microcentrifuge tubes were “zeroed” in an analytical balance. Next, 2±0.1 mg Avicel or mannan were added to each tube, and the mass measured and recorded. The volumes needed to be added to each tube were calculated based on the mass. For CMC-Na and PASC, a stock substrate solution of CMC-Na (2%) and PASC (6.11 mg/ml) were used. For lichenin, KGM, WAX, BWX, OSX and xyloglucan, 2% stock solution was used. For LBG and guar gum, 0.5% stock solution was used. Sodium citrate reaction buffer and enzymes were added to each tube beginning with the reaction buffer. The tubes were incubated with constant mixing in a Thermomixer R (Eppendorf) at 75° C. for 16 h. The tubes were centrifuged at 10,000 rpm for 5 min at 4° C. 50 μL of sample supernatant was transferred to a clean 1.5 mL centrifuge tube for the pHBAH assay. 1 mL of a stock solution of glucose was made at a concentration of 100 mM in sodium citrate buffer, and then serial dilutions were made in sodium citrate buffer to the following concentrations (20 mM, 10 mM and 5 mM). 50 mg of pHBAH was dissolved in 50 mL of ice-cold citrate/NaOH solution for a final concentration of 0.1% (w/v), and the solution was kept on ice. 150 μL of pHBAH solution was added to 50 μL of the sample and glucose standard solutions, and the tubes were incubated at 100° C. for 10 min. The tubes were incubated at room temperature for 5 min. The wavelength at 410 nm was measured for the standards and samples. The A410nm and glucose concentrations were plotted against each other, and linear regression was used to fit a line to the data. The correlation coefficient (R2) value was between 0.98 and 1.0. The equation from the standard curve was used to calculate the concentrations of reducing ends in the samples based upon their absorbances.
FIG. 25 shows the enzymatic activity of Cb1952TM1 on cellulose substrates using HPLC analysis. Three different cellulosic substrates were tested: Avicel, CMC-Na and PASC. In each case, in the presence of Cb1952TM1, glucose and cellobiose were released. In the absence of Cb1952TM1, neither glucose nor cellobiose was observed for all the substrates. The results showed that this part of the enzyme or polypeptide (Cb1952) cleaves glucose and cellobiose as end products from cellulosic substrates (Avicel, CMC-Na and PASC).
FIG. 26 shows a time-course hydrolysis of PASC by Cb1952TM1. 100 nanomolar of Cb1952TM1 was incubated with 2.5 mg/ml PASC at 75° C. At different time interval (0, 0.5 min, 2 min, 10 min, 1 h, 4 h and 24 h), samples were taken out and immediately boiled for 10 min to inactivate the enzyme. After centrifugation, the supernatants of the samples were appropriately diluted with water and applied to HPLC analysis. The results show that Cb1952TM1 initially releases glucose, cellobiose, cellotriose and cellotetraose. With increasing time, only glucose and cellobiose were left in the reaction mixture.
FIG. 27 shows the thermostability of Cb1952 wild-type using PASC as substrate for activity measurement. Cb1952 wild-type has 75%, 43%, 17% and 12% activity after incubation at 70° C., 75° C., 80° C. and 85° C. for 24 h, respectively. 500 nM Cb1952 wild-type was kept at different temperatures (70° C., 75° C., 80° C. and 85° C.). The samples were taken out at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately used for enzyme activity measurement. The enzyme activity was measured at pH 5.5 and at 85° C. on a thermomixer. 2.5 mg/ml final concentration of PASC was used for measurement, and 8.31 μl of the protein sample was added to the substrate and mixed by pipetting up and down for several times. The total volume was 100 μl. The reducing ends corresponding to glucose equivalents were measured according to the methods of Lever, M. (supra). The velocity of reaction in 10 minutes was calculated. The velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the velocity of reaction at time 0, then multiplied by 100, respectively.
FIG. 28 shows the thermostability of Cb1952TM1 using PASC as substrate for activity measurement. Cb1952TM1 has 94%, 76%, 18% and 13% activity after incubation at 70° C., 75° C., 80° C. and 85° C. for 24 h, respectively. 500 nM Cb1952TM1 was kept at different temperatures (70° C., 75° C., 80° C. and 85° C.). The samples were taken out at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately used for enzyme activity measurement. The enzyme activity was measured at pH 5.5 and at 85° C. on a thermomixer. 2.5 mg/ml final concentration of PASC was used for measurement, and 8.31 μl of the protein sample was added to the substrate and mixed by pipetting up and down for several times. The total volume was 100 μl. The reducing ends corresponding to glucose equivalents were measured according to the methods of Lever, M. (supra). The velocity of reaction in 10 minutes was calculated. The velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the velocity of reaction at time 0, then multiplied by 100, respectively.
Discussion of Results with Cb1952 Polypeptides
Cb1952 is the first GH9 cellulase characterized with two tandem CBM3b modules linked to the GH9-CBM3c domains. The CBM3b module (TM7) binds to insoluble cellulose (FIGS. 80A and 80B). Deletion of one CBM3b (TM2) from TM1 did not significantly affect the binding to these substrates (FIGS. 80A and 80B). Correspondingly, the specific activities and kinetic parameters of TM1 and TM2 are similar for cellulose substrates (Table 1). Further deletion of another CBM3b (TM3) reduced both the binding to the insoluble substrates and the specific activities for Avicel and filter paper (Table 1). Therefore, the CBM3b modules facilitate the deconstruction of crystalline cellulose by Cb1952.
Cb1952 and its truncation mutants, especially TM3, retained considerable activities after incubation at 75° C. for 24 h. For other hyperthermophilic endoglucanases, Cel5A of Thermoanaerobacter tengcongensis has above 80% residual activity after incubation at 60° C. for 24 h (24), the Avicelase I of Clostridium stercorarium has above 60% residual activity after incubation at 80° C. for 12 h (6), and the CelB of Caldicellulosiruptor saccharolyticus has a half-life of 29 h at 70° C. (35). The thermostability property of the multi-functional enzyme can facilitate recycling during its use in releasing fermentable sugars from cellulosic substrates. Introduction of an enzyme recycling step in cellulosic ethanol production can significantly reduce the cost of production of the value added product.
The C. bescii Cb1954 (CelA) (ORF1954, GenBank accession number ACM60955) is the first cellulase characterized from this bacterium (49). It is the most highly secreted cellulase when C. bescii is grown on Avicel medium (25). Similar but not identical to Cb1952, Cb1954 is composed of an N-terminally located GH9 module, a C-terminally located GH48 module, and three CBM3 modules between the two catalytic domains. The specific activity of Cb1952 on Avicel (10.15 μmol sugar/min/μmol enzyme) was much lower than that of the full-length Cb1954/CelA (55.0 μmol sugar/min/μmol enzyme), but only slightly lower than that of its truncation mutant CelA′ containing the GH9 catalytic module and CBMs (18.0 μmol sugar/min/μmol enzyme) (49). In a comparison with other hyperthermostable endoglucanases, this specific activity of Cb1952 was lower than those of Cel5A of Thermoanaerobacter tengcongensis (60.0 μmol sugar/min/μmol enzyme) (24) and Avicelase I of Clostridium stercorarium (30.2 μmol sugar/min/μmol enzyme) (6), comparable to that of EGPh of Pyrococcus horikoshii (12.7 μmol sugar/min/μmol enzyme) (1), but much higher than that of the C. saccharolyticus CelB (0.4 μmol sugar/min/μmol enzyme) (41). The specific activity of Cb1952 on filter paper was comparable to those of CelB of Thermotoga neapolitana (20.8 μmol sugar/min/μmol enzyme) (5), Cel5A of Thermoanaerobacter tengcongensis (18.5 μmol sugar/min/μmol enzyme) (24), and EglA of Pyrococcus furious (18.7 μmol sugar/min/μmol enzyme) (3), but much higher than those of CelA of Thermotoga neapolitana (3.2 μmol sugar/min/μmol enzyme) (5) and CelB of C. saccharolyticus (1.8 μmol sugar/min/μmol enzyme) (41). Note that the assay conditions (reaction temperature, buffer, reaction period, method for measuring reducing sugar, and lab equipment) for these specific activities may vary among the enzymes described above. Nevertheless, Cb1952 is an effective enzyme for releasing fermentable sugars from cellulosic substrates at high temperatures.
Interestingly, seven out of the nine genes in the gene cluster in which the Cb1952 encoding gene is located also contain CBM3b modules identical to or highly similar to (identity >98%) the CBM3b of Cb1952. Six of the polypeptides in the gene cluster have either two or three tandem CBM3b repeats. It is reasonable to postulate that these CBM3b modules aid in plant cell wall hydrolysis.
The mannanase activity of Cb1952 was mainly located in the GH5 module; however, limited mannanase activity was also observed with the construct containing the GH9 domain as the catalytic module. In most cases, family 9 glycoside hydrolases are described as endoglucanase (10, 16), cellobiohydrolase (36), 1,4-β-D-glucan glucohydrolase (33), β-glucosidase (30), and exo-β-glucosaminidase (14). The Bacillus licheniformis Cel9 is the only member of this family reported to have mannanase activity (42); however, its kinetic data and hydrolysis pattern on mannose-configured substrates are unknown. The TM1 mutant of Cb1952 showed different hydrolysis patterns compared with the TM5 mutant, in that the GH9 needed a minimal chain length of five and released mannobiose as the shortest end-product, while the GH5 needed a minimal chain length of three and released mannose as the shortest end-product. The ability of the GH9 module of Cb1952 to hydrolyze mannose-configured substrates suggests that the catalytic module can both accommodate the equatorial C-2 hydroxyl of glucose and also tolerate the axial C-2 hydroxyl of mannose.
The absence of a tryptophan for −3 subsite hydrophobic interaction was proposed to destabilize the non-productive binding which might impair the processivity of a GH9 cellulase (31). The mutations of G208 and T298 into aromatic residues (TM3G208WG, TM3G208W, and TM3T298F), however, did not change the processivity of TM3 as reflected by the unaltered ratios of soluble versus insoluble reducing ends. The specific activities of the mutants on crystalline cellulose (Avicel and filter paper) were also comparable to that of the parental TM3. However, for non-crystalline PASC, all turnover numbers of the mutants were increased by roughly 2 folds while the catalytic efficiencies remained unchanged. The different structures of crystalline and non-crystalline cellulose might affect the performance of these enzymes. One of the mutants, TM3G208WG, increased its substrate specificity for locust bean gum by 35 folds (TM3: [kcat/Km]LBG/[kcat/Km]PASC=7 0.4×10−3, TM3G208WG: [kcat/Km]LBG/[kcat/Km]PASC=0.26), suggesting that residues for subsite −3 interaction might be involved in substrate selection.
CBM3c has been proposed to bind loosely to the cellulose ligand and feed a cellulose chain into the GH9 catalytic module (34). However, no biochemical data was provided for this binding. In the co-crystal structure of family 9 cellulase in complex with cello-oligosaccharides, the binding of the cello-oligosaccharide to CBM3c has not been observed so far (26, 34). Our results suggest that a CBM3c can indeed bind to insoluble cellulose although the binding appeared weak. A sequence alignment of Cb1952 with its homologs revealed that considerable differences exist in the amino acid residues proposed to interact with the ligand (19, 22, 23) between Cb1952 CBM3c with its homologues. The conserved Q553, R557, E559, and R563 residues in ThefuCel9A proposed to interact with the ligand are correspondingly replaced by E545, K549, Q561, and K565, respectively, in the CBM3c of Cb1952 (FIG. 82). This observation may be akin to the fine-tuning demonstrated in a Caldanaerobius polysaccharolyticus family 16 CBM by mutating one polar residue (Q121 to E121) involved in hydrogen bonding with the ligand (37). The E545, K549, Q561, and K565 residues can also be found in four CBM3 modules from the related organisms C. kronotskyensis, C. saccharolyticus, C. obsidiansis, and Caldicellulosiruptor sp. Tok7B.1 (FIG. 82). A three-dimensional structure of a CBM3c in complex with a ligand is still lacking, which hinders accurate designation of residues important for ligand binding. Due to the diversity of CBM3c modules (19), one may postulate that other variants of CBM3c might exist which could hold a cello-oligosaccharide tightly enough to capture this complex in a crystal.
Example 10: Endo-Glucanase/Mannanase Cb1953
A putative endoglucanase, Cb1953WT, was identified in Caldicellulosiruptor bescii. The enzyme is the gene product of cb1953, where Cb stands for Caldicellulosiruptor bescii. The endoglucanase cleaves mostly cellobiose from cellulose. The Cb1953WT protein is 1391 amino acids long and has a molecular weight of 153.6 kDa (His-tag+Cb1953 protein). The Cb1953WT has two Glycoside Hydrolase (GH) family 5 catalytic domain and 3 carbohydrate binding proteins (FIG. 29). Two truncated mutants were made, as shown in FIG. 29, to determine the activity in each GH5 module. For the truncated mutants, Cb1953TM1 (961 amino acids, 103.9 kDa) has N-terminal GH 5 catalytic domains with 3 carbohydrate binding modules, whereas Cb1953TM2 (1108 amino acids, 121.7 kDa) has C-terminal GH5 catalytic domains with 3 carbohydrate binding modules as like shown in FIG. 29.
PCR Amplification of Cb1953WT
The genes were amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using PrimeSTAR DNA Polymerase (TAKARA). The Cb1953WT, Cb1953TM1, and Cb1953TM2 coding sequences were amplified using the following respective primer set:









Cb1953WTForward:



(SEQ ID NO: 56)



5′-GAC GAC GAC AAG ATG GCT ACA TCT AAT



GATGGA GTA GTG AAG -3′


 



Cb1953WTReverse:



(SEQ ID NO: 57)



5′-GAG GAG AAG CCC GGT TAA TTT TGC GGC TGG



AAC TGG CGC TGG TTC -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
5
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










Cloning of Cb1953TM1










Cb1953TM1Forward:



(SEQ ID NO: 56)



5′-GAC GAC GAC AAG ATG GCT ACA TCT AAT



GATGGA GTA GTG AAG -3′


 



Cb1953TM1Reverse:



(SEQ ID NO: 58)



5′-GAG GAG AAG CCC GGT TAT GGC ATT GGT ATT



ACT GTC TGC ACC GG -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
4
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










Cloning of Cb1953TM2










Cb1953TM2Forward:



(SEQ ID NO: 59)



5′-GAC GAC GAC AAG ATG



GGTGCCTCTTCAGTACCTACTTCAACACC -3′


 



Cb1953TM2Reverse:



(SEQ ID NO: 57)



5′- GAG GAG AAG CCC GGT TAA TTT TGC GGC TGG



AAC TGG CGC TGG TTC -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
4
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










After the PCR amplification described above, the products of Cb1953WT, Cb1953TM1, and Cb1953TM2 were confirmed by 1% agarose gel electrophoresis. The DNA corresponding to the expected band on the gel was cut out and applied to a Qiagen Gel Extraction kit to extract the DNA out of the gel.
The Novagen pET-46 Ek/LIC kit was used to treat each purified DNA and ligate it into the pET-46 Ek/LIC vector. The treatment of the purified DNA was as follows:














Unit




Reaction
(μl)
Incubation







0.1 pmol purified PCR product
X




10X T4 DNA Polymerase buffer
1




25 mM dATP
1




100 mM DTT
  0.5




Nuclease-free water
7.3-X




2.5 U/μl T4 DNA Polymerase
  0.2




Total
10 
22° C.
30 min









After the reaction, the enzyme was inactivated by incubation at 75° C. for 20 min.

The following protocol was used to anneal the insert into the pET-46 Ek/LIC vector:














Unit




Reaction
(μl)
Incubation


















pET-46 Ek/LIC vector
0.5




T4 DNA Polymerase treated EK/LIC insert
1




Total
1.5
22° C.
5 min









Then add 0.5 μl 25 mM EDTA. Mix by stirring with pipet tip. Incubate at 22° C. for 5 min.

The ligation mixture for Cb1953-pET-46 Ek/LIC was introduced into E. coli JM109 by electroporation, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., four colonies were selected and each was used to inoculate 6 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours, and minipreps (QIAGEN) were made from the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to check the size of the plasmid DNA. After confirmation of insertion of the gene into the plasmid, the inserts were sequenced to confirm the integrity of their sequences.
For gene expression, one of the plasmids was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol (100 μg/ml) and ampicillin (50 μg/ml) and incubated at 37° C. overnight. Five to six colonies were inoculated into 3 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 4 hours. One mL of the culture was added to 500 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.25. The inducer, IPTG, was then added at 0.1 mM final concentration, and the culturing continued at 16° C. overnight.
Gene and Protein Sequences of Cb1953WT, Cb1953TM1, and Cb1953TM2
Wild Type Cb1953 Amino Acid Sequence
The wild-type Cb1953 amino acid sequence is disclosed in SEQ ID NO: 60. The signal peptide of Cb1953, corresponding to amino acid numbers 1-38 of SEQ ID NO: 60 was removed during all PCR amplifications. Thus, the expressed wild-type Cb1953 protein did not contain amino acid numbers 1-38 of SEQ ID NO: 60. The amino acid sequence of the wild-type Cb1953 protein without the signal peptide is disclosed in SEQ ID NO: 61.
The procedure of cloning the gene for wild-type Cb1953 (without the signal peptide) into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The wild-type Cb1953 amino acid sequence (without the signal peptide) with the short peptide is disclosed in SEQ ID NO: 65. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 65.
Wild Type Cb1953 Nucleotide Sequence
The wild-type Cb1953 nucleotide sequence is disclosed in SEQ ID NO: 62. The signal peptide of Cb1953, corresponding to nucleotide numbers 1-114 of SEQ ID NO: 62 was removed during all PCR amplifications. Thus, the nucleotide sequence used to express wild-type Cb1953 protein did not contain nucleotide numbers 1-114 of SEQ ID NO: 62. The nucleotide sequence encoding the wild-type Cb1953 protein without the signal peptide is disclosed in SEQ ID NO: 63.
The wild-type Cb1953 nucleotide sequence (without the signal peptide) with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 64. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 64.
Cb1953TM1 Amino Acid Sequence
The Cb1953TM1 amino acid sequence is disclosed in SEQ ID NO: 122. The procedure of cloning the gene for Cb1953TM1 into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The Cb1953TM1 amino acid sequence with the short peptide from pET-46 Ek/LIC is disclosed in SEQ ID NO: 67. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 67.
Cb1953TM1 Nucleotide Sequence
The Cb1953TM1 nucleotide sequence is disclosed in SEQ ID NO: 123. The Cb1953TM1 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 66. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 66.
Cb1953TM2 Amino Acid Sequence
The Cb1953TM2 amino acid sequence is disclosed in SEQ ID NO: 111. The procedure of cloning the gene for Cb1953TM2 into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The Cb1953TM2 amino acid sequence with the short peptide is disclosed in SEQ ID NO: 69. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 69.
Cb1953TM2 Nucleotide Sequence
The Cb1953TM2 nucleotide sequence is disclosed in SEQ ID NO: 110. The Cb1953TM2 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 68. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 68.
Purification of Cb1953WT, Cb1953TM1, and Cb1953TM2 Proteins
The Cb1953WT, Cb1953TM1, Cb1953TM2 were expressed in E. coli BL-21 CodonPlus (DE3) RIL competent cells by heat shock. The recombinant cells were then grown overnight in LB agar plates supplemented with ampicillin (100 μg/mL) and chloramphenicol (50 μg/ml) at 37° C. After 8 h, the starter cultures were diluted into fresh LB supplemented with ampicillin (100 μg/mL) and chloramphenicol (50 μg/ml) at 37° C. with aeration until the absorbance at 600 nm reached 0.5. Gene expression was then induced by addition of IPTG at a final concentration of 0.1 mM and the temperature for culturing was lowered to 16° C. After 16 hours, the cells were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (25 mM Tris-HCL pH 7.8, 750 mM of NaCl, 5% glycerol, 20 mM imidazole, 1.25% Tween-20). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed three times to remove the unbound proteins. The bound protein (6-Histidine-tagged target proteins) was then eluted from the resin with an elution buffer (50 mM Tris-HCL, pH7.5, 250 mM imidazole). The eluted fractions were then heat-treated at 65° C. for 30 minutes and then centrifuged to remove the precipitated proteins. The proteins were then purified by gel filtration chromatography (HiLoad 16/20 Superdex 200, GE Healthcare) with a Tris-HCl elution buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). Aliquots of eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins bands were visualized by staining with Coomassie brilliant blue G-250 (FIG. 30).
Enzyme Activity
FIG. 31 shows the zymogram of Cb1953WT, Cb1953TM1, Cb1953TM2 on carboxylmethyl cellulose (CMC). The gel was prepared as in standard dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with CMC substrate (final 0.1%, w/v). After electrophoretic fractionation of the proteins, gels were washed twice in distilled water and incubated in 30 mL of refolding buffer (20 mM citrate buffer, pH 6.0, 2.5% Triton X-100, 2 mM dithiothreitol, 2.5 mM CaCl2) for 1 hour at 25° C. and then held overnight in fresh buffer at 37° C. The gel was washed twice in 50 mM Citrate buffer (pH 6.0) and then the results were visualized by staining with 0.1% Congo red and destaining with 1M NaCl. As shown in FIG. 31, Cb1953WT and Cb1953TM2 showed significant white bands at the positions of their expected sizes indicating cellulase activity, but not Cb1953TM1 protein.
FIGS. 32 and 33 show the enzymatic activity of Cb1953WT, Cb1953TM1, and Cb1953TM2 on natural substrates from a reducing sugar assay. Seven different substrates were tested: Avicel, Phosphoric acid swollen cellulose (PASC), carboxylmethyl cellulose (CMC), wheat arabinoxylan (WAX), lichenin, konjac glucomannan, and mannan. Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents. The tubes were incubated with constant mixing in a Thermomixer R (Eppendorf) at 75° C. for 16 h. The tubes were centrifuged at 10,000 rpm for 5 min at 4° C. 50 μL of sample supernatant was transferred to a clean 1.5 mL centrifuge tube for the pHBAH assay. The wavelength at 410 nm was measured for the standards and samples. The A410nm and glucose concentrations were plotted against each other, and linear regression was used to fit a line to the data. The reactions were resolved by thin layer chromatography (TLC), The mobile phase consisted of n-butanol:acetic acid:H2O, 10:5:1 (vol/vol/vol) and 10 cm×20 cm plates were used. The reducing sugar assay (FIG. 32) and TLC (FIG. 33) results show that Cb1953WT and Cb1953TM2 have cellulase activity whereas Cb1953TM1 has mannanase activity. Through the zymogram, reducing assay, and TLC analysis on various substrates, we concluded that the C-terminal GH5 in Cb1953WT functions as a cellulase whereas the N-terminal GH5 functions as mannase.
FIG. 34 shows the time course of enzymatic activity of Cb1953TM2 on PASC using HPLC analysis. For analysis of the products of hydrolysis, the samples were analyzed by high performance anion-exchange chromatography (HPAEC). For HPAEC analyses, 100 μL of each diluted sample was injected onto a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.). For the TLC and HPLC analysis, glucose and five different cellooligosaccharides (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose) were used as standards. In the reaction, Cb1953 started to release cellooligosaccharides (C2-C4) and then glucose was released later. The results showed that this enzyme releases mainly cellobiose from PASC.
FIGS. 35 and 36 show the thermostability of Cb1953WT and Cb1953TM2 on PASC. 50 nM Cb1953WT and Cb1953TM2 were kept at different temperatures (70° C., 75° C., 80° C., 85° C. and 90° C.). The samples were taken out at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately used in enzyme activity measurement. The enzyme activity was measured at 85° C. using Cary 300 UV-Vis spectrophotometer (Varian). The initial velocity of reaction in the first minute was calculated. The initial velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the initial velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the initial velocity of reaction at time 0, then multiplied by 100. From the results, Cb1953WT (FIG. 35) and Cb1953TM2 (FIG. 36) were quite stable at 70° C. and 75° C., maintaining activity of 75 ̃90% of heat non-treated proteins.
FIG. 37 shows the kinetic studies of Cb1953WT and Cb1953TM2 on PASC. 0.05 μM of purified Cb1953WT or Cb1953TM2 in 50 mM Na2HPO4—HCl, pH 6.0, and 150 mM NaCl was incubated with various concentrations of phosphoric acid swollen cellulose (PASC), and the initial rate of hydrolysis was plotted against substrate concentration. The kinetic parameters (Km: 7.603 mg/mL, kcat: 7.513 s−1 and kcat/Km: 0.988 s−1 mL/mg for Cb1953WT and Km: 3.032 mg/mL, kcat: 5.411 s−1 and kcat/Km: 1.785 s−1 mL/mg for Cb1953TM2) were determined by fitting the data to the Michaelis-Menten equation (Graph Pad Prism v5.01).
Example 11: Endocellulase Cb1954
A putative endoglucanase, Cb1954, was identified in Caldicellulosiruptor bescii, where Cb stands for Caldicellulosiruptor bescii. The protein has a Glycoside Hydrolase (GH) family 9 catalytic domain (putative cellulase domain), three family 3 carbohydrate binding modules (CBMs) and one GH48 catalytic domain (FIG. 38). The Cb1954 wild-type is 1746 amino acids long and has a predicted molecular mass of 193.6 kDa (His-tag+Cb1954 wild-type protein). The enzyme Cb1954TM3 and Cb1954TM5 are the truncational mutants of the gene product of Cb1954, where Cb stands for Caldicellulosiruptor bescii. The endocellulase cleaves glucose and cellobiose from cellulose as end products. The Cb1954TM3 protein is 709 amino acids long and has a molecular weight of 78.57 kDa (His-tag+Cb1954TM3 protein). The protein has a Glycoside Hydrolase (GH) family 9 catalytic domain and one family 3 carbohydrate binding module (CBM3). The Cb1954TM5 protein is 1294 amino acids long and has a molecular weight of 142.82 kDa (His-tag+Cb1954TM5 protein). The protein has a Glycoside Hydrolase (GH) family 48 catalytic domain and three family 3 Carbohydrate binding modules (CBM3).
Cloning of Cb1954 Wild-Type
The gene for Cb1954 wild-type was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using PrimeSTAR DNA Polymerase (TAKARA). The Cb1954 wild-type gene was amplified using the following primer set:
The Cb1954 wild-type gene was amplified using the following primer set:









Cb1954 wild-type Forward:



(SEQ ID NO: 70)



5′- GAC GAC GAC AAG ATG CAA GAG GTT AGG



GCT GGT TCG TTT AAC -3′


 



Cb1954 wild-type Reverse:



(SEQ ID NO: 71)



5′- GA GGA GAA GCC CGG TTA TTG ATT GCC



AAA CAG TAT TTC ATA TG -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
6
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










Cloning of Cb1954TM3

The gene for Cb1954 was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using Pfu Turbo® DNA Polymerase. The Cb1954TM3 gene was amplified using the following primer set:









Cb1954TM3Forward:



(SEQ ID NO: 70)



5′- GAC GAC GAC AAG ATG CAA GAG GTT AGG



GCTGGT TCG TTT AAC -3′


 



Cb1954TM3Reverse:



(SEQ ID NO: 72)



5′- GA GGA GAA GCC CGG TTA TAC CTT TAT CTG



TCC ACC TGC TAC-3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL Pfu Turbo ® DNA Polymerase
0.5



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



20 μM Fw Primer
1



20 μM Rv Primer
1



10 mM dNTP Mixture
1



10 × Cloned Pfu Turbo DNA Polymerase Buffer
5



dH2O
40.5



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
2.5
min




Elongation
72° C.
10
min
1 cycle












Last
 4° C.
∞










Cloning of Cb1954TM5

The gene for Cb1954 was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using Pfu Turbo® DNA Polymerase. The Cb1954TM3 gene was amplified using the following primer set:









Cb1954TM5Forward: 5′-



(SEQ ID NO: 73)



GAC GAC GAC AAG ATG TTC AAA GCT ATT GAA



ACT CCA ACA AAC -3′


 



Cb1954TM5Reverse:



(SEQ ID NO: 71)



5′- GA GGA GAA GCC CGG TTA TTG ATT GCC AAA



CAG TAT TTC ATA TG -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL Pfu Turbo ® DNA Polymerase
0.5



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



20 μM Fw Primer
1



20 μM Rv Primer
1



10 mM dNTP Mixture
1



10 × Cloned Pfu Turbo DNA Polymerase Buffer
5



dH2O
40.5



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
4
min




Elongation
72° C.
10
min
1 cycle












Last
 4° C.
∞










After the PCR described above, the amplification of Cb1954 wild-type, Cb1954TM3 and Cb1954TM5 gene was confirmed by 1% agarose gel electrophoresis. The DNA corresponding to the expected band on the gel was cut out and the amplified fragment was extracted using the Qiagen Gel Extraction kit.
A Novagen pET-46 Ek/LIC kit was used to treat the purified DNA and ligate it into the pET-46 Ek/LIC vector. The treatment of the purified DNA was as follows:














Unit




Reaction
(μl)
Incubation







0.1 pmol purified PCR product
X




10X T4 DNA Polymerase buffer
1




25 mM dATP
1




100 mM DTT
  0.5




Nuclease-free water
7.3-X




2.5 U/μl T4 DNA Polymerase
  0.2




Total
10 
22° C.
30 min









After the reaction, the enzyme was inactivated by incubation at 75° C. for 20 min.

The following protocol was used to anneal the insert into the pET-46 Ek/LIC vector.














Unit




Reaction
(μl)
Incubation


















pET-46 Ek/LIC vector
0.5




T4 DNA Polymerase treated EK/LIC insert
1




Total
1.5
22° C.
5 min









Then add 0.5 μl 25 mM EDTA. Mix by stirring with pipet tip. Incubate at 22° C. for 5 min.

Each of the ligation mixture for Cb1954 wild-type-, Cb1954TM3- or Cb1954TM5-pET-46 Ek/LIC was introduced into E. coli NovaBlue competent cells by chemical transformation method, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., four colonies were selected and used to inoculate 6 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours, and minipreps (QIAGEN) were made of the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to check the size of the plasmid DNA. After confirmation that the gene had been inserted into the plasmid, the genes were sequenced to confirm their identities.
For all the constructs of Cb1954, only Cb1954TM3 could be cloned. Thus for the expression of this protein, one of the recombinant plasmids was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol (50 μg/ml) and ampicillin (100 μg/ml) and incubated at 37° C. overnight. Five colonies were inoculated into 3 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 4 hours. One mL of the culture was added to 500 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.25. The inducer, IPTG, was then added at 0.1 mM final concentration, and the culturing continued at 16° C. overnight.
Protein Purification
Cultures were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (50 mM Tris-HCL pH 7.5, 300 mM of NaCl). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed three times to remove the unbound proteins. The bound protein (6-Histidine-tagged Cb1954TM3) was then eluted from the resin with an elution buffer (50 mM Tris-HCL, pH7.5, 250 mM imidazole).
The design of the PCR primers ensured that the protein was fused to 6-histidines encoded in the plasmid. The six histidines will bind to either a nickel-charged resin or a cobalt-charged resin. The bound protein can be displaced from the resin with a buffer containing imidazole. This method facilitates quick purification of the protein of interest.
The Cb1954TM3 gene was expressed in E. coli cells, and the protein was purified in three steps, including a talon resin purification step making use of the 6-histidines encoded by the plasmid, an anion exchange step using Hitrap Q column and a gel filtration step using Hiload 16/60 Superdex 200 column. FIG. 39A shows an SDS-PAGE of purified Cb1954TM3.
Gene and Protein Sequences of Cb1954WT, Cb1954TM3, and Cb1954TM5
Wild Type Cb1954 Amino Acid Sequence
The wild-type Cb1954 endocellulase (EC 3.2.1.4) amino acid sequence is disclosed in SEQ ID NO: 74. The signal peptide of Cb1954, corresponds to amino acid numbers 1-27 of SEQ ID NO: 74. The amino acid sequence of the wild-type Cb1954 protein without the signal peptide is disclosed in SEQ ID NO: 121.
Wild Type Cb1954 Nucleotide Sequence
The wild-type Cb1954 nucleotide sequence is disclosed in SEQ ID NO: 116. The signal peptide of Cb1954 corresponds to nucleotide numbers 1-81 of SEQ ID NO: 116. The nucleotide sequence encoding the wild-type Cb1954 protein without the signal peptide is disclosed in SEQ ID NO: 75.
Cb1954TM3 Amino Acid Sequence
The Cb1954TM3 amino acid sequence is disclosed in SEQ ID NO: 76. The procedure of cloning the gene for Cb1954TM3 into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The Cb1954TM3 amino acid sequence with the short peptide from pET-46 Ek/LIC is disclosed in SEQ ID NO: 81. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 81.
Cb1954TM3 Nucleotide Sequence
The Cb1954TM3 nucleotide sequence is disclosed in SEQ ID NO: 77. The Cb1954TM3 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 80. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 80.
Cb1954TM5 Amino Acid Sequence
The Cb1954TM5 amino acid sequence is disclosed in SEQ ID NO: 78.
Cb1954TM5 Nucleotide Sequence
The Cb1954TM5 nucleotide sequence is disclosed in SEQ ID NO: 79.
Enzyme Activity
FIG. 39B shows the enzymatic activity of Cb1954TM3 on natural substrates from a reducing sugar assay. Three different cellulose substrates were tested: Avicel, sodium carboxymethyl cellulose (CMC-Na) and phosphoric acid swollen cellulose (PASC). Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents. Hydrolysis of PASC was higher than hydrolysis of other substrates.
The concentration of glucose equivalents was determined following enzymatic hydrolysis of Avicel, CMC-Na and PASC, according to the methods of Lever, M. (supra). 1.5 mL microcentrifuge tubes were “zeroed” in an analytical balance. Next, 2±0.1 mg Avicel were added to each tube, and the mass measured and recorded. For CMC-Na and PASC, a stock substrate solution of CMC-Na (2%) and PASC (6.11 mg/ml) were used. Sodium citrate reaction buffer and enzymes were added to each tube beginning with the reaction buffer. The tubes were incubated with constant mixing in a Thermomixer R (Eppendorf) at 75° C. for 16 h. The tubes were centrifuged at 10,000 rpm for 5 min at 4° C. 50 μL of sample supernatant was transferred to a clean 1.5 mL centrifuge tube for the pHBAH assay to determine the reducing ends released by the enzyme. 1 mL of a stock solution of glucose was made at a concentration of 100 mM in sodium citrate buffer, and then serial dilutions were made in sodium citrate buffer to the following concentrations (20 mM, 10 mM and 5 mM). 50 mg of pHBAH was dissolved in 50 mL of ice-cold citrate/NaOH solution for a final concentration of 0.1% (w/v), and the solution was kept on ice. 150 μL of pHBAH solution was added to 50 μL of the sample and glucose standard solutions, and the tubes were incubated at 100° C. for 10 min. The tubes were incubated at room temperature for 5 min. The wavelength at 410 nm was measured for the standards and samples. The A410nm and glucose concentrations were plotted against each other, and linear regression was used to fit a prediction equation to the data. The coefficient of determination (R2) value was between 0.98 and 1.0. The equation from the standard curve was used to calculate the concentrations of reducing ends in the samples based upon their absorbances.
FIG. 40 shows the enzymatic activity of Cb1954TM3 on cellulosic substrates using HPLC analysis. Three different cellulosic substrates were tested: Avicel, CMC-Na and PASC. In each case, in the presence of Cb1954TM3, glucose and cellobiose were released. In the absence of Cb1954TM3, neither glucose nor cellobiose was observed for all the substrates. The results showed that this enzyme releases glucose and cellobiose, and also longer chain oligosaccharides as end products from cellulosic substrates (CMC-Na and PASC).
FIG. 41 shows the thermostability of Cb1954TM3. Cb1954TM3 has 75%, 87%, 64% and 7% activity after incubation at 70° C., 75° C., 80° C. and 85° C. for 24 h, respectively. 500 nM Cb1954TM3 was kept at different temperatures (70° C., 75° C., 80° C. and 85° C.). The enzyme activity was measured at pH 5.5 and at 95° C. on a thermomixer. 2.5 mg/ml final concentration of PASC was used for measurement, and 10 μl of the protein sample was added to the substrate and mixed by pipetting up and down for several times. The total volume was 100 μl. The reducing ends corresponding to glucose equivalents were measured according to the methods of Lever, M. (supra). The velocity of reaction in 10 minutes was calculated. The velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the velocity of reaction at time 0, then multiplied by 100, respectively.
Example 12: Endo-Glucanase Cb1946
A putative endoglucanase, Cb1946WT, was identified in Caldicellulosiruptor bescii. The enzyme is the gene product of Cb1946WT, where Cb stands for Caldicellulosiruptor bescii. The Cb1946WT protein is 1271 amino acids long and has a molecular mass of 139.8 kDa (His-tag+Cb1946 protein). The Cb1946WT has a Glycoside Hydrolase (GH) family 5 catalytic domain at the N-terminal region and Glycoside Hydrolase (GH) family 44 catalytic domain at the C-terminal region and 2 carbohydrate binding modules are positioned between the two catalytic domains (FIG. 42). For the truncated mutants, Cb1946TM1 (653 amino acids, 71.3 kDa) has N-terminal GH5 catalytic domain with 2 carbohydrate binding modules, whereas Cb1946TM2 (1015 amino acids, 111.0 kDa) has C-terminal GH44 catalytic domains with 2 carbohydrate binding modules as shown in FIG. 42.
Cloning of Cb1946WT
The wild type gene and its two truncated mutants (FIG. 42) were amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using PrimeSTAR DNA Polymerase (TAKARA). The nucleotide sequences encoding Cb1946WT, Cb1946TM1, and Cb1946TM2 were amplified using the following primer set and procedures:









Cb1946WT Forward:



(SEQ ID NO: 82)



5′-GAC GAC GAC AAG ATG GCT ACA TCT AAT GAT



GGA GTA GTG AAG -3′


 



Cb1946WT Reverse:



(SEQ ID NO: 83)



5′-GAG GAG AAG CCC GGT TAA TTT AGT TTG TAC



TGA GGT TGA ATA TAA AAC GAT ATG G -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
5
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










Cloning of Cb1946TM1










Cb1946TM1 Forward:



(SEQ ID NO: 82)



5′-GAC GAC GAC AAG ATG GCT ACA TCT AAT GAT



GGA GTA GTG AAG -3′


 



Cb1946TM1 Reverse:



(SEQ ID NO: 84)



5′-GAG GAG AAG CCC GGT TAG TTA AAC CTT ATC



TGT ATC TCC CCT GTG TC -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
4
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










Cloning of Cb1946TM2










Cb1946TM2 Forward:



(SEQ ID NO: 85)



5′-GAC GAC GAC AAG ATG GTA GGG TAC TTG GAC



ATG GTA AAC AAT TGG GA -3′


 



Cb1946TM2 Reverse:



(SEQ ID NO: 83)



5′-GAG GAG AAG CCC GGT TAA TTT AGT TTG TAC



TGA GGT TGA ATA TAA AAC GAT ATG G -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the coding sequence from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
4
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










After the PCR described above, the amplification of Cb1946 gene was confirmed by 1% agarose gel electrophoresis. The DNA corresponding to the expected band on the gel was cut out and applied to a Qiagen Gel Extraction kit to extract the DNA out of the gel.
A Novagen pET-46 Ek/LIC kit was used to treat the purified DNA and ligate it into the pET-46 Ek/LIC vector. The treatment of the purified DNA was as follows:














Unit




Reaction
(μl)
Incubation







0.1 pmol purified PCR product
X




10X T4 DNA Polymerase buffer
1




25 mM dATP
1




100 mM DTT
  0.5




Nuclease-free water
7.3-X




2.5 U/μl T4 DNA Polymerase
  0.2




Total
10 
22° C.
30 min









After the reaction, the enzyme was inactivated by incubation at 75° C. for 20 min.

