# Patent

## Title
Hemicellulose-degrading enzymes

## Abstract

The present disclosure provides methods for the conversion of hemicellulose into fermentable sugars using enzymes isolated from Prevotella bryantii. Hemicellulose-degrading enzymes include an endoxylanase, a β-xylosidase, a bifunctional β-xylosidase and β-glucosidase, a bifunctional arabinofuranosidase and β-xylosidase, a glucuronidase, and an acetyl xylan esterase. The enzymes can be used to release sugars present in hemicellulose for subsequent fermentation to produce value-added products such as ethanol.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a U.S. National Phase patent application of International Application No. PCT/US2010/032589, filed Apr. 27, 2010, which claims priority to U.S. Provisional Patent Application No. 61/173,174, filed Apr. 27, 2009, and U.S. Provisional Patent Application No. 61/245,619, filed Sep. 24, 2009, each of which is hereby incorporated by reference in the present disclosure in its entirety.
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: 658012000300SEQLIST.txt, date recorded: Oct. 26, 2011, size: 313 KB).


FIELD
The present disclosure relates to methods for the degradation of hemicellulose using enzymes including an endoxylanase, a β-xylosidase, a bifunctional β-xylosidase and β-glucosidase, a bifunctional arabinofuranosidase and β-xylosidase, a glucuronidase, and an acetyl xylan esterase. Treatment of hemicellulose with one or more of the enzymes can lead to complete or near-complete degradation of the hemicellulose into its component sugars.
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. The cellulose component of plant matter may be hydrolyzed to glucose (a 6-carbon sugar) by a cellulase system, which usually comprises three important enzymes: an endoglucanase, an exoglucanase, and a beta-glucosidase. These enzymes, however, do not target the hemicellulose component of the plant material.
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. 1a). Several functional groups decorate the backbone, including esters of acetyl (Ac) groups, arabinose, glucuronic acids, and esters of feroryl group. The feroryl groups link the entire structure to lignin.
Hemicellulose constitutes the second largest component of polysaccharides in perennial grasses, such as switchgrass and Miscanthus. 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.
Acid pretreatment is the current standard method for degrading hemicellulose prior to fermentation of its component sugars. This method, however, results in the production of toxic compounds that inhibit future fermentation. Thus, a significant need exists for improved pretreatment methods that can degrade hemicellulose without the production of toxic compounds.
BRIEF SUMMARY
Preferred embodiments of the invention meet this need by providing hemicellulose-degrading enzymes identified in Prevotella bryantii. Use of these enzymes provides a means by which the hemicellulose fraction of biomass can be degraded into fermentable sugars without the production of toxic compounds resulting from acid pretreatment. These enzymes can be utilized alone, in combination, or with other mixtures of enzymes. Methods for the degradation of hemicellulose are also provided herein.
Thus one aspect includes isolated nucleic acids including the nucleotide sequences of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37. Another aspect includes vectors containing any of the preceding isolated nucleic acids. Yet another aspect includes isolated polypeptides including the amino acid sequences of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
One aspect includes compositions containing the nucleotide sequence of SEQ ID NO: 1, and one or more nucleotide sequences selected from the group including SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15. Another aspect includes compositions containing the amino acid sequence of SEQ ID NO: 2, and one or more amino acid sequences selected from the group including SEQ ID NOs: 4, 6, 8, 10, 12, 14, and 16.
Another aspect includes methods for degrading hemicellulose including the steps of providing plant material containing hemicellulose, where the hemicellulose contains a xylose backbone containing β-1,4-linkages and one or more functional groups, and treating the hemicellulose with an enzyme selected from the group including enzymes corresponding to SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38, where the treating cleaves the one or more functional groups from the xylose backbone to form cleaved hemicellulose. In certain embodiments, the method also includes the step of treating the cleaved hemicellulose with a second enzyme corresponding to SEQ ID NO: 2, where the second enzyme cleaves the β-1,4-linkages in the xylose backbone to produce xylose subunits, where the treating results in the degradation of at least 70% of the hemicellulose into functional groups and xylose subunits. In certain embodiments, degradation of the hemicellulose is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complete. Completeness of degradation of hemicellulose refers to the conversion of a hemicellulose substrate to fermentable sugars. In preferred embodiments, the fermentable sugars are 5-carbon sugars. The 5-carbon sugars may be, for example, xylose or arabinose. In certain embodiments that may be combined with the preceding embodiments, the one or more functional groups are arabinose, glucuronyl, or acetyl. In certain embodiments that may be combined with the preceding embodiments, the plant material is Miscanthus, switchgrass, cord grass, rye grass, reed canary 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, willow, aspen, poplar wood, or energy cane. In certain embodiments that may be combined with the preceding embodiments, the treating is conducted at a pH between about 5 and about 6. In certain embodiments that may be combined with the preceding embodiments, the treating is conducted at a temperature between about 25 and about 40° C.
Another aspect includes methods for degrading hemicellulose, including the steps of providing plant material containing hemicellulose, where the hemicellulose contains a xylose backbone containing β-1,4-linkages and one or more functional groups, and contacting the hemicellulose with a transgenic E. coli or yeast that secretes an enzyme selected from the group including SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, where the contacting cleaves the one or more functional groups from the xylose backbone to form cleaved hemicellulose. In certain embodiments, the method also includes the step of contacting the cleaved hemicellulose with a second transgenic E. coli or yeast that secretes an enzyme corresponding to SEQ ID NO: 2, where the contacting cleaves the β-1,4-linkages in the xylose backbone to produce xylose subunits, where the contacting results in the degradation of at least 90% of the hemicellulose into functional groups and xylose subunits. In certain embodiments, degradation of the hemicellulose is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complete.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1, part (a), shows a structural model of a hemicellulose. Part (b) shows that the rate of growth (gradient of curves) of Prevotella bryantii on wheat arabinoxylan is as rapid as its growth rate on glucose.
FIG. 2 shows the protein expression and activity of Pb1893. Part (a) shows the domain architecture of Pb1893, part (b) shows SDS-PAGE of purified Pb1893 from Prevotella bryantii, part (c) shows thin layer chromatography (TLC) analysis of Pb1893 activity, and part (d) shows reducing sugar assays of Pb1893.
FIG. 3 shows the protein expression and activity of Pb1909. Part (a) shows SDS-PAGE of purified Pb1909 from Prevotella bryantii, part (b) shows TLC analysis of Pb1909 activity, and part (c) shows a reducing sugar assay of Pb1909.
FIG. 4 shows protein expression and activity of Pb886 (alternatively named Pb 2351). Part (a) shows SDS-PAGE of purified Pb886 from Prevotella bryantii, part (b) shows screening of Pb886 activity on para-nitrophenyl (pNP) derivatives, part (c) shows the kinetics of pNP substrate hydrolysis, part (d) shows TLC analysis of Pb886 activity on xylo-oligosaccharide substrates, and part (e) shows TLC analysis of Pb886 arabinofuranosidase activity on wheat arabinoxylan (WAX).
FIG. 5 shows the protein expression and activity of Pb911 (alternatively named Pb 2369). Part (a) shows SDS-PAGE of purified Pb911 from Prevotella bryantii, part (b) shows Pb911 activity on pNP substrates, and part (c) shows TLC analysis of Pb911 activity.
FIG. 6 shows the kinetics of xylo-oligosaccharide hydrolysis by Pb911. Part (a) describes the coupled enzyme assay, part (b) shows consumption of xylobiose, part (c) shows rate of release of xylotriose, part (d) shows rate of release of xylotetraose, part (e) shows release of xylopentaose, part (f) shows release of xylohexaose, and part (g) shows the kinetic values for hydrolysis of all five substrates.
FIG. 7 shows the protein expression and activity of Pb1912 (alternatively named Pb 2425). Part (a) shows SDS-PAGE of purified Pb1912 from Prevotella bryantii, part (b) shows TLC analysis of Pb1912 activity, part (c) shows reducing sugar assays of Pb1912, and part (d) shows HPLC analysis of Pb1912 activity with aldouronic acids.
FIG. 8 shows the protein expression and activity of Pb975. Part (a) shows the protein domain architecture of Pb911 and Pb975, part (b) shows SDS-PAGE of purified Pb975 from Prevotella bryantii, part (c) shows Pb975 activity on pNP substrates, part (d) shows TLC analysis of Pb975 activity, and part (e) shows HPLC analysis of Pb975 activity.
FIG. 9 shows the protein expression and activity of Pb1221. Part (a) shows SDS-PAGE of purified Pb1221 from Prevotella bryantii, and part (b) shows steady-state kinetic data for Pb1221.
FIG. 10 shows the protein expression and activity of BACINT 0076. Part (a) shows SDS-PAGE of purified BACINT 0076, part (b) shows xylanase activity of BACINT 0076 on oat-spelt xylan (OSX) and WAX substrates, part (c) shows the specific hydrolysis activity of BACINT 0076 on pNP-β-D-cellobioside, and parts (d) through (g) show the hydrolysis kinetics of other esterase substrates.
FIG. 11 shows the glucose equivalents released from WAX and OSX after treatment with hemicellulase enzyme cocktail.
FIG. 12 shows that increasing the amount of substrate in a reaction leads to an increase in the amount of sugar released. Part (a) shows glucose equivalents released as a function of the amount of substrate, and part (b) shows TLC analysis of the enzyme mix.
FIG. 13 shows hemicellulase activity of the enzyme mix at a pH range of 5 to 6.
FIG. 14 shows the optimum temperature range of the hemicellulose enzyme mix.
FIG. 15 shows the protein expression and activity of Pb398. Part (a) shows the domain architecture of Pb398, part (b) shows SDS-PAGE of purified Pb398 from Prevotella bryantii, and part (c) shows the specific activity of the hydrolysis of pNP substrates.
FIG. 16 shows the subset of genes found to be overexpressed by Prevotella bryantii when grown on WAX as determined by microarray analysis.
FIG. 17 shows the Xylan Utilization System (XUS) gene clusters.
FIG. 18a shows TLC analysis of sugars released from oat-spelt xylan by combinations of the six-enzyme hemicellulase mix with proteins found to be upregulated in the microarray and RNAseq experiments. FIG. 18b shows TLC analysis of sugars released from birch wood xylan. FIG. 18c shows reducing sugar assays for the hydrolysis of oat-spelt xylan by different enzyme mixture combinations. FIG. 18d shows reducing sugar assays for the hydrolysis of birch wood xylan by different enzyme mixture combinations.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present disclosure relates to hemicellulose-degrading enzymes and to methods of using these enzymes for the degradation of hemicellulose into sugars.
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.
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 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 hemicellulose may be fermented for the production of ethanol.
Combinations of enzymes, i.e., an enzyme cocktail, can be tailored to the 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.
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 hemicellulose. Prevotella bryantii contains a complete set of enzymes for degrading hemicellulose 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. Prevotella bryantii grows as rapidly on wheat arabinoxylan (hemicellulose) as on glucose (FIG. 1b).
The experiments described herein suggest that the bacterium has a set of enzymes that function synergistically to release components of the hemicellulose for fermentation and growth. Thus, this set of enzymes represents a group of enzymes that have evolved naturally in this organism to degrade hemicellulose and as such will be a better enzyme system than one that may be put together with components from different organisms.
The present disclosure provides nucleotide and amino acid sequences for enzymes that degrade hemicellulose, including Pb1893, Pb886 (alternatively named Pb2351), Pb911 (alternatively named Pb2369), Pb975 (alternatively named Pb2425), Pb1912, Pb1221, Pb0390 (alternatively named Pb1909), BACINT 0076, Pb398 (alternatively named Pb1917), Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350. Pb1893 functions as an endoxylanase. Pb886 is a bifunctional enzyme that functions as both an arabinofuranosidase and β-xylosidase. Pb911 functions as a β-xylosidase. Pb975 is a bifunctional enzyme that functions as both a β-xylosidase and β-glucosidase. Pb1912 functions as a glucuronidase. Pb1221 functions as an esterase. Pb0390 functions as an endoxylanase and can be used as a substitute for Pb1893. BACINT 0076 functions as an esterase and can be used as a substitute for Pb1221. Pb398 functions as a β-xylosidase. Pb150 functions as an endoxylanase. Pb1906 is predicted to function as an esterase and/or a β-xylosidase. Pb1908 is predicted to function as a β-xylosidase. Pb1911 is predicted to function as an esterase. Pb2001 is predicted to function as an α-glucosidase. Pb2002 is predicted to function as an endo-arabinase. Pb2003 is predicted to function as an α-L-arabinofuranosidase. Pb2004 is predicted to function as an α-xylosidase. Pb2350 is predicted to function as an α-L-arabinofuranosidase. 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.
