Patent Publication Number: US-9404136-B2

Title: A-xylosidase enhanced conversion of plant biomass into fermentable sugars

Description:
This application claims benefit of the filing date of U.S. Provisional Application Ser. No. 61/665,513, filed Jun. 28, 2012, the contents of which are specifically incorporated herein by reference in their entirety. 
    
    
     STATEMENT OF GOVERNMENTAL SUPPORT 
     This invention was made with government support under Grant Nos. DE-FG02-91ER200021 and DE-FC02-07ER64494 by the U.S. Department of Energy. The government has certain rights in the invention. 
    
    
     FIELD OF THE INVENTION 
     This invention is related to the production of biofuels by converting lignocellulosic materials into fermentable sugars. For example, the release of fermentable sugars from a plant biomass may be enhanced using an α-xylosidase enzyme. Such an α-xylosidase enzyme efficiently facilitates degradation of xyloglucan (a major component of biomass from plant cell walls) by cellulase enzymes into xylose and glucose. Without addition of α-xylosidase, commercial cellulase mixtures do not convert xyloglucan to glucose and xylose. 
     BACKGROUND 
     Microbial enzymes exist for catalyzing the depolymerization of plant cell wall polysaccharides allowing the carbon in the plant cell walls to be recycled into free sugars that ultimately are metabolized to CO 2 . An emerging application of microbial enzymes is for conversion (e.g., deconstruction or digestion) of lignocellulosic materials (e.g., corn stover) into fermentable sugars useful for ethanol production. However, while mixtures of microbial enzymes have been isolated from fermentation vats of  Trichoderma , such mixtures are expensive and do not contain optimal amounts or types of enzymes. The high cost of commercially available enzyme mixtures is currently a significant barrier to the development of a viable lignocellulosic biofuel industry. See, e.g., Banerjee et al., Bioenergy Res. 3:82-92 (2010); and Yang et al., Biofuels 2:421-450 (2011). 
     What are needed are methods and compositions that make the lignocellulosic conversion enzyme mixtures more efficient, thereby reducing their cost when expressed as dollars per gallon of ethanol. 
     SUMMARY 
     This invention is related to the production of biofuels by converting lignocellulosic materials into fermentable sugars. α-Xylosidase can be used to improve currently available enzymatic conversion products, and reduce the expense of such conversion so that fermentable sugars from a plant biomass can be obtained more efficiently and less expensively. For example, addition of α-xylosidase to cellulase mixtures can lead to enhanced degradation of xyloglucans into xylose and glucose. Commercially available enzyme mixtures degrade xyloglucans only partially. The resulting product contains significant amounts of disaccharides of glucose and xylose called isoprimeverose, which most microorganisms (e.g., yeast) cannot ferment to fuels such as ethanol. Fermenting microorganisms typically can ferment only free glucose and other monosaccharides such as xylose. