# Patent

## Title
Enzymes for improved biomass conversion

## Abstract

Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/671,208, filed Jul. 13, 2012, the contents of which are incorporated by reference in their entirety.


CONTRACTUAL ORIGIN
The United States Government has rights in this invention under Contract No. DE-AC36-08GO28308 between the United States Department of Energy and Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory.


REFERENCE TO SEQUENCE LISTING
This application contains a Sequence Listing submitted as an electronic text file entitled “12-28_ST25.txt,” having a size in bytes of 64 kb and created on Jul. 12, 2013. Pursuant to 37 CFR §1.52(e)(5), the information contained in the above electronic file is hereby incorporated by reference in its entirety.
BACKGROUND
Lignocellulosic biomass is an abundant source of fermentable sugars, and biofuels derived from these renewable sources represent one of the best alternatives to petroleum-based fuels. Efficient conversion of lignocellulosic biomass, however, remains a challenge due to its inherent recalcitrance. Given the current state of technology of simultaneous saccharification and fermentation (SFF) and the commercial enzyme cocktails available, various chemical and thermal pretreatment steps are required to achieve meaningful conversation of biomass. Another alternative seen as viable and cost competitive for the future is consolidated bioprocessing (CBP), in which case saccharolytic enzyme are produced by the CBP organisms, which also ferment sugars released from biomass to the end product.
In nature, most cellulolytic organisms are of two types: those with non-complexed cellulases, xylanases, and hemicellulases produced by aerobic fungi and most bacteria; and those where cellulases, xylanases, and hemicellulases are complexed on a protein scaffold. This latter case is known only for a few anaerobic bacteria and fungi. In both cases, the enzymes secreted have a wide range of complexity but are mostly equipped with a single catalytic domain. An alternate enzymatic system, midway between the two previous paradigms, is one where the most abundant enzymes secreted are not only multi-modular but possess more than one catalytic domain. This strategy could present several advantages; it allows the synergistic effects between several catalytic domains usually found in cellulosomal systems but also lessen problems that cellulosomes may encounter due to their size.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.
SUMMARY
The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods that are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.
Exemplary embodiments provide methods for degrading cellulose or lignocellulosic biomass by contacting a cellulose containing material or lignocellulosic biomass with an enzyme cocktail comprising a thermostable enzyme comprising a GH9 domain and a GH48 domain and a thermostable β-glucosidase.
In certain embodiments, the thermostable enzyme comprising a GH9 domain and a GH48 domain is from a bacterium of the genus Caldicellulosiruptor, such as Caldicellulosiruptor bescii CelA.
In some embodiments, the thermostable β-glucosidase is from a bacterium of the genus Thermotoga, such as Thermotoga maritima. 
In further embodiments, the enzyme cocktail further comprises a thermostable endoglucanase, which may be from a bacterium of the genus Acidothermus, such as Acidothermus cellulolyticus. One example is Acidothermus cellulolyticus E1.
Also provided are enzyme cocktails comprising Caldicellulosiruptor bescii CelA, a thermostable β-glucosidase, such as a β-glucosidase from the bacterium Thermotoga maritima, and a thermostable endoglucanase, such as Acidothermus cellulolyticus E1.
Further provided are methods for producing a biofuel from lignocellulosic biomass by contacting the lignocellulosic biomass with an enzyme cocktail described herein and converting the sugars to a biofuel by fermentation.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.



BRIEF DESCRIPTION OF THE DRAWINGS
Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.
FIG. 1 shows the nucleotide (A; SEQ ID NO:1) and amino acid (B; SEQ ID NO:2) sequences for CelA from Caldicellulosiruptor bescii. Locations of the GH9 and GH48 domains are indicated in bold and underline, respectively.
FIG. 2 shows the nucleotide (A; SEQ ID NO:3) and amino acid (B; SEQ ID NO:4) sequences for β-glucosidase from Thermotoga maritima. 
FIG. 3 shows the nucleotide (A; SEQ ID NO:5) and amino acid (B; SEQ ID NO:6) sequences for E1 from A. cellulolyticus. 
FIG. 4 shows the avicel conversion by CelA at three different temperatures compared to CBH1/E1 at the same loading (15 mg/g).
FIG. 5 shows the glucan conversion by CelA and C. bescii culture broth on switchgrass substrates (A) or corn stover substrates (B) subjected to various pretreatments.
FIG. 6 shows the conversion of xylan present in native switchgrass by CelA.
FIG. 7 shows a comparison of avicel conversion by CelA alone or in combination with β-glucosidase. Loadings were 15 mg/g CelA and 14 mg/g CelA 1 mg/g+β-glucosidase, respectively.
FIG. 8 shows a comparison of dilute acid pretreated corn stover conversion by CelA alone or in combination with β-glucosidase. Loadings were 15 mg/g CelA and 14 mg/g CelA+1 mg/g β-glucosidase, respectively.
FIG. 9 shows a comparison of avicel conversion by CelA alone or in combination with E1. Loadings were 15 mg/g CelA and 11 mg/g CelA 4 mg/g+E1, respectively.
FIG. 10 shows a comparison of dilute acid pretreated corn stover conversion by CelA alone or in combination with E1. Loadings were 15 mg/g CelA and 11 mg/g CelA 4 mg/g+E1, respectively.
FIG. 11 shows a comparison of avicel conversion by CelA alone or in combination with β-glucosidase and E1. Loadings were 15 mg/g CelA and 10 mg/g CelA 1 mg/g+β-glucosidase+4 mg/g E1, respectively.
FIG. 12 shows a comparison of dilute acid pretreated corn stover conversion by CelA alone or in combination with β-glucosidase and E1. Loadings were 15 mg/g CelA and 10 mg/g CelA 1 mg/g+β-glucosidase+4 mg/g E1, respectively.
FIG. 13 shows a comparison of de-acetylated, dilute acid pretreated corn stover conversion by a CelA mixture (17 mg/g CelA; 2 mg/g E1; 1 mg/g T. maratima β-glucosidase) or a commercial enzyme preparation (Ctec2).
FIG. 14 shows a comparison of de-acetylated, dilute acid pretreated corn stover conversion by a CelA mixture (17 mg/g CelA; 2 mg/g E1; 1 mg/g T. maratima β-glucosidase) or a commercial enzyme preparation (Ctec2).
FIG. 15 shows a comparison of de-acetylated, dilute acid pretreated corn stover conversion by a CelA mixture (17 mg/g CelA; 2 mg/g E1; 1 mg/g T. maratima β-glucosidase) with or without a reducing agent (1 mM cysteine).



DETAILED DESCRIPTION
Disclosed herein are enzymes useful for the hydrolysis of cellulose and the conversion of biomass. While single enzymes disclosed herein digest cellulose and biomass, various combinations of the enzymes show synergistic activities on the substrates. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.
Enzymatic conversion of biomass is currently performed using mixtures of mesophillic enzymes derived from fungi such as T. reesei. These mixtures utilize GH 6 and 7 cellulases to perform most of the cellulose hydrolysis work. An alternative approach is to utilize enzymes from hyperthermophillic bacterial organisms that contain no GH 7 enzymes for either simultaneous saccharification and fermentation (SSF) or single step direct microbial conversion of biomass. These hyperthermophillic systems utilize a combination of GH 9 and GH 48 enzymes to deconstruct cellulose and can operate at extremely high temperatures.
One such system is the cellulase system from Caldicellulosiruptor bescii, which includes the multidomain (GH 9/GH 48) enzyme CelA. As discussed in detail below, the addition of a thermostable beta glucosidase (β-glucosidase) (e.g., from Thermotoga maritima) and/or a thermostable endoglucanase (e.g., from Acidothermus cellulolyticus) to purified enzyme preparations of CelA or to C. bescii culture broths synergistically improves digestion of cellulose and biomass. The addition of these enzymes to an existing enzyme cocktail or a host organism used to produce an enzyme mixture may also significantly improve the performance of these enzyme mixtures, thus improving overall conversion yields, lowering the total enzyme concentrations required to reach a specified level of conversion or reducing the total time required to reach a specified level of conversion.
Without being bound by any particular theory, thermostable endoglucanases and beta-glucosidases may synergistically enhance the function of C. bescii cellulases such as CelA by increasing the number of reducing ends accessible to the CelA enzyme and relieving end product inhibition effects. As used herein, “thermostable” refers to enzymes that exhibit significant activity at elevated temperatures (e.g., over 70° C.) for several days.
Thermostable endoglucanases such as E1 from A. cellulolyticus combined with thermostable β-glucosidases such as those from T. maritima may synergistically improve the activity of GH 9/GH 48 enzymes such as CelA enzymes or cocktails containing CelA enzymes acting on biomass and the overall extent of conversion of biomass by the combined enzymes. The enhanced extent of glucan conversion exhibited by this enzyme combination indicates that there is synergy between the A. cellulolyticus E1 endoglucanase and T. maritima β-glucosidase with the C. bescii CelA enzyme, as well as the whole C. bescii culture broth.
The Caldicellulosiruptor species C. bescii presents several key features that make it an attractive candidate as a consolidated bioprocessing (CBP) microbe. For example, C. bescii grows well on crystalline cellulose as well as unpretreated substrates such as switchgrass and poplar at 75° C. Other species of the genus Caldicellulosiruptor include C. obsidiansis, C. kronotskiensis, C. hydrothermalis, C. owensensis, C. saccharolyticus, C. lactoaceticus, C. acetigenus, and C. kristjanssonii. 
CelA is a complex enzyme containing an N-terminal GH9 endo-beta-1,4-glucanase, three family 3 carbohydrate binding modules (CBMIII), and a C-terminal GH48 exo-beta-1,4-glucanase. CelA is capable of withstanding temperatures over 90° C. and has an optimal activity at the 85-90° C. range making it a good candidate for an industrial process where higher temperatures are desired and where costly steps after pretreatment can be avoided. CelA also combines the strengths of a chain-end forming endoglucanase and an efficient cellobiohydrolase on the same protein. This combination allows CelA to be efficient on highly crystalline cellulose substrates. Also, it outperforms one of the most efficient fungal combinations of Exo/Endo-glucanases on avicel even at the temperature of 60° C., far from ideal for CelA. Finally, CelA exhibits hydrolytic activity on xylan—an attractive feature from a process standpoint where biomass feedstocks used are rich in xylan.
β-glucosidases are a family of exocellulase enzymes that catalyze the cleavage of β(1-4) linkages in substrates such as cellobiose, resulting in the release of glucose. The β-glucosidase from T. maritima (whose sequence is set forth in FIG. 2) is provided as a specific example, but other β-glucosidases may be suitable for use in the enzyme cocktails and methods described herein.
Suitable thermostable β-glucosidases include those derived from bacteria of the genus Thermotoga, including the species T. maritima. Other species of the genus Thermotoga include T. elfii, T. hypogeal, T. lettingae, T. naphthophila, T. neapolitana, T. petrophila, T. subterranean, and T. thermarum. 
Endoglucanases suitable for use in the cocktails and methods include thermostable endoglucanases from organisms of the genus Acidothermus, including A. cellulolyticus, and the E1 endoglucanase from A. cellulolyticus (the sequence of which is presented in FIG. 3).
The components of enzyme cocktails may be varied depending on the nature of the substrate being degraded and the pretreatment protocol applied to the substrate. Exemplary enzyme cocktails may comprise, by weight, 30-95% of a thermostable GH 9/GH 48 enzyme such as CelA, 5-25% of a thermostable β-glucosidase, 5-40% of a thermostable endoglucanase such as E1, and 1-20% of additional enzymes such as xylanases (e.g., the thermostable XynA from A. cellulolyticus) or β-xylosidases. Accessory enzymes may also be included at relatively small percentages of the enzyme cocktail. A thermostable GH 9/GH 48 enzyme such as CelA may be comprise at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the enzyme cocktail. A thermostable β-glucosidase may comprise at least about 5%, 10%, 15%, 20%, or 25% of the enzyme cocktail. A thermostable endoglucanase such as E1 may comprise at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% of the enzyme cocktail. A thermostable xylanase or β-xylosidase may comprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the enzyme cocktail. Additional accessory enzymes may comprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the enzyme cocktail.
The enzyme cocktails exhibit surprisingly improved cellulase activities when compared to the individual enzyme activities or the additive effect of each enzyme. The term “improved activity” refers to an increased rate of conversion of a cellulosic substrate or a specific component thereof. Relative activities can be determined using conventional assays, including those discussed in the Examples below. Additional assays suitable for determining cellulase activity include hydrolysis assays on industrially relevant cellulose-containing substrates such as pretreated corn stover. Hydrolysis assays on crystalline cellulose or amorphous cellulose or on small molecule fluorescent reporters may also be used to determine cellulase activity. In certain embodiments, cellulase activity is expressed as the amount of time or enzyme concentration needed to reach a certain percentage (e.g., 80%) of cellulose conversion to sugars.
Enzymes described herein may be used as purified recombinant enzyme or as culture broths from cells that naturally produce the enzyme or that have been engineered to produce the enzyme. In certain embodiments, enzyme cocktails may achieve cellulose conversions to sugars (as a percentage of the total cellulose in the original substrate) ranging from 50% to 100%, 70% to 100%, or 90% to 100%. In some embodiments, the cellulose conversion exhibited by the enzyme cocktail may be at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