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. See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
As used herein, sequence identity or identity in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical and often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have sequence similarity or similarity. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
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 hemicellulose or an artificial substrate such a pNP.
Nucleotide Sequences Encoding Hemicellulose-Degrading Enzymes
The present disclosure provides nucleotide sequences encoding the hemicellulose-degrading enzymes Pb1893 (SEQ ID NO: 1), Pb886 (SEQ ID NO: 3), Pb911 (SEQ ID NO: 5), Pb975 (SEQ ID NO: 7), Pb1912 (SEQ ID NO: 9), Pb1221 (SEQ ID NO: 11), Pb0390 (SEQ ID NO: 13), and BACINT 0076 (SEQ ID NO: 15), Pb398 (SEQ ID NO: 17), Pb150 (SEQ ID NO: 19), Pb1894 (SEQ ID NO: 21), Pb1906 (SEQ ID NO: 23), Pb1908 (SEQ ID NO: 25), Pb1911 (SEQ ID NO: 27), PB2001 (SEQ ID NO: 29), PB2002 (SEQ ID NO: 31), Pb2003 (SEQ ID NO: 33), Pb2004 (SEQ ID NO: 35), Pb2350 (SEQ ID NO: 37), 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 Pb1893, Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350.
Nucleotide sequences of the present disclosure may encode one or more glycosyl hydrolase (GH) domains. Nucleotide sequence may also encode a carbohydrate binding module (CBM). The CBM module may interrupt a GH domain or be located in between two GH domains. In certain embodiments, the GH domain sequence is conserved in nucleotide variants.
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.
The present disclosure further provides compositions including the isolated nucleotide sequence encoding Pb1893 alone or in combination with one or more of the isolated nucleotide sequences encoding Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350. One composition includes the isolated nucleotide sequences encoding Pb1893 and Pb886. Another composition includes the isolated nucleotide sequences encoding Pb1893 and Pb911. Another composition includes the isolated nucleotide sequences encoding Pb1893 and Pb975. Another composition includes Pb1893 and Pb1912. Another composition includes Pb1893 and Pb1221. Another composition includes Pb1893 and Pb1912. Another composition includes Pb1893 and Pb0390. Another composition includes Pb1893 and BACINT 0076. Another composition includes Pb1893 and Pb398. Another composition includes Pb1893 and Pb150. Another composition includes Pb1893 and Pb1894. Another composition includes Pb1893 and Pb1906. Another composition includes Pb1893 and Pb1908. Another composition includes Pb1893 and Pb1911. Another composition includes Pb1893 and Pb2001. Another composition includes Pb1893 and Pb2002. Another composition includes Pb1893 and Pb2003. Another composition includes Pb1893 and Pb2004. Another composition includes Pb1893 and Pb2350.
Compositions may include vectors or transgenic host cells comprising the nucleotide sequence encoding Pb1893 alone or in combination with one or more of the isolated nucleotide sequences encoding Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350.
The present disclosure further provides for compositions including the isolated nucleotide sequence encoding Pb0390 alone or in combination with one or more of the isolated nucleotide sequences encoding Pb886, Pb911, Pb975, Pb1912, Pb1221, and BACINT 0076. One composition includes the isolated nucleotide sequences encoding Pb0390 and Pb886. Another composition includes the isolated nucleotide sequences encoding Pb0390 and Pb911. Another composition includes the isolated nucleotide sequences encoding Pb0390 and Pb975. Another composition includes Pb0390 and Pb1912. Another composition includes Pb0390 and Pb1221. Another composition includes Pb0390 and Pb1912. Another composition includes Pb0390 and BACINT 0076. Another composition includes Pb0390 and Pb398. Another composition includes Pb0390 and Pb150. Another composition includes Pb0390 and Pb1894. Another composition includes Pb0390 and Pb1906. Another composition includes Pb0390 and Pb1908. Another composition includes Pb0390 and Pb1911. Another composition includes Pb0390 and Pb2001. Another composition includes Pb0390 and Pb2002. Another composition includes Pb0390 and Pb2003. Another composition includes Pb0390 and Pb2004. Another composition includes Pb0390 and Pb2350.
The disclosure further provides for a transformed transgenic host cell comprising one or more of the nucleic acids encoding Pb1893, Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350. 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. 
Amino Acid Sequences Encoding Hemicellulose-Degrading Enzymes
The disclosure also provides for the amino acid sequences encoding the hemicellulose-degrading enzymes Pb1893 (SEQ ID NO: 2), Pb886 (SEQ ID NO: 4), Pb911 (SEQ ID NO: 6), Pb975 (SEQ ID NO: 8), Pb1912 (SEQ ID NO: 10), Pb1221 (SEQ ID NO: 12), Pb0390 (SEQ ID NO: 14), BACINT 0076 (SEQ ID NO: 16), Pb1917 (SEQ ID NO: 18), Pb150 (SEQ ID NO: 20), Pb1894 (SEQ ID NO: 22), Pb1906 (SEQ ID NO: 24), Pb1908 (SEQ ID NO: 26), Pb1911 (SEQ ID NO: 28), Pb2001 (SEQ ID NO: 30), Pb2002 (SEQ ID NO: 32), Pb2003 (SEQ ID NO: 34), Pb2004 (SEQ ID NO: 36), and Pb2350 (SEQ ID NO: 38), 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/sequence similarity to Pb1893, Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350.
Amino acid sequences of the present disclosure may contain one or more glycosyl hydrolase (GH) domains. Amino acid sequences may also contain a carbohydrate binding module (CBM). The CBM module may interrupt a GH domain or be located in between two GH domains. In certain embodiments, the GH domain is conserved in polypeptide variants.
The polypeptides can be expressed in and purified from their native host, Prevotella bryantii. 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. In other embodiments, the isolated or recombinant polypeptide is thermostable. In certain embodiments, the isolated or recombinant polypeptide is stable at about 25 to 40° C.
The present disclosure provides for compositions including the amino acid sequence encoding Pb1893 alone or in combination with one or more of the amino acid sequences encoding Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, and BACINT 0076. One composition includes the amino acid sequences encoding Pb1893 and Pb886. Another composition includes the amino acid sequences encoding Pb1893 and Pb911. Another composition includes the amino acid sequences encoding Pb1893 and Pb975. Another composition includes the amino acid sequences encoding Pb1893 and Pb1912. Another composition includes the amino acid sequences encoding Pb1893 and Pb1221. Another composition includes the amino acid sequences encoding Pb1893 and Pb0390. Another composition includes the amino acid sequences encoding Pb1893 and BACINT 0076. Another composition includes the amino acid sequences encoding Pb1893 and Pb398. Another composition includes the amino acid sequences encoding Pb1893 and Pb150. Another composition includes the amino acid sequences encoding Pb1893 and Pb1894. Another composition includes the amino acid sequences encoding Pb1893 and Pb1906. Another composition includes the amino acid sequences encoding Pb1893 and Pb1908. Another composition includes the amino acid sequences encoding Pb1893 and Pb1911. Another composition includes the amino acid sequences encoding Pb1893 and Pb2001. Another composition includes the amino acid sequences encoding Pb1893 and Pb2002. Another composition includes the amino acid sequences encoding Pb1893 and Pb2003. Another composition includes the amino acid sequences encoding Pb1893 and Pb2004. Another composition includes the amino acid sequences encoding Pb1893 and Pb2350.
The present disclosure also provides compositions including the amino acid sequence encoding Pb0390 alone or in combination with one or more of the amino acid sequences encoding Pb886, Pb911, Pb975, Pb1912, Pb1221, and BACINT 0076. One composition includes the amino acid sequences encoding Pb0390 and Pb886. Another composition includes the amino acid sequences encoding Pb0390 and Pb911. Another composition includes the amino acid sequences encoding Pb0390 and Pb975. Another composition includes the amino acid sequences encoding Pb0390 and Pb1912. Another composition includes the amino acid sequences encoding Pb0390 and Pb1221. Another composition includes the amino acid sequences encoding Pb0390 and BACINT 0076. Another composition includes the amino acid sequences encoding Pb0390 and Pb398. Another composition includes the amino acid sequences encoding Pb0390 and Pb150. Another composition includes the amino acid sequences encoding Pb0390 and Pb1894. Another composition includes the amino acid sequences encoding Pb0390 and Pb1906. Another composition includes the amino acid sequences encoding Pb0390 and Pb1908. Another composition includes the amino acid sequences encoding Pb0390 and Pb1911. Another composition includes the amino acid sequences encoding Pb0390 and Pb2001. Another composition includes the amino acid sequences encoding Pb0390 and Pb2002. Another composition includes the amino acid sequences encoding Pb0390 and Pb2003. Another composition includes the amino acid sequences encoding Pb0390 and Pb2004. Another composition includes the amino acid sequences encoding Pb0390 and Pb2350.
Compositions may include a transgenic host cell comprising one or more of the amino acid sequences encoding Pb1893, Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350. The one or more polypeptides may be secreted from the transgenic host cell.
Compositions comprising a solution of polypeptides are also provided. Compositions comprising lyophilized polypeptides are provided. Compositions may be stable and suitable for storage over long periods of time. In certain embodiments, the compositions are stable for six months to one year. In other embodiments, the compositions are stable for longer than one year.
Treatment Methods
The above-described 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 Pb1893, Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350. Pb0390 may be used as a substitute for Pb1893. In one embodiment, hemicellulose is treated with Pb1893 in combination with one or more enzymes including Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350. BACINT 0076 may be used as a substitute for Pb1221.
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 Pb1893 or a variant cleaves β-1,4-xylose linkages in the xylose backbone. Treatment of hemicellulose with Pb886 or a variant cleaves arabinofuranose linkages and β-1,4-xylose linkages. Treatment of hemicellulose with Pb911 or a variant cleaves β-1,4-xylose linkages. Treatment of hemicellulose with Pb975 or a variant cleaves β-1,4-xylose linkages. Treatment of hemicellulose with Pb1912 or a variant cleaves glucuronic acid linkages. Treatment of hemicellulose with Pb1221 or a variant cleaves ester linkages. Treatment of hemicellulose with Pb0390 or a variant cleaves β-1,4-xylose linkages in the xylose backbone. Treatment of hemicellulose with BACINT 0076 or a variant cleaves ester linkages. Treatment of hemicellulose with Pb398 cleaves β-1,4-xylose linkages. 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 Pb1893, Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350.
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 Pb1893, Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350. The transgenic host cell may be E. coli or yeast. The transgenic host cell may contain a vector encoding Pb1893, Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, Pb2350, or variants thereof. In some embodiments, the hemicellulose is degraded by treating with Pb1893 or a variant alone, or in combination with one or more of Pb886, Pb911, Pb975, Pb1912, Pb1221, Pb0390, BACINT 0076, Pb398, Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350, 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, 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, willow, aspen, poplar wood, and energy cane. The methods may also be practiced on isolated hemicellulose.
In certain embodiments, enzymes are provided at a concentration of at least 0.5 μM per enzyme for every 15 mg of substrate. In certain embodiments, the substrate consists of 15 mg of hemicellulose.
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 6 and at or between a temperature between about 25 and about 40° C.
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, the enzyme cocktail 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 invention are used to generate biofuels or specialty chemicals. The compositions and methods of the invention 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 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. Hemicellulose-degrading enzymes from the rumen bacteria Prevotella bryantii were purified and characterized for hemicellulose degradation activity. The amino acid and nucleotide sequences for the identified enzymes are provided. Enzyme activity for each enzyme was determined by thin layer chromatography (TLC) or reducing sugar assays.
SUMMARY OF EXAMPLES
Example 1: Endoxylanases Pb1893 and Pb0390/Pb1909
Example 2: Arabinofuranosidase/β-xylosidase Pb886/Pb2351
Example 3: β-xylosidase/β-glucosidase Pb911
Example 4: Glucuronidase Pb1912
Example 5: β-xylosidase/β-glucosidase Pb975
Example 6: Acetyl Xylan Esterases Pb1221 and BACINT 0076
Example 7: Synergistic Activity of Hemicellulase Enzyme Cocktail
Example 8: β-xylosidase Pb398/Pb1917
Example 9: Sequence Alignments
Example 10: Transcriptional Analysis of P. bryantii Grown on Polysaccharide and Monosaccharide Substrates
Example 11: Improvement of Hemicellulase Enzyme Cocktail
Example 1
Endoxylanase Pb1893 (SEQ ID NOs: 1 & 2)
An endoxylanase, Pb1893, was identified in Prevotella bryantii. The enzyme is the gene product of Pb1893, where Pb stands for P. bryantii. 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 Pb1893 protein is 561 amino acids long and has a molecular weight of 62.3 kDa (His-tag+truncated Pb1893 protein). The protein has a unique architecture due to a putative carbohydrate binding module (CBM) inserted within the Glycoside Hydrolase (GH) family 10 catalytic domain (FIG. 2a).
Cloning of Pb1893
The gene for Pb1893 was amplified from Prevotella bryantii B14 genomic DNA by PCR using PrimeSTAR HS DNA polymerase (TaKaRa). The Pb1893 gene was amplified using the following primer set:









Pb1893ForNdeI



(SEQ ID NO: 39)



5′-CATATGGACCAGGATATTCCTGGTTTCACAACGGATGAGC-3′



 



Pb1893RevXhoI



(SEQ ID NO: 40)



5′-CTCGAGTTACTCCTGTTTCAAACCTTCACAGAAGCCTAC-3′






The polymerase chain reaction mixture contained the following:










PCR reaction



















2.5 U/mL PrimeSTAR DNA polymerase
0.5




 19 ng/mL P. bryantii gDNA
1



 10 mM Fw Primer
1



 10 mM Rv Primer
1



2.5 mM dNTP Mixture
4



5× PrimeSTAR Buffer
10



dH2O
32.5



Total
50
mL










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.
90 sec



Last
 4° C.
∞










After the PCR reaction described above, the amplification of Pb1893 gene was confirmed by 1% agarose gel electrophoresis. GoTaq DNA polymerase (Promega) was then added to the reaction mixture to add adenine bases to the 3′ terminus of the amplified gene product to facilitate cloning into a TA cloning vector. The reaction for addition of the nucleotide was as follows:











Reaction
Incubation


















5 U/mL GoTaq DNA polymerase
 1
72° C.
15 min


Reaction Mixture
49


Total
50 mL









After the reaction, the following ligation reaction was carried out to clone the amplified Pb1893 gene into the pGEM-T Easy vector (Promega):
















Ligation

Incubation






















3 U/mL T4 DNA ligase
1

4° C.
O/N



Reaction Mixture
3



2× Rapid Ligation Buffer
5



pGEM-T Easy vector
1



Total
10
mL










The ligation mixtures for Pb1893-pGEM-T Easy were introduced into E. coli JM109 by heat shock method, and the cells were plated on LB-ampicillin-X-gal-IPTG. After overnight incubation at 37° C., two white colonies were selected and used to inoculate 3 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. In order to abolish the NdeI site in the wild-type Pb1893 gene, one nucleotide substitution was introduced (CATATG->CATACG) into the gene in the pGEM-T Easy plasmid by PCR-mediated mutagenesis using the following primer set and the reaction mixture below:









Pb1893mtFor



(SEQ ID NO: 41)



5′-GACGAAATCAATTCATACGGTACGTTGAAG-3′



 



Pb1893mtRev



(SEQ ID NO: 42)



5′-CTTCAACGTACCGTATGAATTGATTTCGTC-3′
















PCR Reaction



















2.5 U/mL PrimeSTAR DNA polymerase
0.5




 10 ng/mL Pb1893-pGEM-T Easy
3



 10 mM Fw Primer
1



 10 mM Rv Primer
1



2.5 mM dNTP Mixture
4



5× PrimeSTAR Buffer
10



dH2O
30.5



Total
50
mL










The PCR cycling that introduced the mutation into the gene for Pb1893 was as follows. The mutation was designed to abolish a restriction site, but maintain the amino acid at that position in the polypeptide.










PCR Protocol




















Denature
98° C.
10 sec
16 Cycles



Anneal
53° C.
 5 sec



Elongate
72° C.
4 min 35 sec



Last
 4° C.
∞










After the PCR reaction described above, the amplification of Pb1893-pGEM-T Easy was confirmed by 1% agarose gel electrophoresis. The restriction enzyme DpnI (NEB) was then added to the reaction mixture to degrade the parental plasmid DNA, which harbored the original gene containing the targeted restriction site (NdeI). The reaction was as follows:
















Reaction

Incubation





















20 U/mL DpnI
 1
37° C.
3 hour



Reaction Mixture
49



Total
50 mL










The DpnI-treated Pb1893-pGEM-T Easy was introduced into E. coli JM109 by the heat shock method, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., six colonies were selected and used to inoculate 3 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 to extract plasmids. The plasmids were then electrophoresed on a 1% agarose gel to check the size of plasmid DNA. Next, the gene was sequenced to ensure that the mutagenesis was successful and that the NdeI site was abolished. The cloned gene was excised by NdeI-XhoI and separated by 1% agarose gel. The reaction to cleave the gene out of the plasmid (pGEMT vector) was as follows:
















Digestion

Incubation






















pDNA (total 2300 ng)
16

37° C.
3 hour



10× NEBuffer 4
2



20 U/mL NdeI
1



20 U/mL XhoI
1



Total
20
mL










After the digestion to remove the gene out of the PGEM-T vector, the gene was purified from an agarose gel by a gel extraction kit (QIAGEN) and inserted by DNA ligation into the corresponding sites of pET28a harboring an ampicillin resistance gene (Novagen). The ligation reaction was as follows:











Ligation
Incubation



















2000 U/mL T4 DNA ligase (NEB)
1

16° C.
O/N


 17 ng/mL digested gene fragment
1.6


 23 ng/mL digested pET28a vector
1.3


10× T4 DNA Ligase Buffer (NEB)
2


dH2O
14.1


Total
20
mL









The ligation mixtures for Pb1893-pET28a 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 0.5 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 Pb1893) was then eluted from the resin with an elution buffer composed of the lysis buffer supplemented with 150 mM imidazole.
The gene product of Pb1893 was expressed in a truncated form. The first 20 amino acids, which represent a signal peptide, were removed. In the native organism, P. bryantii, the signal peptide facilitates transport of the PB1893 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, 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.
The Pb1893 (ENDO-1,4-BETA-XYLANASE A PRECURSOR (EC 3.2.1.8)) amino acid sequence is found in SEQ ID NO: 43. The first 20 amino acids comprise the signal peptide, which was removed. The corresponding nucleotide sequence is found in SEQ ID NO: 44. Thus, the sequence encoding the signal peptide was not present in the gene cloned to make Pb1893.
The procedure of cloning the gene for Pb1893 into the plasmid pET28a led to fusion of the gene to a short nucleotide sequence encoding a peptide that contains six histidines. The short peptide comprises the first 21 amino acids of SEQ ID NO: 46 (pET28a-Pb1893). The corresponding nucleotide sequence of pET28a-Pb1893 is SEQ ID NO: 45.
In the protein sequence SEQ ID NO: 48, the sequence before DQD at residues 22-24 originates from the plasmid. The histidines (6-H) facilitated protein purification as described above.
The PB1893 gene was expressed in E. coli cells, and the protein was purified in a single step, making use of the 6-histidines encoded by the plasmid. FIG. 2b shows an SDS-PAGE of purified Pb1893. The molecular markers are in the lane marked M.
Enzyme Activity
The enzymatic activity of Pb1893 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). 50 μL of sample supernatant (substrate reacted with enzyme) was transferred to a clean 1.5 mL centrifuge tube and 100 μL of ethyl alcohol were added. The tube was 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. A marker mixture was made by combining 0.8 μL of 12.5 mg/mL xylose, 1 μL of 12.5 mg/mL xylobiose, 1 μL of 12.5 mg/mL xylotriose, 1.5 μL of 12.5 mg/mL xylotetraose, and 8.6 μL of ethyl alcohol. All sugars were purchased from Megazyme. The samples and maker mixture were evaporated with a concentrator. 2.5 mL of dH2O were added to the tubes. 0.5 to 1 μL of the sample solution was spotted on a TLC plate. The spots were dried and the TLC plate was developed in a developing tank for 3 to 4 hours. 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 Pb1893 on natural substrates using TLC analysis. Two different hemicellulosic substrates were tested: oat-spelt xylan (OSX) and wheat arabinoxylan (WAX). In each case, in the presence of Pb1893 (+), 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 more the complex substrates (OSX and WAX).
The concentration of glucose equivalents was determined following enzymatic hydrolysis of wheat arabinoxylan (WAX) 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 WAX 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 citrate 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 18 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. 750 μL of pHBAH solution was added to 250 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. 2d shows the enzymatic activity of Pb1893 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 two 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-Layer, M. 1972.). Incubation of enzymes with the substrates led to release of products that were quantified as a concentration of glucose equivalents. Hydrolysis of WAX (wheat arabinoxylan) was higher than hydrolysis of oat-spelt xylan (OSX).
Pb0390(Pb1909)
An additional endoxylanase from Prevotella bryantii, Pb0390 (alternatively named Pb1909), was identified that may be used as a substitute for Pb1893 in any enzyme mixture. The protein was expressed and purified as described above. Purified Pb1909 is shown in FIG. 3a. Activity was determined by TLC and reducing sugar assays performed as described above for Pb1893 (FIGS. 3b and 3c). The protein sequence is provided as SEQ ID NO: 14. The nucleic acid sequence including sequence encoding a predicted signal peptide for Pb0390 glycosyl hydrolase family 10 (PEG1909) is SEQ ID NO: 49. The corresponding amino acid sequence is SEQ ID NO: 50.
Example 2
Arabinofuranosidase/Beta-Xylosidase Pb886 (Alternatively Named Pb2351) (SEQ ID NOs: 3 & 4)
An arabinofuranosidase, Pb886, was identified in Prevotella bryantii. This enzyme was also found to have β-xylosidase activity and thus is a bifunctional enzyme. As shown below, the first enzymatic activity (arabinofuranosidase) was higher than the second activity. Arabinofuranosidases cleave the arabinose side chains that decorate the β-1,4 xylose main chain. Since it was also shown to have beta-xylosidase activity, Pb886 must also attack the short chain xylo-oligosaccharides generated by endoxylanases (e.g., Pb1893) to generate xylose monomers. The protein is 560 amino acids in length and has a molecular weight of 62.9 kDa (His-tag+truncated Pb886 protein). It is a GH 43 arabinoxylan arabinofuranohydrolase.
Cloning of Pb886
The gene for Pb886 was amplified from Prevotella bryantii B14 genomic DNA by PCR using PrimeSTAR HS DNA polymerase (TaKaRa). The Pb886 gene was amplified using the following primer set:







Pb886ForNdeI


(SEQ ID NO: 51)


5′-CATATGCAGGATGCTGTTTTCCAGAATTTTAAGTATACTGG-3′


 


Pb886RevXhoI


(SEQ ID NO: 52)


5′-CTCGAGTTATTTCACAGCATAAAGTCCGATTACCGC-3′






The PCR amplification method used to amplify the gene from the P. bryantii genome was as follows:










PCR mixture



















2.5 U/mL PrimeSTAR DNA polymerase
0.5




 19 ng/mL P. bryantii gDNA
1



 10 mM Fw Primer
1



 10 mM Rv Primer
1



2.5 mM dNTP Mixture
4



5× PrimeSTAR Buffer
10



dH2O
32.5



Total
50
mL




















PCR Protocol




















Denature
98° C.
10 sec
30 Cycles



Anneal
55° C.
 5 sec



Elongate
72° C.
90 sec



Last
 4° C.
∞










After the PCR amplification described above, the product (Pb886 gene) was confirmed by 1% agarose gel electrophoresis. GoTaq DNA polymerase (Promega) was then added to the reaction mixture to add adenine bases to the 3′ terminus of the amplified gene product. The reaction was as follows:











Reaction
Incubation


















5 U/mL GoTaq DNA polymerase
 1
72° C.
15 min


Reaction Mixture
49


Total
50 mL









After the reaction, the PCR product was ligated into the TA-cloning vector pGEM-T Easy (Promega) through the following reaction:
















Ligation

Incubation






















3 U/mL T4 DNA ligase
1

4° C.
O/N



Reaction Mixture (PCR product)
3



2× Rapid Ligation Buffer
5



pGEM-T Easy vector
1



Total
10
mL










The ligation mixture for Pb886-pGEM-T Easy was introduced into E. coli JM109 by heat shock method, and the cells were plated on LB-ampicillin-X-gal-IPTG. After overnight incubation at 37° C., two white colonies were selected and used to inoculate 3 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 from the miniprep were then electrophoresed on a 1% agarose gel to check the size of the plasmid DNA. The plasmids with inserts were sequenced to confirm the integrity of the coding sequence. The cloned gene was excised by NdeI-XhoI using the reaction below and separated by 1% agarose gel.












Digestion
Incubation



















Plasmid DNA (1900 ng) with insert
16

37° C.
3 hour


10x NEBuffer 4
2


20 U/mL NdeI
1


20 U/mL XhoI
1


Total
20
mL









The gene, released from the pGEM-T vector, was purified by a gel extraction kit (QIAGEN) and inserted into the corresponding sites of pET28a (Novagen) through the following reaction.












Ligation
Incubation



















2000 U/mL T4 DNA ligase (NEB)
1

16° C.
O/N


17 ng/mL digested gene fragment
1.6


22 ng/mL digested pET28a vector
1.5


10x T4 DNA Ligase Buffer (NEB)
2


dH2O
13.9


Total
20
mL









The ligation mixture for Pb886-pET28a was introduced into E. coli JM109 by heat shock method, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., one colony was 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 plasmid was then electrophoresed on a 1% agarose gel to confirm the size of plasmid/insert DNA.
SEQ ID NO: 53 contains the amino acid sequence of XYLOSIDASE-ARABINOSIDASE (EC 3.2.1.37(xyl), EC 3.2.1.55(ara)) including a predicted signal peptide in the first 19 residues. The signal peptide sequence was excluded in the cloning. The corresponding nucleotide sequence is SEQ ID NO: 54.
Pb886 was expressed in pET2a with an amino terminal six histidine tag to facilitate protein purification. The amino acid sequence of pET28a-Pb886 is SEQ ID NO: 55. The corresponding nucleotide sequence is SEQ ID NO: 56.
The nucleotide and amino acid sequences of Pb886 together with the N-terminal six histidines to facilitate protein purification are found in SEQ ID NO: 57 (DNA) and SEQ ID NO: 58 (protein).
Purification and Enzyme Activity
Pb886 protein was expressed and purified as described in Example 1. FIG. 4a shows an SDS-PAGE of purified P. bryantii Pb886 protein. FIG. 4b shows the results of a screening assay of Pb886 activity on para-nitrophenyl (pNP) substrates. Higher arabinofuranosidase activity was detected with pNP substrates. There was also a significant activity on xylose linked by β-1,4-linkages.
Enzyme-catalyzed hydrolysis of para-nitrophenyl (pNP)-linked monosaccharide substrates was assayed using a thermostated Synergy II multi-mode microplate reader from BioTek Instruments Inc. (Winooski, Vt.). A library of pNP substrates were screened for activity including: pNP-α-L-arabinopyranoside, pNP-α-L-arabinofuranoside, pNP-β-D-fucopyranoside, pNP-α-L-fucopyranoside, pNP-α-D-galactopyranoside, pNP-β-D-galactopyranoside, pNP-α-D-glucopyranoside, pNP-β-D-glucopyranoside, pNP-β-D-maltopyranoside, pNP-α-D-maltopyranoside, pNP-α-D-mannopyranoside, pNP-β-D-mannopyranoside, pNP-α-L-rhamnopyranoside, pNP-β-D-xylopyranoside, pNP-β-D-cellobioside. The substrates (1 mM) in citrate buffer (100 μL; 50 mM sodium citrate, 150 mM NaCl, pH 5.5) were incubated at 37° C. in the presence or absence of Pb886 (0.2 μM) for 30 min, and the level of pNP release was determined by continuously monitoring the absorbance at 400 nm. The pathlength correction feature of the instrument was employed to convert the absorbance values recorded to correspond to a 1 cm pathlength. The extinction coefficient for pNP at pH 5.5 and a wavelength of 400 nm was measured as 0.673 mM−1cm−1.
FIG. 4c illustrates the kinetics of pNP substrate hydrolysis. The activity of Pb886 was verified with para-nitrophenyl derivatives of xylose and arabinose as indicated in the x-axes. These substrates are artificial substrates for the enzyme. Kinetic studies of Pb886 were performed using a thermostated Synergy II multi-mode microplate reader from BioTek Instruments Inc. (Winooski, Vt.). pNP-β-D-Xylopyranoside (0-46.25 mM) or pNP-α-L-arabinofuranoside (0-25 mM) were incubated in a citrate reaction buffer (100 μL; 50 mM sodium citrate, 150 mM NaCl, pH 5.5) at 37° C. in a 96-well flat bottom microtiter plate, and reactions were initiated by the addition of Pb886 (0.2 μM). Hydrolysis of pNP-β-D-xylopyranoside and pNP-α-L-arabinofuranoside were continuously monitored by recording the UV signal at 400 nm. Initial rate data were then plotted against the substrate concentration, and kinetic values were estimated by applying a nonlinear curve fit using GraphPad Prism v5.02 from GraphPad Software (San Diego, Calif.). The extinction coefficient for para-nitrophenol at pH 5.5 and a wavelength of 400 nm was measured as 0.673 mM−1cm−1.
The activity of Pb886 on β-1,4-linked xylose chains, which mimic more of the natural substrate (xylan), were further demonstrated with thin layer chromatography (TLC) as described in Example 1. Xylose (monomer) was released from short-chain xylo-oligomers (X2 and X3) more effectively than from longer chains (X4, X5, X6). FIG. 4d shows the activity of Pb886 on xylo-oligosaccharide substrates (β-xylosidase activity). FIG. 4e shows arabinofuranosidase activity of Pb886 on wheat arabinoxylan, a natural substrate. To clearly demonstrate that Pb886 released arabinose from a natural substrate such as wheat arabinoxylan (WAX), arabinose alone (A1 in lane 2) was compared to standards based on different xylose chains (lane 1). As shown in FIG. 4e, without enzyme (−), no product was generated (lane 3). In lane 4, which contains the Pb886 enzyme (+), a product with a migration pattern similar to arabinose was released from WAX. These results demonstrate that the enzyme released arabinose from a hemicellulose substrate.
Example 3
Beta-Xylosidase/Beta-Glucosidase Pb911 (SEQ ID NOs: 5 & 6)
A β-xylosidase, Pb911, was identified in P. bryantii. During the degradation of hemicellulose, β-xylosidases convert xylo-oligosaccharides produced by an endoxylanase to their monomeric sugars (xylose). P. bryantii has four genes encoding β-xylosidase-like enzymes. All four genes were cloned and expressed as soluble proteins. As shown below, one of these enzymes, Pb911, was initially screened on pNP substrates. The results suggested that Pb911 releases mostly xylose from a β-1,4-linked pNP and to a lesser extent arabinose from arabinose linked to pNP. The protein is 776 amino acids in length and has a molecular weight of 86.3 kDa (His-tag+truncated Pb911 protein). This protein is a GH 3 protein.
Cloning of Pb911
The gene for Pb911 was amplified and cloned into the Novagen pET-15b vector using the following primer sets Takara PrimeSTAR HS DNA Polymerase:









Pb911ForNdeI



(SEQ ID NO: 59)



5′-GCGCCATATGCAAACTATACTTATTAATCAGCAGG-3′



 



Pb911RevXhoI



(SEQ ID NO: 60)



5′-CGCGCTCGAGTCACTTGATGACTTCAG-3′






Pb911 (Amino Acid Sequence)

SEQ ID NO: 61 contains the protein sequence of ORF00911 xylosidase-arabinosidase (Prevotella bryantii) in the first 19 amino acids that were predicted to constitute a signal peptide. The corresponding nucleotide sequence is SEQ ID NO: 62.
The thymine at base 669 in SEQ ID NO: 63 (Prevotella bryantii B14 ORF0911) was a potential point mutation in the gene, but since it did not change the codon, the gene was expressed without any attempt to correct it. There was a silent mutation introduced at position 221 of the corresponding amino acid sequence (SEQ ID NO: 64). This mutation did not result in a change in the amino acid sequence.
The pET15b expressing Pb911 contained the nucleotide sequence SEQ ID NO: 65 and encoded the amino acid sequence SEQ ID NO: 66. The six histidines at the N-terminus were used to facilitate protein purification.
Purification and Enzyme Activity
Pb911 protein was expressed and purified according to the methods described in Example 1. FIG. 5a shows an SDS-PAGE of purified Pb911. FIG. 5b shows that P. bryantii Pb911 hydrolyzed xylose linked to pNP in beta-1,4 linkages. The activity of Pb911 on pNP substrates was measured according to the methods described in Example 2.
TLC performed as described in Example 1 showed that xylo-oligosaccharides, from disaccharides (two xylose sugars) to hexaose (six xylose monomers in a chain) chains, were converted to the monosaccharide xylose by Pb911 (FIG. 5c). The data suggested that Pb911 can convert xylo-oligosaccharides released by an endoxylanase into xylose.
A coupled enzyme assay was used to measure the kinetics of xylo-oligosaccharide hydrolysis by Pb911 (FIG. 6). To continuously monitor the hydrolysis of xylo-oligosaccharides by Pb911 for subsequent determination of kinetic parameters with these natural substrates, a coupled enzyme assay was employed based upon the xylose assay kit from Megazyme (Bray, Ireland). This assay system includes a xylose mutarotase (XMR) enzyme, which catalyzes the interconversion of the α- and β-anomers of D-xylose, and a β-D-xylose dehydrogenase (β-XDH) enzyme, which couples xylose oxidation to the reduction of β-nicotinamide adenine dinucleotide (NAD+). The NADH production can be monitored continuously using a spectrophotometer tuned to a wavelength of 340 nm. The reactions were prepared in 100 μL final volumes in independent wells of a 96-well microtiter plate. The reaction components included XMR (41 μg/mL, final concentration), β-XDH (1.2 U/mL, final concentration), NAD (2 mM, final concentration), adenosine 5′-triphosphate (24 μM, final concentration), xylo-oligosaccharides (X2-X6) (50 μM-10 mM, final concentrations), and all of these components were diluted in a sodium citrate reaction buffer (50 mM sodium citrate, 150 mM NaCl, pH 5.5). The plates were equilibrated to 37° C., and the reactions were initiated by the addition of Xyl3A (9 nM, final concentration). The level of NADH production was measured by continuously monitoring the absorbance at 340 nm using a Synergy II multimode plate reader from BioTek Instruments Inc. (Winooski, Vt.). The pathlength correction feature of the instrument was employed to convert the absorbance values recorded to correspond to a 1 cm pathlength.
Table 1 shows the kinetic values for the activity of wild type and mutant Pb911 enzymes. Pb911 showed improved steady-state kinetic parameters with pNP-β-D-xylopyranoside as compared to pNP-β-D-glucopyranoside, suggesting that xylose-linked chains may be better substrates for this enzyme. Analysis of mutant Pb911 proteins demonstrated that the glutamate 115 residue of the protein is important for determining substrate specificity.







TABLE 1







Steady state kinetic parameters for wild type and mutant enzymes.










pNP-β-D-xylopyranoside
pNP-β-D-glucopyranoside














kcat
KM
kcat/KM
kcat
KM
kcat/KM



(s−1)
(mM)
(s−1 · mM−1)
(s−1)
(mM)
(s−1 · mM−1)

















Pb911 WT
250 ± 10
8.4 ± 1 
30 ± 4 
19 ± 0.6
22 ± 1
0.86 ± 0.05


Pb911 E115A
 0.35 ± 0.02
28 ± 3
0.013 ± 0.002
0.087 ± 0.005 
39 ± 4
0.0022 ± 0.0003


Pb911 E115D
87 ± 5
39 ± 4
2.2 ± 0.2
16 ± 0.4
17 ± 1
0.94 ± 0.06









Example 4
Glucuronidase Pb1912 (SEQ ID NOs: 9 & 10)
Alpha-glucuronidase removes side chains of glucuronic acids which, if present in hemicellulose or lignocellulose, will limit release of sugars for fermentation. Attempts to express the P. bryantii gene coding for this protein in E. coli were unsuccessful. The entire gene was synthesized based on the codons commonly used by E. coli while ensuring that the polypeptide encoded by the “synthesized gene” was the same as the P. bryantii α-glucuronidase. The production of Pb1912, the P. bryantii glucuronidase, is shown in FIG. 7a. The protein is 625 amino acids in length and has a molecular weight of 71.5 kDa (His-tag+truncated Pb1912 protein).
Cloning of Pb1912
The gene for Pb1912 was artificially synthesized by GenScript Corporation (Piscataway, N.J.) to optimize codon usage. The synthesized gene was cloned into pUC57 vector (hereafter, designated as Pb1912-pUC57). The Pb1912 gene was amplified by PCR using PrimeSTAR HS DNA polymerase (TaKaRa) and subcloned into pET46 Ek/LIC vector using Ek/LIC Cloning Kits (Novagen). The primer sequences are below:









Pb1912For



(SEQ ID NO: 67)



5′-GACGACGACAAGATGGAAGATGGCCATCAGCTG-3′



 



Pb1912Rev



(SEQ ID NO: 68)



5′-GAGGAGAAGCCCGGTTTATTCAATCGGCATTTT-3′
















PCR mixture



















2.5 U/mL PrimeSTAR DNA polymerase
0.5




0.2 ng/mL Pb1912-pUC57
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
mL




















PCR Protocol




















Denature
98° C.
 10 sec




Anneal
55° C.
 5 sec
30 Cycles



Elongate
72° C.
105 sec



Last
 4° C.
∞










After the PCR amplification described above, the amplification of Pb1912 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
mL









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 mL










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 mL










The annealing mixtures for Pb1912-pET46 Ek/LIC were 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.
The first 28 residues of the Pb1912 (ALPHA-GLUCURONIDASE (EC 3.2.1.139) amino acid sequence comprise a predicted signal peptide (SEQ ID NO: 69).
Expression of engineered Pb1912 in pET46EK/LIC led to a protein with an N-terminal six histidine tag to facilitate protein purification. The amino acid sequence of pET46Ek/LIC-Pb1912 is found in SEQ ID NO: 70. The corresponding engineered nucleotide sequence optimized for E. coli is found in SEQ ID NO: 71.
The pET15b expressing the engineered Pb1912 contains following nucleotide sequence in SEQ ID NO: 72 that codes for the corresponding amino acid sequence SEQ ID NO: 73. The six histidines at the N-terminus facilitated protein purification.
Purification and Enzyme Activity
Pb1912 protein was expressed and purified according to the methods described in Example 1. FIG. 7a shows the SDS-PAGE of purified Pb1912. TLC analysis performed as described in Example 1 demonstrated the synergistic activity of Pb1912 and Enzyme Mix 1 (endoxylanase (Pb1893), arabinofuranosidase (Pb886), and beta-xylosidase (Pb911) on the hydrolysis of glucuronic acid (4-O-methyl-D-glucurono-D-xylan) (FIG. 7b). The addition of Pb1912 to the mixture increased the release of glucose equivalents by about 14% (FIG. 7c). In the presence of Enzyme Mix 1, a large amount of xylose (X1) was released, but two products remained near the bottom of the TLC plate. The addition of glucuronidase (Pb1912) resulted in the disappearance of the top product with the concomitant appearance of other products (streak) (FIG. 7b). The data, therefore, suggested that the glucuronidase enhanced hydrolysis of xylo-oligosaccharides into smaller or shorter products. However, another larger product (seen as a spot) was still remaining after the addition of Pb1912. HPLC analysis of products released following the incubation of Pb1912 with aldouronic acid mixtures demonstrated that another P. bryantii protein helped to remove this product. After incubating Pb1912 with aldouronic acids, xylo-oligosaccharides and glucuronic acid were liberated, although there were still some peaks that Pb1912 was not able to degrade (FIG. 7d). The results clearly showed the synergistic action of the glucuronidase with the enzyme mix to improve the release of sugars from a glucuronic acid substrate.
For analysis of aldouronic acid hydrolysis, the enzymes (0.5 μM, final concentration) were incubated with the aldouronic acid mixture from Megazyme (60 μg/mL, final concentration) in citrate buffer (50 mM sodium citrate, 150 mM NaCl, pH 5.5) at 37° C. After 16 hours, 100 μL aliquots were removed, and the reactions were terminated by the addition of 300 μL 0.1 M NaOH. The oligosaccharide composition of these neutral and acidic oligosaccharide mixtures following enzymatic hydrolysis were then assessed by high performance anion exchange chromatography (HPAEC-PAD) with a System Gold® HPLC instrument from Beckman Coulter (Fullerton, Calif.) equipped with a CarboPacTM PA1 guard column (4×50 mm) and a CarboPacTM PA1 analytical column (4×250 mm) from Dionex Corporation (Sunnyvale, Calif.) and a Coulochem® III electrochemical detector from ESA Biosciences (Chelmsford, Mass.). Monomeric xylose (X1) and xylo-oligosaccharides (X2-X6) were used as standards. Oligosaccharides were resolved using a mobile phase of 100 mM NaOH with a linear gradient ending with 125 mM NaOAc over 25 min.
Example 5
β-Xylosidase/β-Glucosidase Pb975 (SEQ ID NOs: 7 & 8)
Another β-xylosidase-like enzyme, Pb975, was identified in P. bryantii. In this example, it is shown that Pb975 has both the capacity to release xylose from β-1,4-linked xylose (β-xylosidase activity) and glucose from β-1,4-linked glucose (β-glucosidase activity) units. The gene product of Pb975 is different from Pb911 because Pb975 was found to contain an insertion sequence commonly referred to as the protective antigen, or PA14. The domain architectures of Pb911 and Pb975 are shown in FIG. 8a. It has been predicted that these insertion sequences, which assume a β-barrel shape, engage in binding rather than in a catalytic role (Ridgen et al. 2004. Trends in Biochemical Sciences 29:335-339). Enzymatic activities were demonstrated for Pb975 using pNP (artificial) substrates. Furthermore, adding Pb975 to the enzyme mix, together with glucuronidase, resulted in the removal of the final large product that remained, even upon addition of glucuronidase.
Cloning of Pb975
The gene for Pb975 was amplified and cloned into the Novagen pET-15b vector using the following primer sets with Takara PrimeSTAR HS DNA Polymerase:









Pb975For#2



(SEQ ID NO: 74)



5′-GCGCCATATGATGAAAAGTAAACAACTAATAAC-3′



 



Pb975PADnRev



(SEQ ID NO: 75)



5′-CGCGCTCGAGTTATTTTAGGTAAATAATTAATTTTTTC-3′






The gene product contained an N-terminal predicted signal peptide as underlined in the sequence below. The signal peptide was eliminated from the protein during expression. The protein is 857 amino acids in length with a molecular weight of 95.7 kDa.
The following conditions were used for amplification of the preceding genes by polymerase chain reaction using PrimeSTAR HS DNA Polymerase from Takara:











PrimeSTAR











# of rxns
1

PCR Protocol















Buffer
10

Denature
98° C.
10 sec


PrimeSTAR
0.5
30 Cycles
Anneal
55° C.
15 sec


2.5 mM dNTPs
4

Elongate
72° C.
 3 min


Genomic DNA
1

Last
 4° C.
∞


Primers (5 μM Mix)
2.5


H2O
32


Total
50









Five microliters of the PCR product was then electrophoresed on a 1% agarose gel, and the bands were stained with ethidium bromide and visualized with a UV transilluminator. Next, the PCR products were purified using a PCR purification kit from QIAGEN and restriction enzyme digestions were prepared for the two PCR products as follows:














Digestion













# of rxns
1

Incubation
















10X Buffer 4
2
3 h
37° C.



ORF0975 PCR
10



Product



NdeI
1



XhoI
1



H2O
6



Total
20










The following day, the two enzyme digestions were purified using a PCR purification kit from QIAGEN, and the digested product was eluted in 20 uL of water. The following ligation was prepared with previously digested pET-15b vector:














Ligation













# of rxns
1

Incubation
















10X Buffer
1
16 h
4° C.



pET-15b
2



T4 Ligase
1



PCR Product
6



Total
10










The ligation mixtures for ORF0975-pET15b were introduced into E. coli DH5 alpha by electroporation, and the cells were plated on LB-ampicillin. After overnight incubation at 37° C., approximately 50 colonies were identified on each plate. Five colonies were selected and used to inoculate 3 mL cultures of LB-amp, and the cultures were grown at 37° C. with vigorous aeration for 8 hours. Minipreps were made of the cell cultures. The plasmids were then electrophoresed on a 1% agarose gel and stained with ethidium bromide. Three plasmids were sequenced to confirm the integrity of inserts (Pb975). The correct plasmid was used for gene expression.
The amino acid sequence of ORF00975 xylosidase-arabinosidase (Prevotella bryantii) including the predicted signal peptide is SEQ ID NO: 76. The corresponding nucleotide sequence is SEQ ID NO: 77.
Pb975 (Amino Acid Sequence)
The sequence of the expressed Pb975 protein is found in SEQ ID NO: 78 (Prevotella bryantii B14 ORF0975). The N-terminal tag contains a 6-histidine tag to facilitate protein purification. The corresponding nucleotide sequence is SEQ ID NO: 79.
The pET15b expressing Pb975 contained the nucleotide sequence in SEQ ID NO: 80 that encodes for the corresponding and amino acid sequence SEQ ID NO: 81. The six histidines to facilitate protein purification are underlined.
Purification and Enzyme Activity
Pb975 protein was expressed and purified as described in Example 1. FIG. 8b shows an SDS-PAGE of purified Pb975. FIG. 8c shows hydrolysis of pNP-substrates by Pb975. FIG. 8d shows the synergistic effect of Pb975 when combined with Enzyme Mix 1 and Pb1912. Pb975 helped to hydrolyze the remaining product shown in lane 3. Addition of Pb975 to the enzyme mix (lane 4) led to hydrolysis of the large product, close to the bottom of the plate, in lane 3. A concomitant increase in the amount of xylose (X1) released was observed in lane 4. FIG. 8e shows the synergistic effect of Pb975 in the hydrolysis of aldouronic acids. Pb975 helped to hydrolyze several peaks that were not hydrolyzed by Pb911 and Pb1912. The addition of Pb975 to the mixture increased the release of glucose equivalents by about 10% (FIG. 8d). Experiments were carried out as described in previous examples.
Example 6
Acetyl Xylan Esterase Pb1221 (SEQ ID NOs: 11 & 12)
An acetyl xylan esterase, Pb1221, was identified in P. bryantii. Hemicellulose usually contains acetyl groups as side chains. The linkage of the acetyl groups to the main chain is through ester bonds. The side-chain acetyl groups may inhibit hydrolysis of the main chain in hemicellulose by influencing the enzyme/substrate interaction at the active site. The side-chain acetyl groups can be cleaved by use of an enzyme called acetyl xylan esterase. As described below, Pb1221, which is one of several esterases in P. bryantii, was demonstrated to exhibit acetyl xylan esterase activity.
Cloning of Pb1221
The gene for the acetyl xylan esterase was amplified using the two primers shown below (PEG1221F and PEG1221R).









PEG1221F (Forward PCR primer)



(SEQ ID NO: 82)



5′-CATATGAAGACGACTATTGATGAACATTGGGTAGG-3′



 



PEG1221R (Reverse PCR primer)



(SEQ ID NO: 83)



5′-CTCGAGCTATCGCAGAATTTCAGCAGCATACTGTC-3′






The forward primer PEG1221F was designed to remove the signal peptide from the protein. The PCR product was initially cloned into the TA-cloning vector, pGEM-T, and sequenced to confirm the integrity of the coding sequence. Since the primers incorporated NdeI and XhoI sites, the gene was released from the TA-cloning plasmid and sub-cloned into a pET28a plasmid that had been digested with the same restriction enzymes (NdeI/XhoI). The product was transformed into E. coli JM109, and the cells were plated onto LB plates supplemented with ampicillin at 100 μg/mL and incubated at 37° C. overnight. The next day, colonies were picked and grown in LB broth supplemented with ampicillin (100 μg/mL) and cultured to saturation. Plasmids were then extracted (QIAGEN), and the inserts were sequenced to confirm the integrity of the esterase coding sequence. The NdeI site placed the gene in frame with the 6-Histidine tag encoded by the plasmid. Therefore, when the gene was expressed, the product was a N-terminally His-tagged protein.
For expression of the gene, the pET2a plasmid containing the gene was transformed into E. coli BL21 codon plus DE3 RIL, and the cells were plated onto LB plates supplemented with ampicillin and chloramphenicol at 100 μg/mL and 50 μg/mL, respectively, and incubated at 37° C. overnight. A single colony was picked and cultured (37° C.) in LB broth supplemented with the two antibiotics at the concentrations stated above until the O.D. at 600 nm reached 0.3. Gene expression was induced by adding 0.1 mM IPTG to the cell culture. The temperature was dropped to 16° C. and culturing was continued overnight. The cell pellet was collected through centrifugation, and proteins were released from the cells through lysis with a French press. The protein was purified through the one-step protein purification method described in Example 1 using a cobalt-charged affinity resin (TALON affinity resin, CLONTECH).
The first 20 residues of SEQ ID NO: 85 (fig|666666.450.peg.1221 [Prevotella bryantii B14] [putative esterase]) code for a putative signal peptide. This sequence was deleted during expression. The corresponding nucleotide sequence is SEQ ID NO: 84.
The nucleotide sequence of Pb1221 in PET-28a is found in SEQ ID NO: 86. An N-terminal tag containing six histidines was used to facilitate protein purification. The corresponding amino acid sequence is SEQ ID NO: 87.
The pET28a plasmid expressing Pb1221 contains the nucleotide sequence SEQ ID NO: 88 that encodes for the corresponding amino acid sequence SEQ ID NO: 89. Six histidines at the N-terminus were used to facilitate protein purification.
Purification and Enzyme Activity
Pb1221 was expressed and purified according to the methods described in Example 1. FIG. 9a shows an SDS-PAGE of purified Pb1221. Steady-state kinetic data for Pb1221 was determined at pH 7.0 and 40° C. (FIG. 9b). Kinetic properties of Pb1221 with 1-naphtyl acetate, 1-naphtyl phosphate and 1-naphtyl propionate as substrates were determined at 40° C. and pH 7.0 (NaH2PO4—Na2HPO4, 50 mM). The substrate concentrations ranged from 10 to 300 μM. The reactions were initiated by the addition of Pb1221 (0.5 μM, final concentration) and performed for 10 minutes at a final volume of 200 μL. The reaction was stopped by addition of 150 μL of Fast Garnet GBC (7 mM) and lauryl sulfate (10%, w/v) and incubated for 15 min at room temperature. The products were then determined by absorbance at 560 nm with 1-naphthol as a standard.
BACINT 0076 (Alternatively Named Bi4505)
A putative xylanase-esterase from Bacteroides intestinalis that can substitute for Pb1221 in hemicellulose enzyme-degrading mixtures was identified. The following primers were used to clone the gene (the nucleotide sequence in bold was necessary to allow cloning into pET46 as the expression vector)]:









BACINT 0076For



(SEQ ID NO: 90)



5′-GACGACGACAAGATGCAAAGCGGCGAAACAG-3′



 



BACINT 0076Rev



(SEQ ID NO: 91)



5′-GAGGAGAAGCCCGGTTATTTGAAAAGCAACTGTG-3′






SEQ ID NO: 92 contains amino acid sequence of BACINT 0076 [Bacteroides intestinalis DSM 17393] [xylanase-esterase]. The first 32 residues represent the predicted signal peptide that was removed during cloning. SEQ ID NO: 93 contains the corresponding nucleotide sequence.
Purification and Enzyme Activity
BACINT 0076 was expressed and purified according to the methods described in Example 1 (FIG. 10a). Xylanase activity of BACINT 0076 was determined to be greater with WAX substrate compared to OSX (FIG. 10b). The specific hydrolysis activity of the enzyme on pNP-β-D-cellobioside as well as the hydrolysis kinetics of other pNP substrates were also determined (FIG. 10c-g). Experiments were performed as described in Examples 1 and 2.
Example 7
Synergistic Activity of Hemicellulase Enzyme Cocktail
The enzymes described in Examples 1-6 were combined into a hemicellulase enzyme cocktail. The synergistic activities of the enzymes in the hemicellulase cocktail are illustrated in FIG. 11. The individual enzymes and their mixtures were reacted with either wheat arabinoxylan (WAX) or oat-spelt xylan (OSX) in 50 mM sodium citrate buffer pH 5.5. “All” includes each of the enzymes in the mixture at 0.5 μM concentration: Pb1893 (endoxylanase), Pb911 (β-xylosidase), Pb975 (β-xylosidase/β-glucosidase), Pb886 (arabinofuranosidase/β-xylosidase), Pb1912 (glucuronidase), and Pb1221 (acetyl xylan esterase).
The addition of higher amounts of wheat arabinoxylan substrate led to an increased release of sugar. The enzyme mixture (ALL, with each enzyme at 0.5 μM) was tested to determine if adding more substrate to the reaction mixture would lead to more or less products released (FIG. 12). Wheat arabinoxylan (WAX) was added to the reaction in amounts ranging from 2.5 milligrams to 40 milligrams. The amount of product released was determined as glucose equivalents according to the methods described in Example 1 and is presented as a histogram and line graph (FIG. 12a). TLC was used to visualize the contents of the released products (FIG. 12b). As more substrate was added, more products were released. In each case, most of the products were the five carbon sugars xylose and arabinose. Table 2 contains the calculated amount of xylose and arabinose released. Under these conditions, a substrate concentration of about 30 mg/reaction was optimal since at that concentration almost 90% of substrate was converted to fermentable sugars. The amount of enzyme mixture can be increased to increase product yield.







TABLE 2







Xylose and arabinose released with increasing amount of substrate









WAX (mg)
















2.5
5
7.5
10
15
20
30
40



















% degradation
91.7
98.9
97.4
99.7
91.7
90.4
88.5
80.1


total sugar
2.3
4.9
7.3
10.0
13.8
18.1
26.6
32.0


(mg)


Xylose* (mg)
1.4
2.9
4.3
5.9
8.1
10.7
15.7
18.9


Arabinose*
0.9
2.0
3.0
4.1
5.6
7.4
10.9
13.1


(mg)





*Ara:Xyl = 41:59






The optimal pH and reaction temperature were also investigated. The enzyme mixture (All, with each enzyme at 0.5 μM) was reacted with WAX at 37° C. overnight in 50 mM sodium citrate at various pHs (FIG. 13). To determine optimum temperature, the enzyme mixture was reacted overnight with WAX (5 mg per tube) in 50 mM sodium citrate (pH 5.5) at various temperatures (FIG. 13).
Example 8
Beta-Xylosidase Pb398 (SEQ ID NOs: 17 & 18)
An additional enzyme that may be used in the enzyme cocktail is a β-xylosidase. The xylo-oligosaccharides that are produced by an endoxylanase (e.g., Pb1893) must be converted to the monomeric sugar, xylose. This process can be achieved with a β-xylosidase. Prevotella bryantii has four genes encoding β-xylosidase-like enzymes. All four genes were cloned and expressed as soluble proteins. As described below, the enzyme Pb398 (alternatively named Pb1917) was initially screened on pNP substrates. The results suggested that Pb398 releases mostly xylose from a β-linked pNP. The protein is 878 amino acids in length and has a molecular weight of 98.6 kDa (His-tag+truncated Pb398 protein). This protein is a GH 3 protein. Its domain architecture is shown in FIG. 15a. 
Cloning of Pb398
The Pb398 gene was amplified by PCR using PrimeSTAR HS DNA polymerase (TaKaRa) and sub-cloned into pET46 Ek/LIC vector using Ek/LIC Cloning Kits (Novagen). The primer sequences and PCR protocol are below:









Pb398For



(SEQ ID NO: 94)



5′-GACGACGACAAGATGCTCATCTGCGCTGCTGAAAAG-3′



 



Pb398Rev



(SEQ ID NO: 95)



5′-GAGGAGAAGCCCGGTTAATTTAACGTATAATGTATCTG-3′
















PCR mixture



















2.5 U/mL PrimeSTAR DNA polymerase
0.5




Genomic DNA
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
mL




















PCR Protocol




















Denature
98° C.
 10 sec




Anneal
55° C.
 5 sec
30 Cycles



Elongate
72° C.
105 sec



Last
 4° C.
∞










After the PCR amplification described above, the amplification of Pb398 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
mL









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 mL










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 mL










The annealing mixtures for Pb398-pET46 Ek/LIC were 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 confirmed by nucleotide sequencing.
Pb398 (Amino Acid Sequence)
The first 22 amino acids of SEQ ID NO: 96 (Prevotella bryantii ORF00398 xylosidase) were predicted to constitute a signal peptide. SEQ ID NO: 97 contains the corresponding nucleotide sequence.
The cytosine at bases 2232 and 2436 of SEQ ID NO: 98 was a potential point mutation in the gene, but since it did not change the codon, the gene was expressed without any attempt to correct it. A silent mutation was introduced at codon positions 744 and 812 of SEQ ID NO: 99. This mutation did not result in a change in the amino acid sequence.
The pET46b vector expressing Pb398 contained the nucleotide sequence SEQ ID NO: 100 and encodes the amino acid sequence SEQ ID NO: 101. The N-terminal six histidines were used to facilitate protein purification.
Purification and Enzyme Activity
Pb398 was expressed and purified according to the methods described in Example 1. FIG. 15b shows an SDS-PAGE of purified Pb398. FIG. 15c shows the results of enzymatic activity assays performed with Pb398 on pNP substrates according to the methods described in Example 2. Pb398 hydrolyzed xylose linked to pNP in a beta linkage.
Example 9
Sequence Alignments
Sequence alignments were performed with proteins from the Genbank database. Alignments were carried out with the GenBank default settings.
Pb1893 and Prevotella bryantii XynC (GenBank accession No. CAB01855) were found to share 99% identity.
Pb0390 and Prevotella bryantii endo-1,4-beta xylanase (GenBank accession No. P48789) were found to share 99% identity.
Pb886 and a Prevotella copri hypothetical protein (GenBank accession No. EEF20542) were found to share 66% identity.
Pb911 and a Prevotella copri hypothetical protein (GenBank accession No. ED052403) were found to share 76% identity.
Pb1912 and a Prevotella copri DSM 18205 hypothetical protein (GenBank accession No. ZP03458538) were found to share 60% identity.
Pb975 and a Bacteroides cellulosilyticus hypothetical protein (GenBank accession No. ZP03677923) were found to share 53% identity.
Pb1221 and a Prevotella copri DSM18205 hypothetical protein (EEF18747) were found to share 67% identity.
Bacteroides intestinalis hypothetical protein was found to share 100% identity with itself (GenBank accession No. EDV07678), 97% identity with Bacteroides cellulosilyticus DSM 14838 hypothetical protein (GenBank accession No. EEF91240), and 92% identity with Bacteroides eggerthii DSM 20697 hypothetical protein (EEC53451).
Example 10
Transcriptional Analysis of Prevotella bryantii B14 Grown on Polysaccharide and Monosaccharide Substrates
Prevotella bryantii B14 was grown anaerobically in a defined medium previously described by Griswold and Mackie (Griswold, K. E. and Mackie, R. I. 1997. Journal of Dairy Science. 80(1): 167-175.). Media preparations with two different carbohydrate sources at 0.15% w/v final concentrations were used for comparison of gene expression profiles. For one media preparation, medium viscosity wheat arabinoxylan was used from Megazyme (Bray, Ireland). This polymeric xylan substrate contains only xylose and arabinose sugars in a proportion of Ara:Xyl=41:59. In the second medium, the soluble sugars, xylose, and arabinose were used in the same proportion as that found in the polymeric wheat arabinoxylan substrate. Thus, comparison of growth patterns and gene expression profiles between the two media preparations is directly related to depolymerization of the xylan growth substrate as the energetic potential in both media preparations is equal.
Triplicate 30 mL cultures of Prevotella bryantii B14 were grown in each of the aforementioned media preparations to an optical density of 0.2, and then the cells were mixed with two volumes of Bacteria RNAprotect from QIAGEN (Valencia, Calif.). The bacteria were harvested by centrifugation. Total bacterial RNA was purified using an RNeasy Mini kit from QIAGEN with an optional on column DNase digestion and stored at −80° C. until used for microarray or cDNA sequencing analysis.
Custom 4×72 k microarray gene chips were designed and printed by Roche NimbleGen (Madison, Wis.) including 9 oligonucleotide probes in triplicate for each of the 2589 putative coding sequences within the Prevotella bryantii B14 genome. Total bacterial RNA was converted to double stranded cDNA using a superscript double stranded cDNA synthesis kit from Invitrogen (Carlsbad, Calif.). The cDNA was labeled with Cy3 dye by using a one-color labeling kit from Roche NimbleGen. The labeled oligonucleotides were hybridized with the microarray slides for 16 hours at 42° C. The slides were then washed and then scanned with a microarray scanner from Agilent (Santa Clara, Calif.). The high resolution .tiff images were then analyzed with NimbleScan software, and data analysis was performed using CLC Genomics Workbench version 3 from CLC bio (Cambridge, Mass.).
For cDNA sequencing, 1 μg of total bacterial RNA was depleted of bacterial ribosomal RNA using a RiboMinus kit from Invitrogen. Library preparation was then performed using reagents and protocols from Illumina (San Diego, Calif.), and the cDNA was sequenced using an Illumina genome analyzer. Two RNA samples were used from each growth medium, and each sample was run in an independent lane on the genome analyzer instrument. The cDNA sequencing data was then assembled onto the P. bryantii B14 reference genome using CLC Genomics Workbench version 3.
The microarray gene expression data is shown in FIG. 16. This data shows that a subset of genes was overexpressed by Prevotella bryantii B14 when grown on polymeric wheat arabinoxylan relative to the soluble monosaccharides, xylose and arabinose. Among the genes that were upregulated on wheat arabinoxylan compared to xylose/arabinose are Pb1893, Pb1909 (Pb0390), Pb1912, Pb1917 (Pb398), and Pb2351 (Pb886). Our biochemical data suggested that these enzymes are involved in deconstruction of polymeric xylan substrates to their constituent monosaccharides (FIGS. 2, 3, 4, 7, and 15), and these results provide validation that the microarray experiment identified genes that are involved in depolymerization of hemicellulosic polysaccharides.
Additional genes that were overexpressed in the experiment were not biochemically characterized in our studies (Pb150, Pb1894, Pb1906, Pb1908, Pb1911, Pb2001, Pb2002, Pb2003, Pb2004, and Pb2350). Although the biochemical function of these genes is currently unknown, the fact that they were overexpressed under these experimental conditions indicated that these genes and their corresponding gene products are important for the depolymerization of hemicellulose. Such genes can be cloned, and the proteins expressed for use in enzyme mixtures.
The RNAseq analysis of transcript structure is shown in FIG. 17. Many bacteria coordinate the expression of genes that have related functions by expressing multiple genes from a polycistronic mRNA molecule (i.e., an operon). The RNAseq analysis showed that many of the genes that are overexpressed by Prevotella bryantii B14 during growth on polymeric wheat arabinoxylan relative to the constituent monosaccharides arabinose and xylose are arranged in operons. Specifically, Pb1893, Pb1894, Pb1895, Pb1896, and Pb1897 were found to be co-transcribed on a single messenger RNA (FIG. 17). In addition, Pb1911, Pb1910, Pb1909, and Pb1908 were also found to be co-transcribed on a single messenger RNA molecule (FIG. 17). These observations indicated that the aforementioned genes are very likely involved in the depolymerization of hemicellulose by Prevotella bryantii B14. These two regions of the chromosome for P. bryantii thus represent two xylan utilization systems (XUS) as indicated in FIG. 17.
The transcriptomic studies described herein indicated that the following additional genes find use in depolymerizing hemicellulose: Pb150 (SEQ ID NOs: 19 and 20), Pb1894 (SEQ ID NOs: 21 and 22), Pb1906 (SEQ ID NOs: 23 and 24), Pb1908 (SEQ ID NOs: 25 and 26), Pb1911 (SEQ ID NOs: 27 and 28), Pb2001 (SEQ ID NOs: 29 and 30), Pb2002 (SEQ ID NOs: 31 and 32), Pb2003 (SEQ ID NOs: 33 and 34), Pb2004 (SEQ ID NOs: 35 and 36), and Pb2350 (SEQ ID NOs: 37 and 38). These proteins can be used alone or in combination with the other proteins described herein.
Example 11
Improvement of Hemicellulase Enzyme Cocktail
Eight genes that were upregulated in the microarray and RNAseq experiments when P. bryantii cells were grown on arabinoxylan (Pb1909/Pb390, Pb2350, Pb1908, Pb150, Pb1917/Pb398, Pb2003, Pb2002, and Pb2001) were expressed and tested together with the six proteins (Pb1893, Pb911, Pb975, Pb886, Pb1912, Pb1221) originally constituting the mesophilic hemicellulase enzyme mix. Supplementation of the hemicellulase mix with the eight new proteins led to a significant release of reducing sugars on oat-spelt xylan and birch wood xylan (FIG. 18).
Each enzyme from the group of eight enzymes was tested individually with the original mixture of six hemicellulases. These experiments demonstrated that Pb1909 and Pb150 were responsible for the significant effects observed from the addition of the group of eight new enzymes (FIG. 18a and b). Therefore, adding Pb1909 and Pb150 to the original mix of six enzymes will improve release of sugars from hemicellulose.

## Claims

1. An isolated cDNA comprising a nucleotide sequence, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, and 13.

2. A vector comprising the isolated cDNA of claim 1.

3. A composition comprising the isolated cDNA of claim 1.

4. The composition of claim 3, wherein the composition further comprises the nucleotide sequence of SEQ ID NO: 1.

5. 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 an enzyme selected from of the group consisting of the enzyme comprising the amino acid sequence of SEQ ID NO: 4, the enzyme comprising the amino acid sequence of SEQ ID NO: 6, the enzyme comprising the amino acid sequence of SEQ ID NO: 8, the enzyme comprising the amino acid sequence of SEQ ID NO: 10, the enzyme comprising the amino acid sequence of SEQ ID NO: 12, and the enzyme comprising the amino acid sequence of SEQ ID NO: 14, wherein said treating cleaves said one or more functional groups from said xylose backbone to form cleaved hemicellulose.

6. The method of claim 5, wherein the enzyme is secreted by a transgenic E. coli or yeast.

7. The method of claim 5, comprising the further step of:
c) treating said cleaved hemicellulose with a second enzyme, wherein said second enzyme comprises the amino acid sequence of SEQ ID NO: 2 and cleaves said β-1,4-linkages in said xylose backbone to produce xylose subunits,
wherein said treating results in the degradation of at least 90% of said hemicellulose into said functional groups and said xylose subunits.

8. The method of claim 7, wherein said degradation of said hemicellulose is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complete.

9. The method of claim 7, wherein the second enzyme is secreted by a transgenic E. coli or yeast.

10. The method of claim 5, wherein said one or more functional groups are selected from the group consisting of arabinose, glucuronyl, and acetyl.

11. The method of claim 5, wherein said plant material is selected from the group consisting of Miscanthus, switchgrass, cord grass, rye grass, reed canary 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, willow, aspen, poplar wood, and energy cane.

12. The method of claim 5, wherein said treating is conducted at a pH between 5 and 6.

13. The method of claim 5, wherein said treating is conducted at a temperature between 25 and 40° C.