The following protocol was used to anneal the insert into the pET-46 Ek/LIC vector.














Unit




Reaction
(μl)
Incubation


















pET-46 Ek/LIC vector
0.5




T4 DNA Polymerase treated EK/LIC insert
1




Total
1.5
22° C.
5 min









Then add 0.5 μl 25 mM EDTA. Mix by stirring with pipet tip. Incubate at 22° C. for 5 min.

The ligation mixture for Cb1946-pET-46 Ek/LIC was introduced into E. coli JM109 by electroporation, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., four colonies were selected and used to inoculate 6 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours, and minipreps (QIAGEN) were made from the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to confirm the size of the plasmid DNA. After confirmation of the insert in the plasmid, the gene or coding sequences were sequenced to confirm their identity and integrity.
For gene expression, one of the plasmids was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol (50 μg/ml) and ampicillin (100 μg/ml) and incubated at 37° C. overnight. Five colonies were inoculated into 3 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 4 hours. One mL of the culture was added to 500 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.25. The inducer, IPTG, was then added at 0.1 mM final concentration, and the culturing continued at 16° C. overnight.
Gene and Protein Sequences of Cb1946WT, Cb1946TM1, and Cb1946TM2
Cb1946 Wild-Type Amino Acid Sequence
The wild-type Cb1946 amino acid sequence is disclosed in SEQ ID NO: 86. The signal peptide of Cb1946, corresponding to amino acid numbers 1-38 of SEQ ID NO: 86 was removed during all PCR amplifications. Thus, the expressed wild-type Cb1946 protein did not contain amino acid numbers 1-38 of SEQ ID NO: 86. The amino acid sequence of the wild-type Cb1946 protein without the signal peptide is disclosed in SEQ ID NO: 87.
The procedure of cloning the gene for wild-type Cb1946 (without the signal peptide) into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The wild-type Cb1946 amino acid sequence (without the signal peptide) with the short peptide is disclosed in SEQ ID NO: 91. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 91.
Cb1946 Wild-Type Nucleotide Sequence
The wild-type Cb1946 nucleotide sequence is disclosed in SEQ ID NO: 88. The signal peptide of Cb1946, corresponding to nucleotide numbers 1-114 of SEQ ID NO: 88 was removed during all PCR amplifications. Thus, the nucleotide sequence used to express wild-type Cb1946 protein did not contain nucleotide numbers 1-114 of SEQ ID NO: 88. The nucleotide sequence encoding the wild-type Cb1946 protein without the signal peptide is disclosed in SEQ ID NO: 89.
The wild-type Cb1946 nucleotide sequence (without the signal peptide) with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 90. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 90.
Cb1946TM1 Amino Acid Sequence
The Cb1946TM1 amino acid sequence is disclosed in SEQ ID NO: 117. The procedure of cloning the gene for Cb into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The Cb1946TM1 amino acid sequence with the short peptide is disclosed in SEQ ID NO: 93. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 93.
Cb1946TM1 Nucleotide Sequence
The Cb1946TM1 nucleotide sequence is disclosed in SEQ ID NO: 118. The Cb1946TM1 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 92. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 92.
Cb1946TM2 Amino Acid Sequence
The Cb1946TM2 amino acid sequence is disclosed in SEQ ID NO: 113. The procedure of cloning the gene for Cb1946TM2 into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The Cb1946TM2 amino acid sequence with the short peptide is disclosed in SEQ ID NO: 95. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 95.
Cb1946TM2 Nucleotide Sequence
The Cb1946TM2 nucleotide sequence is disclosed in SEQ ID NO: 112. The Cb1946TM2 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 94. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 94.
Purification of Cb1946WT, Cb1946TM1, and Cb1946TM2 Proteins
The Cb1946WT and its truncated mutants Cb1946TM1 and Cb1946TM2 were expressed in E. coli BL-21 CodonPlus (DE3) RIL competent cells by heat shock. The recombinant cells were then grown overnight in LB agars supplemented with ampicillin (100 μg/mL) and chloramphenicol (50 μg/ml) at 37° C. After 8 h, the starter cultures were diluted into fresh LB supplemented with ampicillin (100 μg/mL) and chloramphenicol (50 μg/ml) at 37° C. with aeration until the absorbance at 600 nm reached 0.5. Gene expression was then induced by addition of IPTG at a final concentration of 0.1 mM and the temperature for culturing was lowered to 16° C. After 16 hours, the cells were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (25 mM Tris-HCL pH 7.8, 750 mM of NaCl, 5% glycerol, 20 mM imidazole, 1.25% Tween-20). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed three times to remove the unbound proteins. The bound protein (6-Histidine-tagged target proteins) was then eluted from the resin with an elution buffer (50 mM Tris-HCL, pH7.5, 250 mM imidazole). The eluted fractions was then heat-treated at 65° C. for 30 minutes and then centrifuged to remove the precipitated proteins. The proteins were then purified by gel filtration chromatography (HiLoad 16/20 Superdex 200, GE Healthcare) with a Tris-HCl elution buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). Aliquots of eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins bands were visualized by staining with Coomassie brilliant blue G-250 (FIG. 43).
Enzyme Activity
FIG. 44 shows the zymogram of Cb1946WT, Cb1946TM1, and Cb1946TM2 on carboxylmethyl cellulose (CMC) agar plate. The agar plate was prepared with CMC substrate (final 0.25%, w/v). After spotting 1 μg of each protein on agar-CMC plates, the plate was incubated at 37° C. overnight and then the gel was visualized by staining with 0.1% Congo red and destaining with 1M NaCl. As shown in FIG. 44, Cb1946WT and Cb1946TM2 showed significant halos on the agar plate indicating cellulase activity, but not Cb1953TM1 proteins.
FIG. 45 shows the enzymatic activity of Cb1946WT, Cb1946TM1, Cb1946TM2 on phosphoric acid swollen cellulose (PASC). Each enzyme (final 0.5 μM) was reacted with phosphoric acid swollen cellulose (PASC) at 1% final concentration in 50 mM citrate-150 mM NaCl, pH 6.0 at 75° C. for 16 hours. The reactions were resolved by thin layer chromatography (TLC). The mobile phase consisted of n-butanol:acetic acid:H2O, 10:5:1 (vol/vol/vol) and 10 cm×20 cm plates were used. For more quantitative analysis of the products of hydrolysis, the samples were analyzed by high performance anion-exchange chromatography (HPAEC) (FIG. 46). For HPAEC analyses, 100 μL of each diluted sample was injected into a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.). For the TLC and HPLC analysis, glucose and five different cellooligosaccharides were used: cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose as standards. Based on the results of TLC and HPLC, Cb1953WT and Cb1953TM2 showed significant release of products such as glucose, cellobiose, cellotriose, and cellotetraose from PASC substrate, indicating that Cb1946WT and Cb1953TM2 have cellulase activities, but not Cb1953TM1.
Example 13: Endocellulase Cb629
An endocellulase, Cb629, was identified in Caldicellulosiruptor bescii. The enzyme Cb629TM1 is the truncational mutant of the gene product of cb629, where Cb stands for Caldicellulosiruptor bescii. The endocellulase initially cleaves glucose, cellobiose and cellotriose from cellulose. The Cb629TM1 protein is 562 amino acids long and has a molecular weight of 63.7 kDa (His-tag+Cb629TM1 protein). The protein has a Glycoside Hydrolase (GH) family 5 catalytic domain and a Carbohydrate Binding Module (CBM) family 17_28 domain (FIG. 47). In addition there is a N-terminal signal peptide (SP) for secretion and three surface layer homology (SLH) modules likely used in anchoring the enzyme to the cell surface. Since the SP and SLH are non-catalytic, they were cleaved from the polypeptide through the PCR amplification described below and the gene product was named Cb629TM1.
Cloning of Cb629TM1
The gene for Cb629TM1 was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using PrimeSTAR DNA Polymerase (TAKARA). The Cb629TM1 gene was amplified using the following primer set:









Cb629TM1Forward:



(SEQ ID NO: 96)



5′- GAC GAC GAC AAG ATG CAG AGC ATA CTG TAT



GAA AAG G -3′


 



Cb629TM1Reverse:



(SEQ ID NO: 97)



5′- GAG GAG AAG CCC GGT TAC TCA AAA AGG ATA



TTG GTA AAT C -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
1



50 μM Rv Primer
1



10 mM dNTP Mixture
1



5 × PrimeSTAR Buffer
10



dH2O
35.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
2
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










After the PCR amplification described above, the amplification of Cb629TM1 was confirmed by 1% agarose gel electrophoresis. The DNA corresponding to the expected band on the gel was cut out and applied to a Qiagen Gel Extraction kit to extract the DNA out of the gel.
A Novagen pET-46 Ek/LIC kit was used to treat the purified DNA and ligate it into the pET-46 Ek/LIC vector. The treatment of the purified DNA was as follows:














Unit




Reaction
(μl)
Incubation







0.1 pmol purified PCR product
X




10X T4 DNA Polymerase buffer
1




25 mM dATP
1




100 mM DTT
  0.5




Nuclease-free water
7.3-X




2.5 U/μl T4 DNA Polymerase
  0.2




Total
10 
22° C.
30 min









After the reaction, inactivate the enzyme by incubating at 75° C. for 20 min.

The following protocol was used to anneal the insert into the pET-46 Ek/LIC vector.














Unit




Reaction
(μl)
Incubation


















pET-46 Ek/LIC vector
0.5




T4 DNA Polymerase treated EK/LIC insert
1




Total
1.5
22° C.
5 min









Then add 0.5 μl 25 mM EDTA. Mix by stirring with pipet tip. Incubate at 22° C. for 5 min.

The ligation mixture for Cb629TM1-pET-46 Ek/LIC were introduced into E. coli NovaBlue competent cells by chemical transformation method, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., four colonies were selected and each was used to inoculate 6 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours, and minipreps (QIAGEN) were made of the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to check the size of the plasmid DNA. After confirmation that the gene had been inserted into the plasmid, the genes were sequenced to confirm the integrity of the coding sequence.
For gene expression, one of the plasmids was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol (50 μg/ml) and ampicillin (100 μg/ml) and incubated at 37° C. overnight. Five to six colonies were inoculated into 10 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 6 hours. Ten mL of the culture was added to 1000 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.3. The inducer, IPTG, was then added at 0.1 mM final concentration, and the culturing continued at 16° C. overnight.
Protein Purification
Cultures were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (50 mM Tris-HCL pH 7.5, 300 mM of NaCl). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed three times to remove the unbound proteins. The bound protein (6-Histidine-tagged Cb629TM1) was then eluted from the resin with an elution buffer (50 mM Tris-HCL, pH7.5, 250 mM imidazole).
The design of the PCR primers ensured that the protein was fused to 6-histidines encoded in the plasmid. The six histidines will bind to either a nickel-charged resin or a cobalt-charged resin. The bound protein can be displaced from the resin with a buffer containing imidazole. This method facilitates quick purification of the protein of interest.
Gene and Protein Sequences of Cb629WT and Cb629TM1
Cb629 Wild-Type Amino Acid Sequence
The wild-type Cb629 endocellulase (EC 3.2.1.4) amino acid sequence is disclosed in SEQ ID NO: 98. The signal peptide of Cb629 corresponds to amino acid numbers 1-29 of SEQ ID NO: 98. The amino acid sequence of the wild-type Cb629 protein without the signal peptide is disclosed in SEQ ID NO: 119.
Cb629 Wild-Type Nucleotide Sequence
The wild-type Cb629 nucleotide sequence is disclosed in SEQ ID NO: 99. The signal peptide of Cb629 corresponds to nucleotide numbers 1-87 of SEQ ID NO: 99. The nucleotide sequence encoding the wild-type Cb629 protein without the signal peptide is disclosed in SEQ ID NO: 120.
Cb629TM1 Amino Acid Sequence
The Cb629TM1 endocellulase (EC 3.2.1.4) amino acid sequence is disclosed in SEQ ID NO: 100. The procedure of cloning the gene for Cb629TM1 into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The Cb629TM1 amino acid sequence with the short peptide is disclosed in SEQ ID NO: 103. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 103.
Cb629TM1 Nucleotide Sequence
The Cb629TM1 nucleotide sequence is disclosed in SEQ ID NO: 101. The Cb629TM1 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 102. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 102.
The Cb629TM1 coding sequence was expressed in E. coli cells, and the protein was purified in one step, i.e. the talon resin purification step making use of the 6-histidines encoded by the plasmid. FIG. 48 shows an SDS-PAGE of purified Cb629TM1.
Enzyme Activity
FIG. 49 shows the enzymatic activity of Cb629TM1 on substrates with products determined through a reducing sugar assay. Three different cellulose substrates were tested: Avicel, sodium carboxymethyl cellulose (CMC-Na) and phosphoric acid swollen cellulose (PASC). Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents. Hydrolysis of PASC was higher than hydrolysis of the other substrates.
The concentration of glucose equivalents was determined following enzymatic hydrolysis of Avicel, CMC-Na and PASC, according to the methods of Lever, M. (supra). 1.5 mL microcentrifuge tubes were “zeroed” in an analytical balance. Next, 2±0.1 mg Avicel were added to each tube, and the mass measured and recorded. The volumes that should be added to each tube were calculated based on the mass. For CMC-Na and PASC, a stock substrate solution of CMC-Na (2%) and PASC (6.11 mg/ml) were used. Sodium citrate reaction buffer and enzymes were added to each tube beginning with the reaction buffer. The tubes were incubated with constant mixing in a Thermomixer R (Eppendorf) at 75° C. for 16 h. The tubes were centrifuged at 10,000 rpm for 5 min at 4° C. 50 μL of sample supernatant was transferred to a clean 1.5 mL centrifuge tube for the pHBAH assay. 1 mL of a stock solution of glucose was made at a concentration of 100 mM in sodium citrate buffer, and then serial dilutions were made in sodium citrate buffer to the following concentrations (20 mM, 10 mM and 5 mM). 50 mg of pHBAH was dissolved in 50 mL of ice-cold citrate/NaOH solution for a final concentration of 0.1% (w/v), and the solution was kept on ice. 150 μL of pHBAH solution was added to 50 μL of the sample and glucose standard solutions, and the tubes were incubated at 100° C. for 10 min. The tubes were incubated at room temperature for 5 min. The wavelength at 410 nm was measured for the standards and samples. The A410nm and glucose concentrations were plotted against each other, and linear regression was used to fit a line to the data. The coefficient of determination (R2) value was between 0.98 and 1.0. The equation from the standard curve was used to calculate the concentrations of reducing ends in the samples based on absorbance data.
FIG. 50 shows the enzymatic activity of Cb629TM1 on substrates using HPLC analysis. Three different cellulosic substrates were tested: Avicel, CMC-Na and PASC. In each case, in the presence of Cb629TM1, glucose and cellobiose were released. In the absence of Cb629TM1, neither glucose nor cellobiose was observed from all the substrates. The results showed that this enzyme releases glucose and cellobiose as end products from cellulosic substrates (Avicel, CMC-Na and PASC).
FIG. 51 shows that this enzyme is also able to release mostly disaccharides (cellobiose) and glucose from cello-oligosaccharide. The enzyme does not cleave hydrolyze cellobiose (G2 in the figure).
FIG. 52 shows the thermostability of Cb629TM1. Cb629TM1 has 109%, 99%, 96%, 83% and 34% activity after incubation at 60° C., 65° C., 70° C., 75° C. and 80° C. for 24 h, respectively. 500 nM Cb629TM1 was kept at different temperatures (60° C., 65° C., 70° C., 75° C. and 80° C.). The samples were taken out at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h) and immediately used for enzyme activity measurement. The enzyme activity was measured at pH 5.5 and at 70° C. on a thermomixer. 2.5 mg/ml final concentration of PASC was used for measurement, and 8.31 μl of the protein sample was added to the substrate and mixed by pipetting up and down for several times. The total volume was 100 μl. The reducing ends corresponding to glucose equivalents were measured according to the methods of Lever, M. (supra). The velocity of reaction in 10 minutes was calculated. The velocity of reaction for time 0 was set as 100; then the remaining activities (percentage) for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h were calculated by dividing the velocities of reaction for time 0.5 h, 1 h, 2 h, 4 h, 7 h, 11 h and 24 h by the velocity of reaction at time 0, then multiplied by 100, respectively.
Example 14: β-Glucosidase Cb486
A putative β-glucosidase Cb486, was identified in Caldicellulosiruptor bescii. The enzyme is the gene product of Cb486, where Cb stands for Caldicellulosiruptor bescii. β-glucosidases catalyze the hydrolysis of cellobiose (a disaccharide of glucose) into two units of glucose. The Cb486 protein is 466 amino acids long and has a predicted molecular weight of 54.9 kDa (His-tag+Cb486 protein). The protein has a Glycoside Hydrolase (GH) family 1 catalytic domain (FIG. 53A).
Cloning of Cb486
The gene for Cb486 was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using iProofTM High-Fidelity DNA Polymerase (BIO-RAD). The Cb486 gene was amplified using the following primer set:









Cb486Forward:



(SEQ ID NO: 104)



5′-GAC GAC GAC AAG ATG AGT TTA CCA AAA GGA TTT



CTG TGG GGT GC -3′


 



Cb1172Reverse:



(SEQ ID NO: 105)



5′-GAG GAG AAG CCC GGT TAT GAG TTT TCC TTT ATA



TAC TGC TG -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2 U/μL iProoF TM High-Fidelity DNA Polymerase
0.5



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



50 μM Fw Primer
0.5



50 μM Rv Primer
0.5



10 mM dNTP Mixture
1



5 × iProof HF Buffer
10



dH2O
36.5



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
98° C.
30
sec
1 cycle



Denaturing
98° C.
10
sec
35 cycles



Annealing
62° C.
30
sec




Elongation
72° C.
2
min




Elongation
72° C.
10
min
1 cycle












Last
 4° C.
∞










After the PCR described above, the amplification of the gene for Cb486 was confirmed by 1% agarose gel electrophoresis. The DNA corresponding to the expected band on the gel was cut out and applied to a Qiagen Gel Extraction kit to extract the DNA out of the gel.
A Novagen pET-46 Ek/LIC kit was used to treat the purified DNA and ligate it into the pET-46 Ek/LIC vector. The treatment of the purified DNA was as follows:














Unit




Reaction
(μl)
Incubation







0.1 pmol purified PCR product
X




10X T4 DNA Polymerase buffer
1




25 mM dATP
1




100 mM DTT
  0.5




Nuclease-free water
7.3-X




2.5 U/μl T4 DNA Polymerase
  0.2




Total
10 
22° C.
30 min









After the reaction, the enzyme was inactivated by incubation at 75° C. for 20 min.

The following protocol was used to anneal the insert into the pET-46 Ek/LIC vector.














Unit




Reaction
(μl)
Incubation


















pET-46 Ek/LIC vector
0.5




T4 DNA Polymerase treated EK/LIC insert
1




Total
1.5
22° C.
5 min









Then add 0.5 μl 25 mM EDTA. Mix by stirring with pipet tip. Incubate at 22° C. for 5 min.

The ligation mixture for Cb486-pET-46 Ek/LIC was introduced into E. coli JM109 by electroporation, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., four colonies were selected and each was used to inoculate 6 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours, and minipreps (QIAGEN) were made of the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to check the size of the plasmid DNA. After confirmation that the gene had been inserted into the plasmid, the inserts were sequenced to confirm their identity and integrity of the sequence.
For gene expression, one of the plasmids was transformed into E. coli BL21 codon plus DE3 RIL by the heat shock method and plated on LB plates supplemented with chloramphenicol (100 μg/ml) and ampicillin (50 μg/ml) and incubated at 37° C. overnight. Five to six colonies were inoculated into 3 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 4 hours. One mL of the culture was added to 500 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.25. The inducer, IPTG, was then added at 0.1 mM final concentration, and the culturing continued at 16° C. overnight.
Protein Purification
Cultures were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (25 mM Tris-HCL pH 7.8, 750 mM of NaCl, 5% glycerol, 20 mM imidazole, 1.25% Tween-20). The proteins in the cells were released through a French pressure cell. After centrifugation to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed three times to remove the unbound proteins. The bound protein (6-Histidine-tagged Cb486) was then eluted from the resin with an elution buffer (50 mM Tris-HCL, pH7.5, 250 mM imidazole).
The gene product of Cb486 was expressed in its full-length form. The design of the PCR primers ensured that the protein was fused to 6-histidines encoded in the plasmid. The six histidines will bind to either a nickel-charged resin or a cobalt-charged resin. The bound protein can be displaced from the resin with a buffer containing imidazole. This method facilitates quick purification of the protein of interest.
Gene and Protein Sequences of Cb486WT
Cb486 Wild-Type Amino Acid Sequence
The wild-type Cb486 β-glucosidase (EC 3.2.1.21) amino acid sequence is disclosed in SEQ ID NO: 106. The procedure of cloning the gene for wild-type Cb486 into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The wild-type Cb486 amino acid sequence with the short peptide is disclosed in SEQ ID NO: 109. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 109.
Cb486 Wild-Type Nucleotide Sequence
The wild-type Cb486 nucleotide sequence is disclosed in SEQ ID NO: 107. The wild-type Cb486 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 108. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 108.
The Cb486 gene was expressed in E. coli cells, and the protein was purified in one step, using the talon resin purification step making use of the 6-histidines encoded by the plasmid. FIG. 53B shows an SDS-PAGE of purified Cb486.
Enzyme Activity
FIG. 54 shows the enzymatic activity of Cb486 on xylo-oligosaccharides (X2-X6) through Thin Layer Chromatography (TLC) analysis. The following xylo-oligosaccharides (X2-X6) were tested: xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose. This was done by an overnight hydrolysis of the xylo-oligosaccharides followed by resolving of the products with TLC. In each case, in the presence of Cb486, xylose and xylobiose were released. In the absence of Cb486, only minor amount of xylose was observed for xylobiose; no products of hydrolysis were released for other xylo-oligosaccharides. The results showed that this enzyme releases xylose and xylobiose from xylo-oligosaccharides (xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose).
FIG. 55 shows that this enzyme is also capable of cleaving cello-oligosaccharides from cellobiose (2 glucose units joined by beta 1,4-linkage) to cellohexaose (six glucose units linked together by beta 1,4-linkages) to glucose. Thus this enzyme when coupled with an endoglucanase that release short chains of glucose should be able to convert the short chains to the monosaccharides glucose. The multi-functional activity (cleavage of different linkages) should make this enzyme an important enzyme in enzyme mixes used in hydrolyzing complex polysaccharides.
FIGS. 56A and 56B show the pH and temperature profiles, respectively of the activity of Cb486.
Example 15: Cellulase Mixture from Caldicellulosiruptor bescii for the Hydrolysis of Miscanthus 
Based on the analyses above, a cellulase mixture containing Cb629TM1, Cb486, Cb1946TM2, Cb1952TM1, Cb1953TM2, and Cb1954TM3 was reconstituted to represent Caldicellulosiruptor bescii cellulases (FIG. 57). A previously reconstituted hemicellulase of Caldicellulosiruptor bescii (FIG. 58) was also prepared to test synergistic effects with the cellulase mixture. All enzyme mixtures (each 0.5 μM) were reacted with 2%, 5%, and 8% pretreated (autoclaved Miscanthus & 1% NaOH treated+microwaved Miscanthus) samples in 50 mM citrate-150 mM NaCl buffer (pH 6.5) at 75° C. overnight with shaking.
The reactions were resolved by thin layer chromatography (TLC). The mobile phase consisted of n-butanol:acetic acid:H2O, 10:5:1 (vol/vol/vol), and 10 cm×20 cm plates were used. (FIGS. 59, 61, 63, and 65).
For further analysis of the products of hydrolysis, the 8% substrate reaction samples were analyzed by high performance anion-exchange chromatography (HPAEC) (FIGS. 60, 62, 64, and 66; i.e. FIG. 60 is HPAEC data of samples from FIG. 59, FIG. 62 is HPAEC data of samples from FIG. 61, etc.). For HPAEC analyses, 100 μL of each diluted sample was injected onto a System Gold HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with CarboPacTM PA1 guard (4×50 mm) and analytical (4×250 mm) columns from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem III electrochemical detector from ESA Biosciences (Chelmsford, Mass.).
For the TLC (FIGS. 59, 61, 63, and 65) and HPLC (FIGS. 60, 62, 64, and 66) analysis, glucose (C1) and five different cellooligosaccharides were used: cellobiose (C2), cellotriose (C3), cellotetraose (C4), cellopentaose (C5), and cellohexaose (C6) as standards. For the separation of xylose and glucose, Aminex HPX-87H column (300×7.8 mm, BioRad) was used with LC-20AT HPLC (SHIMADZU) with 5 mM sulfuric acid as mobile phase and 0.6 mL/mL flow rate at 65° C.
Based on TLC and HPLC data in FIGS. 59-62, in the presence of both cellulases and hemicellulases, the cellulase and hemicellulase mixtures released more glucose and xylose synergistically on pretreated Miscanthus samples than the amount of glucose released by the same cellulase mixture alone or the amount of xylose released by the same hemicellulase mixture alone. For example, as shown in FIG. 60, more glucose was released from the microwave pretreated Miscanthus by the cellulase mixture while in the presence of the hemicellulase mixture (lane 4, C1 peak;  ̃11 mM) than when the cellulase mixture acted on Miscanthus alone (lane 2, C1 peak;  ̃7 mM). Also, as shown in FIG. 60, more xylose was released from the pretreated Miscanthus by the hemicellulase mixture while in the presence of the cellulase mixture (lane 4, X1 peak;  ̃6 mM) than when the hemicellulase mixture acted on Miscanthus alone (lane 2, X1 peak;  ̃3 mM). As shown in FIGS. 61 and 62, synergistic effects between the cellulase and hemicellulase mixtures were also obtained with the autoclave pretreated Miscanthus. Thus, the results provided herein show the surprising result that an enzyme cocktail containing a cellulase mixture disclosed herein and a hemicellulase mixture disclosed herein shows synergistic activity between the cellulase and hemicellulase mixtures.
The results in FIGS. 59-62 also show that more products were released from the microwave pretreated Miscanthus (FIGS. 59 and 60) than the autoclave pretreated Miscanthus samples (FIGS. 61 and 62).
In FIGS. 63-66, the enzyme mixture without Cb486 (β-glucosidase) was tested on both pretreated samples. The results show that the enzyme mixtures released mainly cellobiose in the mix without β-glucosidase (Cb486). The results in lane 4 of FIG. 63 and FIG. 64 shows that the mixture of hemicellulase and cellulose without the beta-glucosidase will lead to xylose and mostly cellobiose from the microwaved sample. Similar data is obtained for the same experiment but with autoclaved Miscanthus as the substrate (FIGS. 65 and 66).
Example 16: Heat Shock Protein Cb1581
A small heat shock protein, Cb1581, was identified in Caldicellulosiruptor bescii. The protein is the gene product of Cb1581, where Cb stands for Caldicellulosiruptor bescii. The protein is 162 amino acids long and has a molecular weight of 19.68 kDa (His-tag+Cb1581 protein).
Cloning of Cb1581
The gene for Cb1581 was amplified from Caldicellulosiruptor bescii DSM 6725T genomic DNA by PCR using PrimeSTAR DNA Polymerase (TAKARA). The cb1581 gene was amplified using the following primer set:









Cb1581Forward: 5′-



(SEQ ID NO: 144)



GACGACGACAAGATGCTCAGAGACATAGTTCCATTTGGC -3′


 



Cb1581Reverse: 5′-



(SEQ ID NO: 145)



GAGGAGAAGCCCGGTTATTCTATATCAATTGTTCTTACATC -3′






The polymerase chain reaction mixture contained the following:










PCR reaction


















2.5 U/μL PrimeSTAR DNA Polymerase
0.4



17 ng/μL Caldicellulosiruptor bescii genomic DNA
1



20 μM Fw Primer
1



20 μM Rv Primer
1



2.5 mM dNTP Mixture
4



5 × PrimeSTAR Buffer
10



dH2O
32.6



Total
50 μL









To amplify the gene from the genomic DNA, the following PCR cycling was used:










PCR protocol





















Denaturing
95° C.
5
min
1 cycle



Denaturing
94° C.
30
sec
35 cycles



Annealing
50° C.
30
sec




Elongation
72° C.
1
min




Elongation
72° C.
7
min
1 cycle












Last
 4° C.
∞










After the PCR reaction described above, the amplification of cb1581 gene was confirmed by 1% agarose gel electrophoresis. The DNA corresponding to the expected band on the gel was cut out and applied to a Qiagen Gel Extraction kit to extract the DNA out of the gel.
A Novagen pET-46 Ek/LIC kit was used to treat the purified DNA and ligate it into the pET-46 Ek/LIC vector. The treatment of the purified DNA was as follows:














Unit




Reaction
(μl)
Incubation







0.1 pmol purified PCR product
X




10X T4 DNA Polymerase buffer
1




25 mM dATP
1




100 mM DTT
  0.5




Nuclease-free water
7.3-X




2.5 U/μl T4 DNA Polymerase
  0.2




Total
10 
22° C.
30 min









After the reaction, inactivate the enzyme by incubating at 75° C. for 20 min.

The following protocol was used to anneal the insert into the pET-46 Ek/LIC vector.














Unit




Reaction
(μl)
Incubation


















pET-46 Ek/LIC vector
0.5




T4 DNA Polymerase treated EK/LIC insert
1




Total
1.5
22° C.
5 min









Then add 0.5 μl 25 mM EDTA. Mix by stirring with pipet tip. Incubate at 22° C. for 5 min.

The ligation mixture for cb1581-pET-46 Ek/LIC were introduced into E. coli XL10-Gold by electroporation method, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., four colonies were selected and used to inoculate 6 mL cultures of LB-ampicillin. The cultures were grown at 37° C. with vigorous aeration for 16 hours, and minipreps (QIAGEN) were made of the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel to check the size of the plasmid DNA. After confirmation that the gene had been inserted into the plasmid, the genes were sequenced to confirm their identity.
For gene expression, one of the plasmids was transformed into E. coli BL21-CodonPlus (DE3)-RIPL by the heat shock method and plated on LB plates supplemented with chloramphenicol (50 μg/ml) and ampicillin (100 μg/ml) and incubated at 37° C. overnight. Five to six colonies were inoculated into 10 ml of LB broth supplemented with the two antibiotics at the same concentration and cultured for 6 hours. Ten mL of the culture was added to 1000 mL of LB broth supplemented with the two antibiotics at the same concentration and cultured at 37° C. until the absorbance at 600 nm reached  ̃0.3. The inducer, IPTG, was then added at 0.1 mM final concentration, and the culturing continued at 16° C. overnight.
Protein Purification
Cultures were centrifuged to collect the cell pellet. The pellet was then suspended in a lysis buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5). The proteins in the cells were released through a French pressure cell. After centrifugation at 10000 rpm for 30 minutes to pellet the cell debris, the supernatant was applied to a cobalt-charged resin (TALON, Clontech) and washed three times to remove the unbound proteins. The bound protein (6-Histidine-tagged Cb1581) was then eluted from the resin with an elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH7.5).
The design of the PCR primers ensured that the protein was fused to 6-histidines encoded in the plasmid. The six histidines will bind to either a nickel-charged resin or a cobalt-charged resin. The bound protein can be displaced from the resin with a buffer containing imidazole. This method facilitates quick purification of the protein of interest.
Gene and Protein Sequences of Cb1581
Cb1581 Wild-Type Amino Acid Sequence
The wild-type Cb1581 amino acid sequence is disclosed in SEQ ID NO: 146. The procedure of cloning the gene for wild-type Cb1581 into the plasmid pET-46 Ek/LIC led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The wild-type Cb486 amino acid sequence with the short peptide is disclosed in SEQ ID NO: 149. The amino acids of the short peptide are amino acids 1-14 of SEQ ID NO: 149.
Cb1581 Wild-Type Nucleotide Sequence
The wild-type Cb1581 nucleotide sequence is disclosed in SEQ ID NO: 147. The wild-type Cb1581 nucleotide sequence with the coding sequence for the short peptide from the plasmid pET-46 Ek/LIC is disclosed in SEQ ID NO: 148. The nucleotides coding for the short peptide nucleotides are nucleotides 1-42 of SEQ ID NO: 148.
The cb1581 gene was expressed in E. coli cells, and the protein was purified in one step, that is, a talon resin purification step making use of the 6-histidines encoded by the plasmid. FIG. 83 shows an SDS-PAGE of purified Cb1581.
Enhancing Enzymatic Hydrolysis of Microwave Pretreated Miscanthus 
FIG. 84 shows the enhancing effect of Cb1581 on enzymatic hydrolysis of microwave pretreated miscanthus at 70° C. (FIG. 84A) or 80° C. (FIG. 84B). The hydrolysis was carried out at pH 6.0 using 0.5 μM each of the cellulase/hemicellulase enzyme mixture in a total volume of 500 μl with 10% miscanthus as the substrate. The enzymes in the mixture include Cb1946TM2, Cb1952TM1, Cb1953TM2, Cb1954TM3, Cb629TM1, Cb486, Cb193, Cb195, Cb2487, Cb1172, Cb909, and Cb162. Two mL microcentrifuge tubes were “zeroed” in an analytical balance. Next, 50±0.2 mg microwave pretreated miscanthus were added to each tube. The tubes were incubated with constant rotation in a EchoThermTMRT11 Variable Speed Rotating Mixers (Torrey Pines Scientific) at 70° C. or 80° C. for 24 h.
The concentration of glucose equivalents was determined following enzymatic hydrolysis of microwave pretreated miscanthus, according to the methods of Lever, M. (A new reaction for colorimetric determination carbohydrates. Anal. Biochem. 1972: 47; 273-279). After the reaction, the tubes were centrifuged at 10,000 rpm for 5 min at 4° C. 45 μL of water and 5 μL of sample supernatant were transferred to a clean 1.5 mL centrifuge tube for the pHBAH assay. 1 mL of a stock solution of glucose was made at a concentration of 100 mM in sodium citrate buffer, and then serial dilutions were made in sodium citrate buffer to the following concentrations (20 mM, 10 mM and 5 mM). 50 mg of pHBAH was dissolved in 50 mL of ice-cold citrate/NaOH solution for a final concentration of 0.1% (w/v), and the solution was kept on ice. 150 μL of pHBAH solution was added to 50 μL of the sample and glucose standard solutions, and the tubes were incubated at 100° C. for 10 min. The tubes were incubated at room temperature for 5 min. The wavelength at 410 nm was measured for the standards and samples. The A410nm and glucose concentrations were plotted against each other, and linear regression was used to fit a line to the data. The correlation coefficient (R2) value was between 0.98 and 1.0. The equation from the standard curve was used to calculate the concentrations of reducing ends in the samples based upon their absorbance. The releasing of sugars is enhanced with the increasing amount of Cb1581 in the reaction mixture at both 70° C. and 80° C.
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## Claims

1. A method for producing a Caldicellulosiruptor bescii endoxylanase and a Caldicellulosiruptor bescii β-xylosidase comprising:
culturing a host cell comprising a nucleic acid encoding the Caldicellulosiruptor bescii endoxylanase of SEQ ID NO: 3, and a nucleic acid encoding the Caldicellulosiruptor bescii β-xylosidase of SEQ ID NO: 27 in a culture medium, under suitable conditions to produce the endoxylanase and the β-xylosidase,
wherein the endoxylanase has a Km for xylan that ranges from about 1.3 mg/mL to about 13.9 mg/mL at a temperature of 85° C. and a pH of 6.0; a Kcat for xylan that ranges from about 93 s−1 to about 7865 s−1 at a temperature of 85° C. and a pH of 6.0; and a Kcat/Km for xylan that ranges from about 33 ml/mg s−1 to about 562 ml/mg s−1 at a temperature of 85° C. and a pH of 6.0,
wherein the β-xylosidase has an optimum temperature of about 90° C., a Km for xylo-oligosaccharides that is about 8.21 mM at a temperature of 90° C. and a pH of 6.0; a Kcat for xylo-oligosaccharides that is about 619 s−1 at a temperature of 90° C. and a pH of 6.0; and a Kcat/Km for xylo-oligosaccharides that is about 75 mM−1 s−1 at a temperature of 90° C. and a pH of 6.0, and
wherein the host cell is an E. coli cell.

2. The method of claim 1, wherein the host cell further comprises one or more recombinant nucleic acids encoding one or more polypeptides selected from the group consisting of: Caldicellulosiruptor bescii endocellulase Cb629 and Caldicellulosiruptor bescii β-glucosidase Cb486 polypeptides.

3. The method of claim 2,
wherein the Caldicellulosiruptor bescii endocellulase Cb629 polypeptide has the sequence of SEQ ID NO: 98 and
wherein the Caldicellulosiruptor bescii β-glucosidase Cb486 polypeptide has the sequence of SEQ ID NO: 106.

4. The method of claim 2, the method further comprising:
a) culturing the host cell in culture media under conditions sufficient to support the expression of said recombinant nucleic acid(s); and
b) collecting one or more cellulases from said media or said host cell.

5. The method of claim 1, wherein the host cell further comprises a recombinant nucleic acid encoding a Caldicellulosiruptor bescii heat shock protein Cb1581 polypeptide.

6. The method of claim 1, wherein the host cell further comprises one or more recombinant nucleic acids selected from the group consisting of:
a) a nucleic acid encoding the Caldicellulosiruptor bescii endoxylanase of SEQ ID NO: 7,
b) a nucleic acid encoding the Caldicellulosiruptor bescii α-arabinofuranosidase of SEQ ID NO: 13,
c) a nucleic acid encoding the Caldicellulosiruptor bescii α-glucuronidase of SEQ ID NO: 19,
d) a nucleic acid encoding the Caldicellulosiruptor bescii acetyl xylan esterase of SEQ ID NO: 33, and
e) a nucleic acid encoding the Caldicellulosiruptor bescii endoxylanase of SEQ ID NO: 37.