Methods for degrading cellulose and materials containing cellulose using the enzymes and enzyme cocktails are also provided herein. For example, the enzyme cocktails may be used in compositions to help degrade (e.g., by liquefaction) a variety of cellulose products (e.g., paper, cotton, etc.) in landfills. The enzyme cocktails may also be used to enhance the cleaning ability of detergents, function as a softening agent or improve the feel of cotton fabrics (e.g., stone washing or biopolishing) or in feed compositions.
Cellulose containing materials may also be degraded to sugars using the enzymes. Ethanol may be subsequently produced from the fermentation of sugars derived from the cellulosic materials. Exemplary cellulose-containing materials include bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, corn fiber, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood (e.g., poplar) chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.
Biofuels such as ethanol may be produced by saccharification and fermentation of lignocellulosic biomass such as trees, herbaceous plants, municipal solid waste and agricultural and forestry residues. Typically, saccharification is carried out by contacting the lignocellulosic biomass with an enzyme cocktail that includes one or more of the enzymes described herein. Such enzyme cocktails may also contain one or more endoglucanases (such as the Family 5 endoglucanase E1 from Acidothermus cellulolyticus) or one or more β-glucosidases (e.g., a β-glucosidase from A. niger) to optimize hydrolysis of the lignocelluloses. Additional suitable endoglucanases include EGI, EGII, EGIII, EGIV, EGV or Cel7B (e.g., Cel7B from T. reesei). Enzyme cocktails may also include accessory enzymes such as hemicellulases, pectinases, oxidative enzymes, and the like.
Enzymes with the ability to degrade carbohydrate-containing materials, such as cellulases with endoglucanase activity, exoglucanase activity, or β-glucosidase activity, or hemicellulases with endoxylanase activity, exoxylanase activity, or β-xylosidase activity may be included in enzyme cocktails. Examples include enzymes that possess cellobiohydrolase, α-glucosidase, xylanase, β-xylosidase, α-galactosidase, β-galactosidase, α-amylase, glucoamylases, arabinofuranosidase, mannanase, β-mannosidase, pectinase, acetyl xylan esterase, acetyl mannan esterase, ferulic acid esterase, coumaric acid esterase, pectin methyl esterase, laminarinase, xyloglucanase, galactanase, glucoamylase, pectate lyase, chitinase, exo-β-D-glucosaminidase, cellobiose dehydrogenase, ligninase, amylase, glucuronidase, ferulic acid esterase, pectin methyl esterase, arabinase, lipase, glucosidase or glucomannanase activities.
A lignocellulosic biomass or other cellulosic feedstock may be subjected to pretreatment at an elevated temperature in the presence of a dilute acid, concentrated acid or dilute alkali solution for a time sufficient to at least partially hydrolyze the hemicellulose components before adding the enzyme cocktail. Additional suitable pretreatment regimens include ammonia fiber expansion (AFEX), treatment with hot water or steam, or lime pretreatment. A lignocellulosic biomass or other cellulosic feedstock may also be de-acetylated before or after the pretreatment regimens listed above.
Lignocellulosic biomass and other cellulose containing materials are contacted with enzymes at a concentration and a temperature for a time sufficient to achieve the desired amount of cellulose degradation. The enzymes and cocktails disclosed herein may be used at any temperature, but are well suited for higher temperature digestions. For example, the enzymes or cocktails may be used at temperatures ranging from about 50° C. to about 60° C., from about 60° C. to about 70° C., from about 70° C. to about 80° C., from about 80° C. to about 90° C., from about 90° C. to about 100° C., from about 50° C. to about 100° C., from about 60° C. to about 90° C., from about 70° C. to about 85° C., or from about 80° C. to about 85° C. Exemplary temperatures include 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 and 90° C.
Suitable times for cellulose degradation range from a few hours to several days, and may be selected to achieve a desired amount of degradation. Exemplary digestion times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours; and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15 days. In some embodiments, digestion times may be one or more weeks.
A reducing agent may also be added to the digestion mixture to improve the cellulose degradation by the enzyme mixture. Exemplary reducing agents include cysteine and dithiothreitol. Reducing agents may be added at concentrations ranging from 1 nM to 100 mM (e.g., 1 mM).
Separate saccharification and fermentation is a process whereby cellulose present in biomass is converted to glucose that is subsequently converted to ethanol by yeast or bacteria strains. Simultaneous saccharification and fermentation is a process whereby cellulose present in biomass is converted to glucose and, at the same time and in the same reactor, converted into ethanol by yeast or bacteria strains. Enzyme cocktails may be added to the biomass prior to or at the same time as the addition of a fermentative organism.
The resulting products after cellulose degradation may also be converted to products other than ethanol. Examples include conversion to higher alcohols, hydrocarbons, or other advanced fuels via biological or chemical pathways, or combination thereof.
“Nucleic acid” or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides. This includes single- and double-stranded molecules (i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids) as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
Nucleic acids referred to herein as “isolated” are nucleic acids that have been removed from their natural milieu or separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing. Isolated nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids that are isolated.
Nucleic acids referred to herein as “recombinant” are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures that rely upon a method of artificial replication, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes. Recombinant nucleic acids also include those that result from recombination events that occur through the natural mechanisms of cells, but are selected for after the introduction to the cells of nucleic acids designed to allow or make probable a desired recombination event. Portions of isolated nucleic acids that code for polypeptides having a certain function can be identified and isolated by, for example, the method disclosed in U.S. Pat. No. 4,952,501.
An isolated nucleic acid molecule can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules can include, for example, genes, natural allelic variants of genes, coding regions or portions thereof, and coding and/or regulatory regions modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a polypeptide or to form stable hybrids under stringent conditions with natural gene isolates. An isolated nucleic acid molecule can include degeneracies. As used herein, nucleotide degeneracy refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies.
Unless so specified, a nucleic acid molecule is not required to encode a protein having protein activity. A nucleic acid molecule can encode a truncated, mutated or inactive protein, for example. In addition, nucleic acid molecules may also be useful as probes and primers for the identification, isolation and/or purification of other nucleic acid molecules, independent of a protein-encoding function.
Suitable nucleic acids include fragments or variants that encode a functional enzyme. For example, a fragment can comprise the minimum nucleotides required to encode a functional cellulase. Nucleic acid variants include nucleic acids with one or more nucleotide additions, deletions, substitutions, including transitions and transversions, insertion, or modifications (e.g., via RNA or DNA analogs). Alterations may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
In certain embodiments, a nucleic acid may be identical to the sequences represented in FIGS. 1-3. In other embodiments, the nucleic acids may be least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences represented in FIG. 1-3, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences represented in FIGS. 1-3. Sequence identity calculations can be performed using computer programs, hybridization methods, or calculations. Exemplary computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, BLASTN, BLASTX, TBLASTX, and FASTA. The BLAST programs are publicly available from NCBI and other sources. For example, nucleotide sequence identity can be determined by comparing query sequences to sequences in publicly available sequence databases (NCBI) using the BLASTN2 algorithm.
Embodiments of the nucleic acids include those that encode the polypeptides that functions as cellulases or functional equivalents thereof. The amino acid sequences of exemplary enzymes are depicted in FIGS. 1-3. A functional equivalent includes fragments or variants of these that exhibit the ability to function as a cellulase. As a result of the degeneracy of the genetic code, many nucleic acid sequences can encode a polypeptide having, for example, the amino acid sequence Shown in FIGS. 1-3. Such functionally equivalent variants are contemplated herein.
Altered or variant nucleic acids can be produced by one of skill in the art using the sequence data illustrated herein and standard techniques known in the art. Variant nucleic acids may be detected and isolated by hybridization under high stringency conditions or moderate stringency conditions, for example, which are chosen to prevent hybridization of nucleic acids having non-complementary sequences. “Stringency conditions” for hybridizations is a term of art that refers to the conditions of temperature and buffer concentration that permit hybridization of a particular nucleic acid to another nucleic acid in which the first nucleic acid may be perfectly complementary to the second, or the first and second may share some degree of complementarity that is less than perfect.
Nucleic acids may be derived from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA, or combinations thereof. Such sequences may comprise genomic DNA, which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions or poly (A) sequences. The sequences, genomic DNA, or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means.
Also disclosed herein are recombinant vectors, including expression vectors, containing nucleic acids encoding enzymes. A “recombinant vector” is a nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice or for introducing such a nucleic acid sequence into a host cell. A recombinant vector may be suitable for use in cloning, sequencing, or otherwise manipulating the nucleic acid sequence of choice, such as by expressing or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences not naturally found adjacent to a nucleic acid sequence of choice, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) that are naturally found adjacent to the nucleic acid sequences of choice or that are useful for expression of the nucleic acid molecules.
The nucleic acids described herein may be used in methods for production of enzymes and enzyme cocktails through incorporation into cells, tissues, or organisms. In some embodiments, a nucleic acid may be incorporated into a vector for expression in suitable host cells. The vector may then be introduced into one or more host cells by any method known in the art. One method to produce an encoded protein includes transforming a host cell with one or more recombinant nucleic acids (such as expression vectors) to form a recombinant cell. The term “transformation” is generally used herein to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell, but can be used interchangeably with the term “transfection.”
Non-limiting examples of suitable host cells include cells from microorganisms such as bacteria, yeast, fungi, and filamentous fungi. Exemplary microorganisms include, but are not limited to, bacteria such as strains of Bacillus brevis, Bacillus megaterium, Bacillus subtilis, Caulobacter crescentus, and Escherichia coli (e.g., BL21 and K12); filamentous fungi from the genera Trichoderma (e.g., T. reesei, T. viride, T. koningii, or T. harzianum), Penicillium (e.g., P. funiculosum), Humicola (e.g., H. insolens), Chrysosporium (e.g., C. lucknowense), Gliocladium, Aspergillus (e.g., A. niger, A. nidulans, A. awamori, or A. aculeatus), Fusarium, Neurospora, Hypocrea (e.g., H. jecorina), and Emericella; and yeasts from the genera Saccharomyces (e.g., S. cerevisiae), Pichia (e.g., P. pastoris), or Kluyveromyces (e.g., K. lactis). Cells from plants such as Arabidopsis, barley, citrus, cotton, maize, poplar, rice, soybean, sugarcane, wheat, switch grass, alfalfa, miscanthus, and trees such as hardwoods and softwoods are also contemplated herein as host cells.
Host cells can be transformed, transfected, or infected as appropriate by any suitable method including electroporation, calcium chloride-, lithium chloride-, lithium acetate/polyene glycol-, calcium phosphate-, DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting, injection, microinjection, microprojectile bombardment, phage infection, viral infection, or other established methods. Alternatively, vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, for example, by injection. Exemplary embodiments include a host cell or population of cells expressing one or more nucleic acid molecules or expression vectors described herein (for example, a genetically modified microorganism). The cells into which nucleic acids have been introduced as described above also include the progeny of such cells.
Vectors may be introduced into host cells such as those from filamentous fungi by direct transformation, in which DNA is mixed with the cells and taken up without any additional manipulation, by conjugation, electroporation, or other means known in the art. Expression vectors may be expressed by filamentous fungi or other host cells episomally or the gene of interest may be inserted into the chromosome of the host cell to produce cells that stably express the gene with or without the need for selective pressure. For example, expression cassettes may be targeted to neutral chromosomal sites by recombination.
Host cells carrying an expression vector (i.e., transformants or clones) may be selected using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule. In prokaryotic hosts, the transformant may be selected, for example, by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
Host cells may be cultured in an appropriate fermentation medium. An appropriate, or effective, fermentation medium refers to any medium in which a host cell, including a genetically modified microorganism, when cultured, is capable of growing or expressing the polypeptides described herein. Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources, but can also include appropriate salts, minerals, metals and other nutrients. Microorganisms and other cells can be cultured in conventional fermentation bioreactors and by any fermentation process, including batch, fed-batch, cell recycle, and continuous fermentation. The pH of the fermentation medium is regulated to a pH suitable for growth of the particular organism. Culture media and conditions for various host cells are known in the art. A wide range of media for culturing filamentous fungi, for example, are available from ATCC. Exemplary culture/fermentation conditions and reagents are provided in the Examples that follow.
The nucleic acid molecules described herein encode the enzymes with amino acid sequences such as those represented by FIGS. 1-3. As used herein, the terms “protein” and “polypeptide” are synonymous. “Peptides” are defined as fragments or portions of polypeptides, preferably fragments or portions having at least one functional activity as the complete polypeptide sequence. “Isolated” proteins or polypeptides are proteins or polypeptides purified to a state beyond that in which they exist in cells. In certain embodiments, they may be at least 10% pure; in others, they may be substantially purified to 80% or 90% purity or greater. Isolated proteins or polypeptides include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides that are isolated. Proteins or polypeptides referred to herein as “recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids.
Proteins or polypeptides encoded by nucleic acids as well as functional portions or variants thereof are also described herein. Polypeptide sequences may be identical to the amino acid sequences presented in FIGS. 1-3, or may include up to a certain integer number of amino acid alterations. Such protein or polypeptide variants retain functionality as cellulases, and include mutants differing by the addition, deletion or substitution of one or more amino acid residues, or modified polypeptides and mutants comprising one or more modified residues. The variant may have one or more conservative changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). Alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
In certain embodiments, the polypeptides may be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences set forth in FIGS. 1-3 and possess enzymatic function. Percent sequence identity can be calculated using computer programs (such as the BLASTP and TBLASTN programs publicly available from NCBI and other sources) or direct sequence comparison. Polypeptide variants can be produced using techniques known in the art including direct modifications to isolated polypeptides, direct synthesis, or modifications to the nucleic acid sequence encoding the polypeptide using, for example, recombinant DNA techniques.
Polypeptides may be retrieved, obtained, or used in “substantially pure” form, a purity that allows for the effective use of the protein in any method described herein or known in the art. For a protein to be most useful in any of the methods described herein or in any method utilizing enzymes of the types described herein, it is most often substantially free of contaminants, other proteins and/or chemicals that might interfere or that would interfere with its use in the method (e.g., that might interfere with enzyme activity), or that at least would be undesirable for inclusion with a protein.
EXAMPLES
Example 1
Bacterial Strains and Growth Conditions
C. bescii DSM 6725 cells were grown at 75° C. under anaerobic conditions on Avicel. Non-avicel bound proteins were pre-affinity purified on a phenyl sepharose column.
Example 2
Purification of CelA
The CelA holoenzyme was initially purified with anion exchange chromatography using a Source 15Q column (GE Healthcare, Piscataway, N.J.). For this chromatography, buffer A was 20 mM Tris, pH 6.8 and buffer B was 20 mM Tris, pH 6.8, with 1 M NaCl. The resulting fraction was then further purified using a source 15PHE hydrophobic interaction column (GE Healthcare, Piscataway, N.J.), with buffer A and buffer C, 20 mM acetate, pH 5.0, with 1 M ammonium sulfate. Relevant fractions were then subjected to size exclusion chromatography using a Sephacryl 300 column (GE Healthcare, Piscataway, N.J.) and eluted with buffer D, 20 mM acetate, pH 5.0, and 100 mM NaCl. The relevant CelA containing fractions were further purified with anion exchange chromatography using a Source 15Q column (GE Healthcare, Piscataway, N.J.) and buffer F, 20 mM Tris-HCL, pH 8.0, and buffer G, 20 mM Tris-HCL, pH 8.0, with 1 M NaCl. The relevant CelA containing fractions were further purified with hydrophobic interaction chromatography using a Source 15ISO column (GE Healthcare, Piscataway, N.J.) with buffer A (20 mM Tris-HCL, pH 8.0) and buffer B (20 mM Tris-HCL, pH 8.0 with 1 M ammonium sulfate. The CelA-containing fractions were once again subjected to size exclusion chromatography using a Sephacryl 300 column (GE Healthcare, Piscataway, N.J.) and eluted with buffer D, 20 mM acetate, pH 5.0 and 100 mM NaCl. The purified fusion proteins were concentrated with a Vivaspin 10K concentrator (Vivaproducts, Littleton, Mass.), and the protein concentration was determined with a Pierce BCA protein assay (Pierce, Rockford, Ill.).
Example 3
Expression of CelA GH48 and GH9 Modules
CelA CBM3-GH48 construct was overexpressed with N-terminal his-tag in E. coli. It was amplified by primers ACACCGGCTAGCAGCAGCACACCTGTAGCAGG (SEQ ID NO:7) and TAGCTTCTCGAGTTATTGATTGCCAAACAGTA (SEQ ID NO:8) (the restriction sites are underlined), and the template of the genomic DNA of C. bescii was employed. The PCR fragment was inserted into the plasmid of pET28b (Novagen, Madison, Wis.), and was overexpressed in the E. coli BL21(DE3) strain (Stratagene, La Jolla, Calif.) at 37° C. with addition of 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG).
The GH9 construct from CelA was overexpressed with N-terminal his-tag in E. coli. It was amplified by primers of ATGCTAGCTAGCGGTTCGTTTAACTATGGGGA (SEQ ID NO:9) and GTCGTTCTCGAGTCATTCAATAGCTTTGAAATCTG (SEQ ID NO:10) (the restriction sites are underlined), and the template of the genomic DNA of C. bescii was employed. The PCR fragment was inserted into the plasmid of pET28b (Novagen, Madison, Wis.), and was overexpressed in the E. coli BL21(DE3) strain (Stratagene, La Jolla, Calif.) at 37° C. with addition of 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG).
Example 4
Purification of CelA GH48 and GH9 Modules
The CelA GH48 component was first desalted into 20 mM acetic acid, pH 5.5, and then ammonium sulfate was added to a concentration of 2 M. This fraction was subjected to hydrophobic interaction chromatography using a Source 15PHE column (GE Healthcare, Piscataway, N.J.). For this chromatography, buffer A was 20 mM acetic acid, pH 5.5, and with 2 M ammonium sulphate, buffer B was 20 mM acetic acid, pH 5.5. The resulting peak was then purified using a source 15Q anion interaction column (GE Healthcare, Piscataway, N.J.), with buffer A (20 mM Tris, pH 6.8) and buffer C (20 mM Tris, pH 6.8, with 1 M NaCl. Finally, CelA-GH48 was separated from minor impurities by size exclusion chromatography using HiLoad Superdex 75 (26/60) (GE Healthcare, Piscataway, N.J.) in buffer H (20 mM acetate, pH 5.0, with 100 mM NaCl). The purified protein was concentrated with a Vivaspin 5K concentrator (Vivaproducts, Littleton, Mass.), and the concentration determined using the Pierce BCA assay (Pierce, Rockford, Ill.).
The C. bescii GH9 module was further purified by first dialyzing it twice against 1.5 L of buffer A (20 mM acetic acid, pH 5.5, 1 mM EDTA with 1 mM DTT) followed by cation exchange chromatography using a Source 15S column (GE Healthcare, Piscataway, N.J.). For this chromatography, buffer A and buffer B with additional 1 M NaCl were used. Finally, minor impurities were removed by size exclusion chromatography using HiLoad Superdex 75 (26/60) (GE Healthcare, Piscataway, N.J.) in 20 mM Tris-HCl, pH 7.0, containing 100 mM NaCl, 5 mM CaCl2, 1 mM EDTA, and 1 mM sodium azide. The purified protein solution was concentrated with a Vivaspin 5K concentrator (Vivaproducts, Littleton, Mass.), and its concentration was measured using a NanoDrop UV spectrophotometer (NanoDrop, Wilmington, Del.).
Example 5
Substrates and Biomass Samples
Avicel (ph101), corn stover, and switchgrass samples were used to evaluate the cellulolytic efficiency of CelA, raw C. bescii broth, and enzyme cocktails. The biomass samples were submitted to several types of pretreatments with increasing severity including diluted acid and ammonia fiber explosion (AFEX) pretreatments for both corn stover and switchgrass; and alkaline peroxide pretreatment for corn stover. The details of each pretreatment condition are described in Table 1.















TABLE 1







Pretreatement conditions





















Temp
Time
Compositional analysis (%)



















Catalyst
(° C.)
(min)
Glucan
Xylan
Galactan
Arabinan
Mannan
Lignin
Ash
Protein





















Dilute acid
% 0.5
150
20
39.73
21.18

3

24.3
7.06



pretreated corn
4SO2H




1.33

ND


1.56


stover













Alkaline
% 2.5
65
60
59
28
0
1.9
0
6.2
1
ND


peroxide
2O2H












pretreated corn













stover













Native
NA
NA
NA
30.2
21.3
ND
3.1
5.3
17.4
6.4
2.3


Switchgrass













Dilute Acid
% 5
190
1
42.59
5.48

0.3
0
21.52
6.09



Pretreated
4SO2H




0.47




ND


Switchgrass













AFEX
g/g 1.52
150
30
30.2
21.3
ND
3.1
5.3
17.4
6.4
2.3


pretreated
biomass












Switchgrass













Avicel ph101
NA
NA
NA
94.94
1.48
0.12
0.31
ND
0.54
0.1
ND


De-acetylated,



63.91
5.01
0.63
0.82

23.93
4.07



dilute acid













pretreated corn













stover









To provide a basis for the maximum theoretical sugar yield achievable from each substrate during enzymatic hydrolysis, portions of each of the pretreated solids samples were dried and subjected to the standard two-stage sulfuric acid hydrolysis method for determining structural carbohydrates in lignocelluloses as described by Sluiter and coworkers (NREL Technical Report: NREL/TP-510-42623 (2006)). In this method, the carbohydrate content of each pretreated sample is calculated from the carbohydrates released.
Example 6
Enzyme Activity Assays
Enzyme activities were determined at 60° C., 75° C., and 85° C. at an enzyme concentration of 15 or 20 mg protein per g glucan. Digestion assays using fungal enzymes were performed at 50° C. and an enzyme concentration of 12 mg/g of Cbh1 from T. reesei and 3 mg/g of E1 from A. cellulolyticus. Both bacterial and fungal digestion assays were done in 20 mM acetate, pH 5.5, containing 10 mM CaCl2, and 100 mM NaCl with continuous mixing.
Digestions were run continuously for seven days and sugar release was monitored. Samples were taken at various time points, enzymes were inactivated by boiling for 15 minutes and samples were then filtered through 0.45 um Acrodisc syringe filters and analyzed for Glucose, Xylose, and Cellobiose by HPLC. Samples were injected at 20 μL and run on an Agilent 1100 HPLC system equipped with a BioRad Aminex HPX-87H 300 mm×7.8 mm column heated to 55° C. A constant flow of 0.6 mL/min was used with 0.1 M H2SO4 in water as the mobile phase to give separation of the analytes. Glucose, xylose, and cellobiose were quantified against independent standard curves. All experiments were performed in triplicate and the resulting extents of conversion are shown as percent glucan converted.
Additionally, CelA and its two recombinant catalytic domains, GH48 and GH9, were tested for activity on 5 mM para-nitrophenol-β-D-xylopyranoside (PnP-X) and para-nitrophenol-β-D-cellobioside (PnP-C) in 20 mM acetate buffer, pH 5.5, 50 mM NaCl at 75° C. The incubation was performed for one hour and then quenched with 50 μl of 1 M calcium carbonate. Absorbance was read at 405 nm.
Example 7
Crystallization
Screening for crystals was done with sitting drop vapor diffusion using a 96-well plate and Crystal Screen HT, PEG ion HT and Grid Screen Salt HT from Hampton Research (Aliso Viejo, Calif.). 50 μL of well solution with drops containing 1 μL of well solution and 1 μL of protein solution were used for screening and a 24-well hanging drop vapor diffusion setup with 1 ml of well solution and drops containing 1 μL of well solution and 1 μL of protein solution was used for optimization of crystallization conditions. The GH9 protein solution contained 9.5 mg/mL of protein, 20 mM Tris pH 7, 100 mM NaCl, 5 mM CaCl2, 1 mM EDTA and 1 mM Na azide. The best crystals for the unliganded GH9 formed in 0.1 M Na cacodylate pH 6.4, 1.8 M ammonium sulfate and 10% dioxane using a 24 well optimization plate. Before flash freezing, the crystal was soaked in a 2 μL cryo solution drop with 10% (v/v) ethylene glycol and 10% (v/v) glycerol in the well solution and incubated for 5 seconds. The crystals of GH9 with cellobiose were obtained using other crystals from the same condition with an excess amount of cellobiose powder in the cryo solution drop and by incubating for 30 seconds. The GH48 protein solution contained 1.8 mg/mL of protein in 20 mM acetic acid pH 5, with 100 mM NaCl. Crystals were grown in 0.1 M tri-sodium citrate, pH 5.8, 20% (w/v) PEG 4000 and 20% (v/v) 2-propanol using a 24 well optimization plate. Before flash freezing, the crystal was incubated for 5 seconds in a 2 μL cryo solution drop containing 10% (v/v) ethylene glycol and 10% (v/v) glycerol in the well solution.
Example 8
X-Ray Diffraction, Structure Determination, and Structure Analysis
Before data collection, all crystals were flash-frozen in a cold nitrogen gas stream at 100 K. Data collection was performed using a Bruker X8 MicroStar X-Ray generator with Helios mirrors and Bruker Platinum 135 CCD detector. Data was indexed and processed with the Bruker Suite of programs version 2008.1-0 (Bruker AXS, Madison, Wis.). Intensities were converted into structure factors and 5% of the reflections were flagged for Rfree calculations using programs F2MTZ, Truncate, CAD and Unique from the CCP4 package of programs. The GH9 structures were solved using molecular replacement program Molrep with PDB entry 1KSD as a model. The GH48 structure was solved using MrBump with PDB entry 1G9G as a model. For all three structures, ARP/wARP version 7.0 and Coot version 0.6.2 was used for multiple cycles of automatic and manual model building. Further refinement and manual correction was performed using REFMACS version 5.6.0117 and Coot. The resulting structures have been deposited to the Protein Data Bank with PDB codes 4DOD (GH9), 4DOE (GH9-CB) and 4EL8 (GH48).
Programs Coot, PyMOL and ICM (molsoft) were used for comparing and analyzing structures. Ramachandran plot statistics were calculated using Molprobity and root mean square deviations (rmsd) of bond lengths and angles were calculated from ideal values of Engh and Huber stereochemical parameters. Wilson B-factor was calculated using CTRUNCATE version 1.0.11. Structural similarity searches were done using pair wise secondary structure matching by PDBefold.
Example 9
Enzymatic Activity on Model Substrates
The cellulolytic performance of purified CelA, isolated from the C. bescii enzyme broth, was examined at 60° C., 75° C., and 85° C. To remove any variability in the enzymatic digestion of biomass substrates such as differences in glucan content or pretreatments, the first enzymatic assays were performed with the model cellulose avicel PH-101. The percentage of glucan released over a seven-day digestion is shown in FIG. 4. CelA exhibited the most activity at the highest temperature tested, 85° C. This single enzyme can convert more than 60% of mostly crystalline cellulose content in 6 days. While lower temperatures impair the performance of CelA, losing close to 10-15% of its activity for every 10-degree temperature drop, its activity remains higher than most single cellulases tested in these conditions. Surprisingly, even at temperature of 60° C. where CelA has lost over 40% of its peak activity, it still outperforms a mixture of the fungal Cel7A from T. reesei (Cbh1) and E1 from A. cellulolyticus. At the same mg/g protein loading, CelA alone is about 2 times as effective compared to a current model mixture of enzymes with the same Exo-endo activity. However, on a molar basis (since CelA is about 4 times larger in terms of molecular weight), there is about 1⁄4 less CelA enzyme present, making CelA about 8 times as active as the Cbh1/E1 combination.
The cellulolytic performance of purified CelA, alone or in combination with E1 from A. cellulolyticus and/or β-glucosidase from T. maritima, on avicel at 75° C. was also tested. Total enzyme loadings were 15 mg protein per g glucan. As shown in FIGS. 7, 9 and 11, the addition of small amounts of β-glucosidase (1 mg per g; FIG. 7) or E1 (4 mg per g; FIG. 9) or both (FIG. 11) to CelA surprisingly enhanced the enzymatic activity of the CelA in a synergistic manner.
Example 10
Enzymatic Activity on Untreated and Pretreated Biomass
The cellulolytic activity of CelA, C. bescii enzyme broth, and enzyme cocktails were tested on native switchgrass samples as well as some with varying pretreatment severity, including dilute acid (DA) and AFEX pretreatments. These enzymatic assays were performed at 75° C. and at a total protein loading of 15 mg of enzyme per g of feedstock. Under these conditions, the maximum glucan conversion of CelA peaks at 30-35% (FIG. 5A) on native switchgrass and dilute acid pretreated switchgrass. CelA activity was the lowest for AFEX pretreated switchgrass were the glucan conversion is 20%. Surprisingly, the activity of CelA on biomass is similar to that of the C. bescii enzyme broth. For comparison with CelA, the commercial enzyme preparation CTec2 was used on DA pretreated switchgrass at its optimal temperature of 50° C. The exact enzyme composition of CTec2 is unknown as it is proprietary; however, the mixture likely contains cellulases, hemicellulases, and beta-glucosidases from modified fungi. CTec2 achieves 63% conversion of glucans after 168 hours and outperforms CelA and C. bescii broth at the same enzyme loading of 15 mg/g.
Similarly, the enzymatic activity of CelA was also tested on a dilute acid (DA) and alkaline peroxide (AP) pretreated corn stover at 75° C. but an enzyme loading of 20 mg of enzyme per g of feedstock (FIG. 5B). CelA performs better on AP corn stover, with a conversion of close to 50%. The same result is observed for the C. bescii enzyme broth.
The ability of CelA to convert xylan found in native switchgrass at 75° C. was also examined. As shown in FIG. 6, CelA can efficiently convert xylan when it is present in untreated biomass. Levels of xylan conversion approached 60% in these assays, which is much higher than expected.
The enzymatic activity of purified CelA, alone or in combination with E1 from A. cellulolyticus and/or β-glucosidase from T. maritima, on DA pretreated corn stover at 75° C. was also tested. Total enzyme loadings were 15 mg protein per g glucan. As with activities seen on avicel, the addition of small amounts of β-glucosidase (1 mg per g; FIG. 8) or E1 (4 mg per g; FIG. 10) or both (FIG. 12) to CelA surprisingly enhanced the enzymatic activity of the CelA in a synergistic manner.
Example 11
Activity of CelA and CelA Components on PnP
The xylanase activity of CelA and its two catalytic units GH48 and GH9 was examined on para-nitrophenol-β-D-xylopyranoside (PnP-X) and para-nitrophenol-β-D-cellobioside (PnP-C). The results shown in Table 2 indicate that CelA and the GH48 module have activity on both substrates whereas the GH9 component has activity on cellulose. CelA was also able to achieve 60% conversion of xylan from native switchgrass, which shows its potential for an industrial process using mild to no pretreatment.









TABLE 2






PnP-X
PnP-C







CelA holoenzyme
+++
++++


CelA GH48
++++
++++


CelA GH9
−
+









Example 12
Crystal Structures of the C. bescii CelA GH9 and GH48 Modules
The structure of the unliganded C. bescii CelA GH9 module was refined to a resolution of 1.7 Å with R and Rfree of 0.155 and 0.179, respectively, and one molecule in the asymmetric unit. The GH9 module with cellobiose was refined to a resolution of 1.56 Å with R and Rfree of 0.148 and 0.175, respectively, and one molecule in the asymmetric unit. GH48 had a resolution of 2.45 Å with R and Rfree of 0.195 and 0.258 with one molecule in the asymmetric unit.
The GH9 module of CelA has an (alpha/alpha)6 barrel fold. The unliganded GH9 structure has one calcium atom, one 1,4-dioxane molecule, 11 ethylene glycol molecules, ten glycerol molecules and four sulfates. The liganded GH9 module has one cellobiose molecule and one cellotriose molecule bound at the active site and one calcium atom, one 1,4-dioxane molecule, 26 ethylene glycol molecules, six glycerol molecules and three sulfates bound elsewhere in the structure.
The GH48 module has an (alpha/alpha)6 barrel fold. The structure revealed one calcium atom, one ethylene glycol and one sulfate. The GH48 structure did not contain the CBM3 module that was included before it in the construct. The CBM3 module may have been cut from the GH48 because of degradation during the crystallization trials. The CelF (PDB entry 1FCE) numbering was used, starting from residue seven (of CelF) that was the first one visible in the electron density. The last three residues and a loop formed by residues 308 to 310 were not modeled due to weak or no electron density. The 308 to 310 loop was special because it actually had some density where the corresponding residues from CelF (PDB entry 1FCE) and CelS (PDB entry 1L1Y) are but the resulting model would be too inaccurate due to possible other overlapping conformations making the density weak and noisy leading to wrong conformations after refinement.
Pairwise secondary-structure matching of structures with at least 70% secondary structure similarity by PDBefold found 19 unique structural matches for the GH9 module (liganded structure was used) and 21 matches for GH48. Structures similar to CelA GH9 included other GH9s and glucuronyl hydrolases with varying sequence identities with a GH9 from Clostridium thermocellum (PDB entry 2XFG) being most similar (71% sequence identity). CelA GH48 was only similar to the different structures of CelF and CelS GH48.
Example 13
Enzymatic Activity, Time of Addition and Reducing Agents
The cellulolytic activities of enzyme cocktails (17 mg/g CelA; 2 mg/g E1; 1 mg/g T. maratima β-glucosidase) were tested on samples of de-acetylated, dilute acid pretreated corn stover in a horizontal screw reactor. Results shown in FIG. 13 demonstrate that up to 80% conversion of this substrate can be achieved using the enzyme cocktails.
CelA enzyme cocktails may also be added to a reactor at a higher temperature than commercial enzyme preparations. Because large reactors take time and water/energy to cool, CelA mixtures can create savings in time and energy by being added earlier in the process (e.g., at 80° C. rather than cooling to 50° C.) without loss of enzymatic activity. FIG. 14 shows the activity of CelA mixture in comparison to a commercial enzyme cocktail.
FIG. 15 shows the increase in overall conversion when a reducing agent is added to the reaction mixture. The addition of 1 mM cysteine improved the conversion by up to 20%.
The Examples discussed above are provided for purposes of illustration and are not intended to be limiting. Still other embodiments and modifications are also contemplated.
While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

## Claims

1. A method for degrading cellulose, comprising contacting a cellulose containing material with an enzyme cocktail comprising a recombinant thermostable enzyme from Caldicellulosiruptor bescii comprising a glycoside hydrolase (GH9) domain and a glycoside hydrolase (GH48) domain and a recombinant thermostable β-glucosidase, wherein at least 50% of the cellulose in the material is converted to at least one sugar.

2. The method of claim 1, wherein the thermostable enzyme comprising a GH9 domain and a GH48 domain is Caldicellulosiruptor bescii CelA.

3. The method of claim 1, wherein the thermostable β-glucosidase is from a bacterium of the genus Thermotoga.

4. The method of claim 1, wherein the enzyme cocktail further comprises a thermostable endoglucanase.

5. The method of claim 4, wherein the thermostable endoglucanase is from a bacterium of the genus Acidothermus.

6. The method of claim 4, wherein the thermostable endoglucanase is Acidothermus cellulolyticus E1.

7. A method for degrading lignocellulosic biomass, comprising contacting the lignocellulosic biomass with an enzyme cocktail comprising a recombinant thermostable enzyme from Caldicellulosiruptor bescii comprising a glycoside hydrolase (GH9) domain and a glycoside hydrolase (GH48) domain and a recombinant thermostable β glucosidase, wherein at least 50% of the cellulose in the biomass is converted to at least one sugar.

8. The method of claim 7, wherein the thermostable enzyme comprising a GH9 domain and a GH48 domain is Caldicellulosiruptor bescii CelA.

9. The method of claim 7, wherein the thermostable β-glucosidase is from a bacterium of the genus Thermotoga.

10. The method of claim 7, wherein the enzyme cocktail further comprises a thermostable endoglucanase.

11. The method of claim 10, wherein the thermostable endoglucanase is from a bacterium of the genus Acidothermus.

12. The method of claim 1, wherein the thermostable enzyme comprising a GH9 domain and a GH48 domain comprises at least 30% of the enzyme cocktail.

13. The method of claim 1, wherein the thermostable enzyme comprising a GH9 domain and a GH48 domain comprises at least 50% of the enzyme cocktail.

14. The method of claim 1, wherein the thermostable enzyme comprising a GH9 domain and a GH48 domain is Caldicellulosiruptor bescii CelA, wherein the thermostable β-glucosidase is from the bacterium Thermotoga maritima; and wherein the CelA comprises at least 30% of the enzyme cocktail and the β-glucosidase comprises at least about 5% of the enzyme cocktail.

15. The method of claim 14, wherein the enzyme cocktail further comprises at least 5% of the thermostable endoglucanase Acidothermus cellulolyticus E1.

16. A method for degrading cellulose, comprising contacting a cellulose containing material with an enzyme cocktail comprising CelA from Caldicellulosiruptor bescii and a thermostable β-glucosidase from Thermotoga maritima; 
wherein the thermostable β-glucosidase comprises about 5% of the enzyme cocktail by weight; and
wherein at least 50% of the cellulose in the material is converted to at least one sugar.

17. The method of claim 16, wherein at least 70% of the cellulose in the material is converted to at least one sugar.

18. The method of claim 16, wherein 50 to 100% of the cellulose in the material is converted to at least one sugar.

19. The method of claim 16, wherein 70 to 100% of the cellulose in the material is converted to at least one sugar.