Abstract:
A novel beta-glucosidase and nucleic acids encoding the beta-glucosidase. Also disclosed are cells, compositions, and methods relating to using the beta-glucosidase to convert ligocellulosic material to fermentable sugars.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the priority of U.S. Provisional Application No. 61/299,007, filed on Jan. 28, 2010. The contents of the application are hereby incorporated by reference in its entirety.  
     
    
     BACKGROUND 
       [0002]    Developments of alternative energy resources (e.g., solar energy, biofuel, hydropower, wind power etc.) have been encouraged for years to cope with arising energy problems related to vastly consumptions of fossil fuels. Among many possible resources, plant biomass is of particular interest as it is renewable. Plant mass contains a high amount of cellulose, a starting material for making biofuel. To convert cellulose to biofuel, it is first degraded to fermentable sugars, such as cellobiose and glucose, by the cellulolytic system of microorganisms. This system includes three major types of hydrolases, i.e., endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21). Although many glucosidases have been isolated from various microorganisms, their efficiencies are not satisfactory. Thus, there is a need for a high efficient glucosidase. 
       SUMMARY 
       [0003]    This invention relates to a novel β-glucosidase, BGL-Cr-D2, which is isolated from a Taiwan-indigenous fungus  Chaetomella raphigera.  Shown in  FIG. 2  are the sequences of the mature BGL-Cr-D2 protein (722 a.a. resides/SEQ ID NO: 1), the corresponding bgl-Cr-D2 cDNA (2166 bps/SEQ ID NO: 2), the signal peptide sequence (SEQ ID NO: 7), the corresponding cDNA (SEQ ID NO: 8), the BGL-Cr-D2 protein with its signal peptide (SEQ ID NO: 4), and the corresponding cDNA (SEQ ID NO: 5). Shown in  FIG. 1  is the genomic DNA region (2851 bps, SEQ ID NO: 3) in  Chaetomella raphigera  encoding the BGL-Cr-D2 enzyme, which contains 12 intron sections, and the genomic DNA region corresponding to SEQ ID NO: 4 (SEQ ID NO: 6). 
         [0004]    Accordingly, one aspect of this invention features an isolated polypeptide containing a sequence exhibiting at least 70% (e.g., 80%, 90%, 95%, or 99%) amino acid identity to SEQ ID NO:1 as determined by the BLAST algorithm. In one example, the polypeptide has a β-glucosidase activity. 
         [0005]    This invention also encompasses (i) an isolated nucleic acid including a nucleotide sequence that encodes the polypeptide described above, and (ii) a host cell containing such an isolated nucleic acid. In one example, the nucleotide sequence contains the sequence of SEQ ID NO: 2, 3, 5, or 6. The isolated nucleic acid of this invention can be an expression vector, in which the nucleotide sequence is operably linked to a suitable promoter sequence (i.e., a sequence capable of initiating transcription in a host cell). 
         [0006]    The term “isolated polypeptide” or “isolated nucleic acid” used herein refers to a polypeptide or nucleic acid substantially free from naturally associated molecules, i.e., the naturally associated molecules constituting at most 20% by dry weight of a preparation containing the polypeptide or nucleic acid. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, and HPLC. 
         [0007]    Also within the scope of this invention are (A) a composition containing the polypeptide described above, an exo-glucanase, an endo-glucanase, and/or a β-glucosidase, and (B) a method of producing a fermentable sugar from a ligocellulosic material. The method includes (i) providing the just-described composition, and (ii) contacting this composition with a lignocellulosic material to produce a fermentable sugar, e.g., glucose, xylose, arabinose, galactose, mannose, rhamnose, surose, fructose, lactose, maltose, trehalose, or cellobiose. The fermentable sugar can be converted to a fermentation product, such as alcohol, by microbial fermentation or enzyme treatment. Examples of the lignocellulosic material used in this method include, but are not limited to, orchard prunings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, oat hulls, sugar cane, corn, corn stover, corn stalks, corn gluten feed, corn cobs, corn husks, corn kernel, fiber from kernels, prairie grass, gamagrass, foxtail, sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, shrubs, grasses, sugar cane bagasse, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings, oat hulls, hard and soft woods, organic waste materials generated from agricultural processes, forestry wood waste, or combinations thereof. If the fermentation product is combustible, the method further contains a step of combusting the combustible fermentation product to produce energy. 
         [0008]    This invention also features a bioreactor containing a lignocellulosic material and a composition, including the polypeptide described above, an exo-glucanase, an endo-glucanase, and/or a β-glucosidase. 
         [0009]    The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. 
     
    
     
       DESCRIPTION OF DRAWINGS 
         [0010]      FIG. 1  is a diagram showing the genomic DNA region (2851 bps, SEQ ID NO: 3) in  Chaetomella raphigera  encoding the BGL-Cr-D2 enzyme, which contains 12 intron sections (as indicated with “White” sections in the diagram and “Small Caps/Gray/Strikethrough” sections in the sequence). A region encoding signal peptide is indicated with “Black” in the diagram and “Underline” in the sequence. 
           [0011]      FIG. 2  is a diagram showing the bgl-Cr-D2 cDNA (2166 bps/SEQ ID NO: 2 excluding signal sequence indicated with underline/SEQ ID NO: 8) and its encoding amino acid sequences (722 a.a. resides/SEQ ID NO: 1, excluding signal peptide/SEQ ID NO: 7) for a Taiwan-indigenous fungus  Chaetomella raphigera.    
           [0012]      FIG. 3  is a diagram of the phylogenetic tree based on amino acid sequences of beta-glucosidase (BGL) from glycoside hydrolase (GH) family 3, showing that the BGL-Cr-D2-Pp secreted from reconstructed  Pichia pastoris  (strain SMD 1168) was related to the beta-D-glucoside glucohydrolase (i.e., BGL) of  T. stipitatua, A. terreus  NIH2624, and  A. fumigates  A1163 (i.e., 64-65% similarity in AA sequences). 
           [0013]      FIG. 4  is a diagram of a vector map of reconstructed pGAPZαC, showing that the bgl-Cr-D2 gene was cloned for further transformation to  P. pastoris  expression system. (In the simplified nucleotide sequence showed below the map, the lengths of shortened regions, i.e., GAP promoter, α-factor signal sequence, and bgl-Cr-D2, do not correlate to the real sizes as scaled in the diagram map.) 
           [0014]      FIGS. 5A and 5B  are diagrams showing growth and pNPGase activity versus time for the colonies of (A)  Pichia pastoris  strain SMD1168 and (B)  P. pastoris  strain SMD1168 carrying bgl-Cr-D2; they show similar growth profile but significant pNPGase activity only for the colony cloned with bgl-Cr-D2. 
           [0015]      FIGS. 6A-C  are photographs showing enzymes sizes and activities of native PAGE (A), MUG zymogram (B) and SDS PAGE (C). (Pp:  Pichia pastoris;  Cr-D2:  Chaetomella raphigera  fungus strain D2; BGL: beta-glucosidase). 
           [0016]      FIG. 7  is a diagram showing that the recombinant enzyme BGL-Cr-D2-Pp was stable over a wide pH range (4-9) based on 20-hour pH stability assessment (incubated the enzyme at 4° C. under each of the particular pH levels for 20 hours prior to performing standard pNPGase activity measurement, i.e., pH5 and 55° C. for 10 mins). The optimal pH level for pNPGase activity of the BGL-Cr-D2-Pp was around 5 (measured pNPGase activity of the enzyme at each of the particular pH levels). 
           [0017]      FIG. 8  is a diagram showing that the recombinant enzyme BGL-Cr-D2-Pp was stable under a temperature range of 4-55° C. based on 4-hour thermostability assessment (incubated the enzyme under each of the particular temperature for 4 hours prior to performing standard pNPGase activity measurement). Optimal temperature for pNPGase activity of the BGL-Cr-D2-Pp was around 70° C. (measured pNPGase activity of the enzyme at each of the particular temperature); but, at this high temperature, the enzyme would become inactive after longer incubation time (e.g., ≧2 hrs,  FIG. 9 ). 
           [0018]      FIG. 9  is a 3-D diagram showing that thermostability of the recombinant β glucosidase BGL-Cr-D2-Pp under temperature ranged from 4 to 75° C. over a 9-days period. Under a temperature ≦50° C., the BGL-Cr-D2-Pp remained stable (relative activity &gt;80%) over 9 days. 
       
    
    
     DETAILED DESCRIPTION 
       [0019]    This invention is based, at least in part, on the unexpected discoveries of a novel beta-glucosidase and its high enzymatic activity or efficiency. Beta-glucosidases (or beta-D-glucoside glucohydrolase, BGL) are a group of the key enzymes for processing lignocellulosic saccharization. This type of enzyme can liberate D-glucose units from the cellulose-hydrolyzing media such as cellobiose, cello-oligosaccharides, and other glucosides after the actions of endo-glucanases (EG) (mainly degrading the amorphous parts of cellulose fiber) and exo-glucanases (or cellobiohydrolases, CBH) (mainly degrading the crystalline cellulose). For industrial-scale production of biofuel from lignocelluloses, finding low-cost cellulases (i.e., CBH, EG, and BGL) with high activity and productivity is essential. 
         [0020]    This invention discloses new discoveries in polypeptides with beta-glucosidase activity, polynucleotides encoding the polypeptides, and enhancement in beta-glucosidase specific activity for recombinant beta-glucosidase expressed from yeast. Explicitly, a Taiwan-indigenous fungus, i.e.,  Chaetomella raphigera  strain D2, was found capable of secreting an active beta-1,4-glucosidase (BGL-Cr-D2). The gene (bgl-Cr-D2, size=2166 bps) encoding this enzyme was cloned from reverse-transcribed cDNA and has been successfully expressed in a yeast expression system using  Pichia pastoris  strain SMD1168 as the host. Recombinant enzyme BGL-Cr-D2-Pp secreted from  P. pastoris  had a higher β-glucosidase activity (based on pNPGase activity measurement) than the native enzyme (approx. 3×) and the benchmark Novozyme-188 (approx. 17×). This invention provides a new recombinant β-glucosidase enzyme that has potential to be utilized together with other cellulases for cellulosic saccharization. 
         [0000]    1. Biofuels from Lignocellulose 
         [0021]    Developments of renewable power resources (e.g., solar energy, biofuel, hydropower, wind power etc.) have been encouraged for years to cope with arising energy problems related to vastly consumptions of fossil fuels. Among the currently-exploited renewable energy forms/sources, biofuel has a unique advantage in reducing agricultural/municipal/industrial organic wastes while generating cleanliness and energy-rich fuels (e.g., methane, ethanol, butanol, and hydrogen). Thus, it has been listed as one of important sustainable energies (Antoni et al., 2007, Appl Microbiol Biotechnol 77:23-35; and Rubin, 2008, Natures 454:841-845). Not only in Taiwan, production of bioethanol from lignocellulose is urgent worldwide as about 5×10 12  tons of cellulose-rich agriculture wastes are produced on the earth every year and supplement of ethanol in gasoline is ongoing or on-its-way in many countries. Prior to further fermentation for ethanol production, various enzyme systems (e.g., ligninase, cellulases, and hemicellulases) are required for complete breakdown of lignocelluloses (containing about 5-30% of lignin, approximately 35-50% of cellulose, and approximately 20-35% of hemicellulose) to release fermentable glucose (Lynd et al., 2002, Microbiol Mol Biol Rev 66:506-577). 
       2. Cellulases 
       [0022]    For bioethanol production from cellulose, efficient saccharification (degradation of polymeric cellulose fibers to release smaller glucose molecules) by cellulases is crucial. Cellulases are a group of enzymes responsible for complex cellulose hydrolysis. Multiple cellulases have been classified into three groups, i.e., (i) exoglucanases (including 1,4-β-D-glucan cellobiohydrolases, CBH, EC 3.2.1.91 and 1,4-β-D-glucan glucanohydrolases, EC 3.2.1.74), (ii) endoglucanases (EG) (EC 3.2.1.4), and (iii) beta-glucosidases (or β-D-glucoside glucohydrolase, BGL) (EC 3.2.1.21) based on structural and functional properties of the enzymes (Coughlan et al., 1988, In Biochemistry and Genetics of Cellulose Degradation, pp. 11-30; and Henrissat et al., 1989, Gene 81:83-95). The CBH mainly act on the crystalline sections at reducing or nonreducing end of cellulose fiber while EG randomly attack the amorphous parts of cellulose. More specifically, these cellulases can cleave the β-1,4-glucan or β-D-glucosidic linkages between glucosyl residues within celluloses to form short-chain media including cellobiose (i.e., disaccharides), other cellodextrins (i.e., cello-oligosaccharides), and glucosides (e.g., alcoholic, cyanogenic, or phenolic glycosides). These short-chain intermediates can be further hydrolyzed to glucose (i.e., monosaccharide) by BGL that primarily catalyzes the transfer of glycosyl group between oxygen nucleophiles (Bhatia et al., 2002, Crit Rev Biotechnol 22(4):375-407). For cellulose saccharification, insufficient BGL activity can result in not only shortage of glucose but also accumulation of cellobiose that is a potent inhibitor for cellulose hydrolysis by CBH and EG (Harris et al., 2007, U.S. Pat. No. 7,244,605B2). Thus, BGL plays an important role in achieving high ethanol yield from cellulose (Lynd et al., 2002, Microbiol Mol Biol Rev 66:506-577; and Hong et al., 2007, Appl Microbiol Biotechnol 73:1331-1339). 
         [0000]    3. Sources of β-glucosidases (BGLs) 
         [0023]    The enzyme BGL is ubiquitous and has been discovered in all the living kingdoms from microbes, insects, and plants to highly-evolved mammals (Esen, 1993, In Biochemistry and Molecular Biology, American Chemical Society, 1-14; Bhatia et al., 2002, Crit Rev Biotechnol 22(4):375-407). One of major BGL-producing sources is from bacteria including some strains of genus  Agrobacterium, Bacillus, Butyrivibrio, Cellovibrio, Clostridium, Erwinia, Pseudomonas, Pyrococcus, Ruminococcus, Streptomyces, Thermotoga, Thermobifida  etc. (Hashimoto et al., 1998, Arch Biochem Biophys 360:1-9; Srivastava et al., 1999, Biotechnol Lett 21:293-297; and Yun et al., 2001, Biosci Biotechnol Biochem 65(9):2028-2032). Fungal strains (molds or yeasts) with BGL-producing ability include those from genus  Aspergillus, Candida, Humicola, Penicillium, Pichia, Saccharomycopsis, Talaromyces, Trichoderma  etc. (Dan et al., 2000, J Biol Chem 275(7):4973-4980; and Dunn-Coleman et al., 2008, U.S. Patent Publication 2008/0095889A1). Among these microbes, fungal species  Aspergillus niger, Aspergillus fumigates,  and  Trichoderma reesei  are the most well-known and efficient BGL producers making them the major sources for commercial BGLs in the world (Dan et al., 2000, J Biol Chem 275(7):4973-4980; Harris et al., 2007, U.S. Pat. No. 7,244,605B2; and Dunn-Coleman et al., 2008, U.S. Patent Publication 2008/0095889A1). In addition, few plants like barley,  Costus , and Maize have been found containing BGL as well. 
         [0024]    Beside above-mentioned native sources, some recombinant BGLs have been expressed in different hosts (e.g.,  E. coli, B. subtilis, P. pastoris, S. cerevisiae, A. kawachii,  and  T. reesei ) (Pandey et al., 1995, J Ferment Bioeng 80(5):446-453; Murray et al., 2004, Protein Expres Purif 38:248-257; and Roy et al., 2005, Biochem Bioph Res Co 336:299-308). For example, bgl genes from  Bacillus  sp. had been successfully cloned and expressed in  E. coli  system (Hashimoto et al., 1998, Arch Biochem Biophys 360:1-9; and Srivastava et al., 1999, Biotechnol Lett 21:293-297). In addition, an  Aspergillus niger  BGL coding gene, bgl1, had been expressed in  P. pastoris  and  S. cerevisiae  (Dan et al., 2000, J Biol Chem 275(7):4973-4980). Clone BGL genes from diverse sources and then express in different hosts has been a practical approach for gaining higher BGL productivity (over-expression) and/or activity (advantageous post-translational modification improve kinetic features of the enzymes). 
       4. Properties of BGLs 
       [0025]    The properties of BGLs are rather diverse by far. In general, BGLs from different orders and kingdoms appear to differ in their specificities for the aglycone part (aryl-, alkyl-, or amino-) linked to the glycosyl group (Bhatia et al., 2002, Crit Rev Biotechnol 22(4):375-407). They have been found to be intracellular, extracellular, cytosol-associated, membrane-bound, or periplasmically-localized. Their catalytic functionalities on hydrolyzing short-chain cellobiose and oligosaccharides are quite similar in cellulosic microorganisms although some other various functions have been noticed in different organisms (e.g., synthesis of glycosyl-bond between different molecules in some microbes, releasing cyanides from cyano-glucoside precursors in insects and plants, and hydrolysis of glycosylceramides for treating Gaucher&#39;s disease in humans) (Bhatia et al., 2002, Crit Rev Biotechnol 22(4):375-407). For cellulose hydrolysis, BGLs are able to hydrolyze their substrates with net retention of anomeric configuration that occurs presumably via a two-step, double-displacement mechanism involving two carboxylic acid residues at key active site (Davies et al., 1998, Comprehensive Biological Catalysis Vol. 1, pp. 119-208; and Withers, 2001, Carbohyd Polym 44:325-337). It has been noted that most of BGLs have different substrate specificity and can act on a wide spectrum of substrates (e.g., pNPG; cellobiose, salicin, MUG, arbutin, aryl-, alkyl-, and methyl-glucosides). In addition, most of BGLs have optimum pH within weak-acid levels and optimum temperature in mesophilic scopes despite some strong-acid and/or thermophilic properties have been reported. The BGL monomers from diverse organism sources have a very broad molecular size range (i.e., about 13-137 kDa) (Gabelsberger et al., 1993, Appl Microbiol Biotechnol 40:44-52; Iwashita et al., 1999, Appl Environ Microbiol 65:5546-5553; Srivastava et al., 1999, Biotechnol Lett 21:293-297; and Dan et al., 2000, J Biol Chem 275(7):4973-4980). Amino acid sequence similarities between BGLs of different organism species also vary widely (10s -90s %). Nevertheless, amino acid bases of aspartic acid (Asp, D) and glutamine acid (Glu, E) have been recognized as catalytic necleophile and catalytic proton donor of BGLs. 
       5. Classification of BGLs 
       [0026]    Based on chemical reactions they catalyze, all BGLs have been assigned to EC 3.2.1.21 category (i.e., Enzyme Commission number of 3-hydrolases, 2-glycosylases, 1-glycosidases, and 21-glucosidase) according to numerical classification scheme for more than 3,000 enzymes published by the IUBMB (International Union of Biochemistry and Molecular Biology) (Webb, 1992, Enzyme nomenclature: recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes, ISBN 0-12-227164-5; and ExPASy, 2009, www.expasy.org/enzyme/). Additionally, BGLs are versatile hydrolytic enzymes that have been further classified according to various criteria (e.g., substrate specificity and sequence identity). At first, BGLs had been grouped into Type I and II (Beguin et al., 1990, Ann Rev Microbiol 44:219-248) or subfamily A and B (Rojas et al., 1995, Biochem Mol Biol Int 35:1223-1231); but lately, these earlier classifications have been mostly replaced by the currently-acceptable scheme based on amino acid and/or nucleotide sequence identity (Henrissat et al., 1996, Biochem J 316:695-696). 
         [0027]    Based on amino acid sequence and folding similarities, the BGLs have been placed in either family 1 or 3 among 115 up-to-date (November 2009) defined GH (glycoside-hydrolases) families with an exception of the glucosylceramidases (i.e., acid β-glucosidase placed in family 30) (Henrissat, 1991, Biochem J 280:309-316; Henrissat et al., 1993, Biochem J 293:781-788; Henrissat et al., 1996, Biochem J 316:695-696; Hong et al., 2007, Appl Microbiol Biotechnol 73:1331-1339; and Cantarel et al., 2009, Nucleic Acids Res. 37:D233-238). Family 1 (GH1) contains β-glucosidases from some archaea, bacteria, fungi, plants, and mammals whereas family 3 (GH3) comprises β-glucosidases of some bacterial, fungal, and plant origins (Henrissat et al., 1996, Biochem J 316:695-696; and Cantarel et al., 2009, Nucleic Acids Res. 37:D233-238). 
       6. Applications 
       [0028]    Beside cellobiose/cellodextrins hydrolysis function used for cellulosic ethanol production, BGLs also can be broadly used in the fields of medicine, agriculture, and food industry wherein many reactions engaging cleavage or synthesis of glycosidic bonds are required. Applications based on hydrolytic activities of BGLs include (1) removal of bitterness from citrus fruit juices, (2) manufacture of low-viscosity gellan foods, (3) detoxification of cassava (the 3 rd -4 t  largest source of calories in the tropics), (4) enhancement of aroma release for benefiting winemaking process, (5) feed additive for enhancing nutrient utilization in single-stomached animals, (6) hydrolysis of genistin to genistein as an antitumor agent, (7) production of melanin from phloridzin for reducing the risk of skin cancer and promoting dark color of hair, (8) production of hydroxytyrosol from oleuropein for preventing coronary heart disease and cancer, (9) hydrolysis of laminarin for the production of yeast extract and the conversion of algal biomass to fermentable sugars, (10) making pigments as natural food dyes in confectionary products, etc. (Bhatia et al., 2002, Crit Rev Biotechnol 22(4):375-407). 
         [0029]    In addition, synthetic activities of BGLs have been widely used in the manufacture of pharmaceuticals, fine chemicals and food ingredients (as summarized by Bhatia et al., 2002, Crit Rev Biotechnol 22(4):375-407). For instance, synthesis of an aromatic n-alkyl glucoside ester that was effective in the treatment of fever, rheumatism, headache, and other ailments (Otto et al., 1998, Biotechnol Lett 20:437-440). Other applications of BGLs also have been reported such as sero-diagnosing histoplasmosis or post-diagnosing hepatic ischemia-reperfusion injury and recovery. Therefore, productions of high quality/quantity BGLs from diverse sources have been fundamental tasks for these multipurpose applications. 
         [0030]    Described herein is an isolated polypeptide including an amino acid sequence at least 70% identical to that of BGL-Cr-D2 (SEQ ID NO:1) or a part thereof having at least 20 (e.g., 30, 50, 80, 100, 150, 200, 250, 300, and 350) contiguous amino acids. This polypeptide has a high beta-glucosidase activity. 
         [0031]    The “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul  Proc. Natl. Acad. Sci. USA  87:2264-68, 1990, modified as in Karlin and Altschul  Proc. Natl. Acad. Sci. USA  90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.  J. Mol. Biol.  215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al.,  Nucleic Acids Res.  25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. 
         [0032]    The isolated polypeptide can be prepared by purification from a suitable microorganism, e.g.,  Chaetomella raphigera.  It also can be prepared via conventional recombinant technology. An example follows. A DNA fragment encoding BGL-Cr-D2 can be prepared by polymerase chain reaction from  Chaetomella raphigera  cells and cloned into an expression vector. Upon insertion, the BGL-Cr-D2-encoding fragment is operably linked to a suitable promoter contained in the expression vector. The resultant DNA construct is then introduced into suitable host cells (e.g.,  E. coli  cells, yeast cells, insect cells, and mammalian cells) for expression of BGL-Cr-D2, which can be purified from the cells by conventional methods. One example of yeast cells is  Pichia pastoris,  e.g.,  Pichia pastoris  strain SMD1168. 
         [0033]    To make a functional equivalent of BGL-Cr-D2, which is also within the scope of this invention, one or more conservative amino acid substitutions can be introduced into SEQ ID NO:1 without disrupting its β-glucosidase activity. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in SEQ ID NO: 1 is preferably replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of SEQ ID NO: 1, such as by saturation mutagenesis, and the resultant mutants can be screened for the β-glucosidase activity to identify mutants that retain the activity as described below in the Example section below. 
         [0034]    Fusion protein technology can be applied to improve expression efficiency and facilitate purification of the polypeptide of this invention. To prepare a fusion protein containing BGL-Cr-D2, a DNA fragment encoding this β-glucosidase can be linked to another DNA fragment encoding a fusion partner, e.g., glutathione-s-transferase (GST), 6×-His epitope tag, or M13 Gene 3 protein. The resultant fusion nucleic acid expresses in suitable host cells a fusion protein that can be isolated by methods known in the art. The isolated fusion protein can be further treated, e.g., by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of this invention. 
         [0035]    Also described herein is an isolated nucleic acid encoding the polypeptide of this invention. A nucleic acid refers to a DNA molecule (e.g., a cDNA or genomic DNA), an RNA molecule, or a DNA/RNA analog, which can be synthesized from nucleotide analogs. In one example, the nucleic acid of this invention is an expression vector in which a DNA fragment encoding the polypeptide is operably linked to a suitable promoter. 
         [0036]    As used herein, the term “promoter” refers to a nucleotide sequence containing elements that initiate the transcription of an operably linked nucleic acid sequence in a desired host microorganism. At a minimum, a promoter contains an RNA polymerase binding site. It can further contain one or more enhancer elements which, by definition, enhance transcription, or one or more regulatory elements that control the on/off status of the promoter. When  E. coli  is used as the host microorganism, representative  E. coli  promoters include, but are not limited to, the β-lactamase and lactose promoter systems (see Chang et al.,  Nature  275:615-624, 1978), the SP6, T3, T5, and T7 RNA polymerase promoters (Studier et al.,  Meth. Enzymol.  185:60-89, 1990), the lambda promoter (Elvin et al.,  Gene  87:123-126, 1990), the trp promoter (Nichols and Yanofsky,  Meth. in Enzymology  101:155-164, 1983), and the Tac and Trc promoters (Russell et al.,  Gene  20:231-243, 1982). When yeast is used as the host microorganism, exemplary yeast promoters include 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter, galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter. Promoters suitable for driving gene expression in other types of cells are also well known in the art. 
         [0037]    A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The vector can be capable of autonomous replication or integrate into a host DNA. Examples of the vector include a plasmid, cosmid, or viral vector. The vector of this invention includes a nucleotide sequence encoding BGL-Cr-D2 in a form suitable for expression of the nucleic acid in a host cell. Preferably the vector includes one or more regulatory sequences operatively linked to the encoding sequence. A “regulatory sequence” includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vector can be introduced into host cells to produce the polypeptide of this invention. 
         [0038]    Also within the scope of this invention is a host cell that contains the above-described nucleic acid. Examples include  E. coli  cells, insect cells (e.g., using baculovirus expression vectors), yeast cells, plant cells, or mammalian cells. See e.g., Goeddel, (1990)  Gene Expression Technology:  Methods in Enzymology 185, Academic Press, San Diego, Calif. To produce a polypeptide of this invention, one can culture a host cell in a medium under conditions permitting expression of the polypeptide encoded by a nucleic acid of this invention, and purify the polypeptide from the cultured cell or the medium of the cell. Alternatively, the nucleic acid of this invention can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. 
         [0039]    Further described herein is a method of converting lignocellulosic material to fermentable products (e.g., fermentable sugars) using a multi-enzyme composition containing the β-glucosidase described herein and other cellulolytic enzymes, such as exo-glucanase and endo-glucosidase. See, e.g., US Application Nos. 20070238155 and 20070250961. The term “cellulolytic enzyme” refers to an enzyme that hydrolyzes cellulose (a polysaccharide consisting of glucose units) into smaller sugar units. See Gilbert H J, Hazlewood G P, 1993  J Gen Microbiol  139:187-194; Olimiya K et al. 1997  Biotechnol Genet Eng Rev.  14:365-414. See also, e.g., US Application 2007016805. This multi-enzyme composition can be obtained from, e.g., a microbial, a plant, or a combination thereof, and will contain enzymes capable of degrading lignocellulosic material. In addition to the cellulolytic enzymes mentioned above, it can further include cellobiohydrolases, endoglucanase, beta.-glucosidases), hemicellulases (such as xylanases, including endoxylanases, exoxylanase, and beta-xylosidase), ligninases, amylases, alpha-arabinofuranosidases, alpha-glucuronidases, alpha-glucuronidases, arabinases, glucuronidases, proteases, esterases (including ferulic acid esterase and acetylxylan esterase), lipases, glucomannanases, or xylogluconases. 
         [0040]    As used herein the term “lignocellulosic material” refers to materials containing cellulose and/or hemicellulose. Generally, these materials also contain xylan, lignin, protein, and carbohydrates, such as starch and sugar. Lignocellulose is found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. The process of converting a complex carbohydrate (such as starch, cellulose, or hemicellulose) into fermentable sugars is also referred to herein as “saccharification.” Fermentable sugars, as used herein, refer to simple sugars, such as glucose, xylose, arabinose, galactose, mannose, rhamnose, surose, fructose, lactose, maltose, trehalose, or cellobiose. Lignocellulosic material can include virgin plant biomass and/or non-virgin plant biomass such as agricultural biomass, commercial organics, construction and demolition debris, municipal solid waste, waste paper, and yard waste. Common forms of lignocellulosic material include trees, shrubs and grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn kernel including fiber from kernels, products and by-products from milling of grains such as corn, rice, wheat, and barley (including wet milling and dry milling), as well as municipal solid waste, waste paper, and yard waste. The lignocellulosic material can also be, but is not limited to, herbaceous material, agricultural residues, forestry residues, and paper mill residues. “Agricultural biomass” includes branches, bushes, canes, corn and corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, short rotation woody crops, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings, oat hulls, hard and soft woods (not including woods with deleterious materials), organic waste materials generated from agricultural processes including farming and forestry activities, specifically including forestry wood waste, or a mixture thereof. 
         [0041]    The fermentable sugar produced in the method described above can be converted to useful value-added fermentation products via enzyme treatment or chemical reaction. Examples of the fermentation product include, but are not limited to amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, lactic acid, and ethanol, including fuel ethanol. Specific value-added fermentation products that may be produced by the methods of the invention include, but not limited to, biofuels (including ethanol and butanol); lactic acid; plastics; specialty chemicals; organic acids, including citric acid, succinic acid and maleic acid; solvents; animal feed supplements; pharmaceuticals; vitamins; amino acids, such as lysine, methionine, tryptophan, threonine, and aspartic acid; chemical feedstocks. The fermentable sugar can also be used for culturing microbes that produce fermentation products, e.g., industrial enzymes, such as proteases, cellulases, amylases, glucanases, lactases, lipases, lyases, oxidoreductases, transferases and xylanases. 
         [0042]    The invention also provides a method of producing energy from lignocellulosic material. This method include providing the multi-enzyme composition described above; contacting the composition with the lignocellulosic material to produce a fermentable product; fermenting the fermentable product to produce a combustible fermentation product, and combusting the combustible fermentation product to produce energy. This method can be performed in a bioreactor that contains all necessary components and may preferably be configured for anaerobic growth of microorganisms. Methods for making and using bioreactors are known in the art. See, e.g., US Application 20080131958. 
         [0043]    The polypeptide and composition described above can also be used in the paper and pulp industry. For example, the polypeptide, which has a high β-glucosidase activity can be used in the deinking and refining of recycled paper. In this application, utilizing of them could reduce the amount of enzyme used per ton of paper substantially, and reduce the time of exposure to the enzyme needed to increase the brightness of the paper. Reducing the concentration of enzyme and the time of exposure to the enzyme in the refining process, correspondingly and desirably reduces the reaction of the cellulase on the fibrils themselves and processing costs. 
         [0044]    The polypeptide of this invention has additional industrial applications. Given its high β-glucosidase activity, this polypeptide can function at a lower amount. The polypeptide, in combination with other enzymes, can be used, with enhanced yields, in extracting juice from fruits, or extracting juice or soup flavorings from vegetables. In combination with protease, it can be used to dissociate dried seaweed, which is then fermented with alcohol to produce vinegar. The polypeptide, mixed with other enzymes, can also serve as a dough conditioner in the baking industry. See, e.g., U.S. Pat. No. 6,602,700. 
         [0045]    Moreover, the polypeptide of this invention can also be used in the textile industry. It can be used to brighten and soften cotton fabrics by removing microfibers on the surface, which causes a dull look of clothes. More specifically, it can be included as an additive in formulating enzyme-containing detergents for soil removal, fabric softening, and color brightening. For example, it can be used as a replacement to pumice in producing blue jeans having a “stone-washed” effect. Enzyme treatment causes less damage to the jean fabric than lengthy exposure to pumice. See U.S. Pat. Nos.5,232,851, 5,677,151, 6,451,063, and 7,226,773. 
         [0046]    In another aspect, the present invention provides a transgenic plant, the genome of which is augmented with a recombinant polynucleotide encoding a polypeptide of this invention operably linked to a promoter sequence. The polynucleotide is optimized for expression in the plant and the polypeptide is produced at a level greater than 5% total soluble protein, greater than 10% total soluble protein or greater than 20% total soluble protein. The polypeptide may be expressed constitutively or tissue-specifically. For example, it may be expressed in a plant tissue selected from the group consisting of stems and leaves. It may also be expressed in a targeted sub-cellular compartment or organelle, such as apoplast, chloroplast, cell wall, or vacuole. The plant may be a monocotyledonous plant or a dicotyledonous plant. In certain embodiments, the plant is a crop plant. The plant may be selected from the group consisting of corn, switchgrass, sorghum, miscanthus, sugarcane, poplar, pine, wheat, rice, soy, cotton, barley, turf grass, tobacco, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus. Methods for making transgenic plants are well known in the art. 
         [0047]    The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety. Further, any mechanism proposed below does not in any way restrict the scope of the claimed invention. 
         [0000]    I. Materials and methods 
       1. Cultivation of Native D2 Fungal Strain 
       [0048]    The native fungal strain (i.e.,  Chaetomella raphigera  D2) was cultured with MR (Mendels-Reese) medium (pH 5.0) to harvest fungal cells for DNA/RNA extractions and their crude enzymes for property analyses (e.g., amino acid sequences, cellulosic activities, and enzyme concentrations). Each one-liter MR medium contains 1 g soy peptone, 1.4 g (NH 4 ) 2 SO 4 , 0.3 g urea, 2.0 g KH 2 PO 4 , 0.34 g CaCl 2 , 0.3 g MgSO 4 .7H 2 O, 5.0 mg FeSO 4 .7H 2 O, 1.6 mg MnSO 4 .7H 2 O, 1.4 mg ZnSO 4 .7H 2 O, 2 mg CoCl 2 .6H 2 O, and 0.72 g cellobiose. Fungal culture was grown at a temperature of 30° C. and a mixing rate of 125 rpm for 4 days prior to gather fungal cells and their crude enzymes. 
       2. Nucleic Acid Extraction and Crude Enzyme Collection 
       [0049]    Genomic DNA of the  C. raphigera  D2 fungus was extracted using Wizard® Genomic DNA Purification Kit (Promega, USA) and fungal RNA was extracted using Plant Total RNA Miniprep Purification Kit (GeneMark, Taiwan). Concentrations of extracted nucleic acid were measured by a spectrophotometer (NanoDrop ND-1000, Thermo Scientific, USA). The DNA and RNA extracts were stored at −20° C. prior to further treatments (e.g., PCR and RT PCR) were conducted. 
         [0050]    Crude enzymes were collected by filtering supernatant of 4-day fungal culture through Whatman No.1 filter paper (Whatman/GE Healthcare, USA), then 0.45 μm Supor® Membrane Disc Filter (PALL, USA), and followed by 30K NMWL (Nominal Molecular Weight Limit) Amico® Ultra-15 Centrifugal Filter (Millipore, USA). Enzyme concentrations were determined based on Bio-Rad Protein Assay using BSA (bovine serum albumin) as calibration standard (Bio-Rad, USA). The filtered enzymes were stored at 4° C. until further analyses were conducted. 
       3. Enzyme Electrophoresis and N-terminal Sequencing 
       [0051]    Enzyme electrophoreses including native PAGE (polyacrylamide gel electrophoresis) and SDS (sodium dodecylsulfate) PAGE were performed based on the protocols suggested by Hoefer&#39;s Protein Electrophoresis Applications Guide (Hoefer Scientific Instruments, 1994). Before Coomassie blue staining for native PAGE, zymogram was carried out by immersing gel with 0.5 mM MUG (4-Methylumbelliferyl β-D-glucopyranoside) solution and incubating at 50° C. for 15 minutes and then followed by observation under UV light (Benoit et al., 1995, Curr Microbiol 30:305-312). Additionally, instead of coomassie blue staining for SDS-PAGE, silver staining using PlusOne Silver Staining Kit (GE Healthcare, USA) was also performed for observing some enzymes with low concentration. 
         [0052]    For N-terminal sequence analysis, the target enzyme (i.e., BGL with MUG activity) was transferred from SDS-PAGE gel to PVDF membrane (iBlot Gel Transfer Stacks, Invitrogen, USA) and analyzed by Applied Biosystems Procise Protein Sequencer model 494 (Applied Biosystems, USA) based on Edman degradative chemistry. Part of the revealed N-terminal sequence, PGDGDWA (SEQ ID NO: 9), was used to design a degenerate primer D2-bgl-NT: CCN GGN GAY GGN GAY TGG GC (SEQ ID NO: 10) for further amplifying target bgl gene from genomic DNA and/or reverse transcribed cDNA. 
       4. Cloning and Sequencing for D2-bgl Gene 
       [0053]    The bgl-Cr-D2 cDNA was first reverse transcribed (RT) from RNA sample using a poly-T primer: GGT TCT TGC CAC AGT CAC GAC TTT TTT TTT TTT TTT TTT (SEQ ID NO: 11) and SuperScript® III Reverse Transcriptase (Invitrogen, USA); and, subsequently amplified using a primer set of D2-bgl-NT and poly-T anchor primer: GGT TCT TGC CAC AGT CAC GAC (SEQ ID NO: 12) and a DNA polymerase (TaKaRa Ex Taq™, TaKaRa Bio Inc, Japan). PCR thermo-cycle conditions were 94° C. for 4 minutes, followed by 30 cycles at 94° C. for 1 minute, 58° C. for 30 seconds, and 72° C. for 3 minutes, and then a final elongation at 72° C. for 5 minutes. The RT-PCR product (i.e., amplicon of bgl-Cr-D2 cDNA) was cloned in pGEM®-T Easy vector (Promega, USA) and transferred into  E. coli  cells (strain DH5α) for preservation and further sequencing. Besides, the bgl-Cr-D2 gene (containing introns) was amplified from genomic DNA sample using a primer set of D2-bgl-f: CCT GGT GAT GGT GAT TGG GCA GC (SEQ ID NO: 13) and D2-bgl-r: ATG TCC ACC TTT CCG AAT ACC TTG GC (SEQ ID NO: 14) and TaKaRa Ex Tag™. The PCR product was also cloned in pGEM®-T Easy vector for sequencing. Intron sections were determined by comparing bgl-Cr-D2 cDNA (without introns) and bgl-Cr-D2 genomic DNA (with introns) sequences. 
         [0054]    Additionally, signal sequence upstream of the bgl-Cr-D2 gene was revealed by randomly digesting genomic DNA with SacI restriction enzyme (NEB, New England Biolabs Inc., USA) and self-ligation with T4 DNA ligase (Promega, USA); and then, followed by inverse PCR using a primer set of D2-bgl233f: CGT TTC GTC CAA AAT GTA ACA GCA T (SEQ ID NO: 15) and D2-bgl232r: GAT GCT TTC ACC GTC AGT TCT GA (SEQ ID NO: 16). The PCR program was as follow: 5 minutes at 95° C., followed by 25 cycles consisting of 1 minute at 95° C., 1 minute at 55° C., and 6 minutes at 72° C. and a final cycle consisting of 10 minutes at 72° C. The inverse PCR product (should contain a sequence in the order of D2-bgl233f+partial D2-bgl+SacI+partial D2 genome+signal sequence of D2-bgl+partial D2-bgl+D2-bgl232r) was cloned in pGEM®-T Easy vector for further sequencing. 
         [0055]    The putative amino acid sequence of BGL-Cr-D2 from revealed bgl-Cr-D2 cDNA was aligned with other GH3 family BGLs using Clustal X software (Thompson et al., 1997, Nucleic Acids Research 25(24): 4876-4882) to analyze phylogenetic relationships. The alignment result was used to build phylogenetic tree that was visualized in TreeView (Page, 1996, Computer Applications in the Biosciences 12:357-358). 
         [0000]    5. Expression in  Pichia pastoris    
         [0056]    The bgl-Cr-D2 cDNA (cloned in the pGEM®-T Easy Vector) was further constructed in pGAPZα C vector for constitutive expression of recombinant BGL-Cr-D2-Pp in  Pichia yeast  (Invitrogen, USA). Briefly, the bgl-Cr-D2 cDNA in the Easy vector was PCR amplified with a primer set of D2-bgl-f-EcoRI: CGC TT G AAT TC G ATG CCT GGT GAT GGT GAT TGG (SEQ ID NO: 17) and D2-bgl-r-NotI: TTC AA G CGG CCG C AT GTC CAC CTT TCC GAA TAC C (SEQ ID NO: 18). This PCR product was joined into the pGAPZα C vector by double digesting amplicon and vector with EcoRI and NotI (NEB, USA) and then ligation with T4 DNA ligase. The constructed pGAPZα C vector carrying bgl-Cr-D2 cDNA was transformed into  E. coli  (strain DH5α) for preservation. Prior to subsequently transforming into  Pichia host,  the pGAPZα C vector with D2-bgl cDNA was purified with Plasmid Miniprep Purification Kit (GeneMark, Taiwan) and linearized with B spHI (NEB, USA). The linear plasmid DNA was transformed into  Pichia pastoris  (strains GS115 and SMD1168) via homologous recombination based on protocol for  Pichia  Expression Kit (Invitrogen, USA). The reconstructed Pichia pastoris yeast cells were grown in YPD zeocin100  medium and the conditions (e.g., 30° C. and 200 rpm) suggested in the Invitrogen protocol.  Pichia  cells and their enzymes were collected at different time points (0-14 days) to assess their growth and enzyme activity/quantity. 
       6. Activity Tests 
       [0057]    Cellulolytic activities of crude enzymes from native D2 strain and recombinant enzymes from Pichia pastoris were tested using different substrates (p-nitrophenyl β-D-glucopyranoside, pNPG; cellobiose; carboxymethylcellulose, CMC; xylan; avicel; and, filter paper). The pNPGase activities of the enzymes were measured based on pNP releasing rate from 2 mM pNPG at 55° C. in 5 minutes. The pNP concentrations were calibrated based on spectrophotometric absorbance at OD 405 . The cellobiase activities were assessed by cellobiose reducing rate and/or glucose producing rate where cellobiose and glucose concentrations were determined by HPLC (high performance liquid chromatography) analyses. Additionally, CMCase (for endo-glucanase activity), xylanase (for xylose hydrolyzing activity), avicelase (for exo-glucanase activity), and FPase (representing total cellulase activity) were measured based on production rate of reducing sugars that were detected by DNS (dinitrosalicyclic acid) method. 
       II. Results 
       [0058]    In this invention, a Taiwan-indigenous fungus  Chaetomella raphigera  strain D2 was first found capable of secreting crude enzyme with significant β-glucosidase activity based on assessment using pNPG (p-nitrophenyl-β-D-glucopyranoside) and cellobiose as substrates. The gene (bgl-Cr-D2) encoding the target β-glucosidase (BGL-Cr-D2) was derived from genomic DNA and reverse-transcribed cDNA. It showed that the bgl-Cr-D2 cDNA had a size of 2166 bps (not including signal sequence and stop codon) and the bgl-Cr-D2 genomic DNA has a size of 2851 bps ( FIGS. 1 &amp; 2 ). Twelve introns were identified after comparing the bgl-Cr-D2 genomic DNA with cDNA. The bgl-Cr-D2 gene would encode 722 bases of amino acid to form the BGL-Cr-D2 ( FIG. 2 ). Based on the comparison of the amino acid sequences for the β-glucosidases derived from glycoside hydrolase (GH) family 3, the newly-found BGL-Cr-D2 was homologous to the β-D-glucoside glucohydrolase of  Talaromyces stipitatua  (65% similarity) and  Aspergillus terreus  (64% similarity) ( FIG. 3 ). Based on the phylogenetic analyses,  C. raphigena  D2,  T. stipitatua,  and  A. terreus  formed a sub-group that could distinguish from other fungal and bacterial groups (e.g.,  Penicillium, Sacharomycopis, Clostridium,  and  Escherichia coli ). 
         [0059]    The bgl-Cr-D2 gene was further cloned into pGAPZαC vector ( FIG. 4 ) and transformed to a yeast expression system using  Pichia pastoris  strain SMD1168 as host cells. When compared the growth and pNPGase activity for the  Pichia pastoris  strain SMD1168 and that carrying bgl-Cr-D2, significant pNPGase activity was observed only for the colony cloned with bgl-Cr-D2 while the growth profiles were similar for the both colonies ( FIG. 5 ). The growth and activity reached plateau levels after incubating at 30° C. for around 4-6 days. Furthermore, the recombinant β-glucosidase (BGL-Cr-D2-Pp) had a molecular size of around 95 kDa ( FIG. 6C ) (larger than theoretical size of 79 kDa due to post-translational glycosylation); and, showed high specific activity based on assessments of saccharifying rate (Table 1) and observations from native PAGE (polyacrylamide gel electrophoresis) (FIG.  6 A)/MUG (4-methylumbelliferyl-β-D-glucopyranoside) zymogram ( FIG. 6B ). 
         [0060]    The recombinant BGL-Cr-D2-Pp remained active after 20-hour incubation under a wide pH range (i.e., pH4-pH9) ( FIG. 7 ). However, the pNPGase activity of this enzyme significantly decreased at pH levels higher than 5.5. Besides, the BGL-Cr-D2-Pp had highest pNPGase activity at a temperature of 75° C.; but, at this high temperature, the activity dropped tremendously after short-term incubation (i.e., 2 hours) ( FIGS. 8 &amp; 9 ). Thus, the pNPGase activities of the recombinant BGL-Cr-D2-Pp were performed at pH 5 and a temperature of 55° C. for 10 minutes. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Comparison of enzyme activities for native BGL-Cr-D2,  Pichia pastoris   
               
               
                 strain SMD1168 secretes, recombinant BGL-Cr-D2-Pp secreted from 
               
               
                   P. pastoris , and Novozyme-188 for different substrates (all 
               
               
                 reactions were performed at pH 5 and 55° C.). 
               
             
          
           
               
                   
                 Reaction 
                   
                   
                   
                   
               
               
                   
                 time 
                 BGL-Cr- 
                 Pp 
                 BGL-Cr- 
               
               
                 Substrates 
                 (minutes) 
                 D2 
                 SMD1168 
                 D2-Pp 
                 N-188 
               
               
                   
               
             
          
           
               
                   
                 Activity (U**/mg BGL) 
               
             
          
           
               
                 2 mM pNPG* 
                 5 
                 1,029.9 
                 41.1 
                 2,963.9 
                 171.7 
               
               
                 2 mM 
                 5 
                 — 
                 — 
                 57.5 
                 11.1 
               
               
                 Cellobiose 
               
             
          
           
               
                   
                 Activity (U/mg enzymes †† ) 
               
             
          
           
               
                 1% CMC †   
                 30 
                 121.1 
                 1.0 
                 0.7 
                 1.1 
               
               
                 1% Xylan 
                 30 
                 186.9 
                 0.0 
                 3.8 
                 3.4 
               
               
                 1% Avicel 
                 120 
                 0.0 
                 0.2 
                 0.0 
                 0.0 
               
               
                 FP ‡   
                 120 
                 0.1 
                 0.0 
                 0.1 
                 0.0 
               
               
                   
               
               
                 *p-nitrophenyl-β-D-glucopyranoside; 
               
               
                   † carboxymethylcellulose; 
               
               
                   ‡ filter paper; 
               
               
                 **1 U = 1 μmole pNP produced from pNPG, glucose produced from cellobiose, or reduced saccharides produced from CMC, xylan, avicel or FP per minute; 
               
               
                   †† total enzymes except BGL. 
               
             
          
         
       
     
       Other Embodiments  
       [0061]    All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. 
         [0062]    A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.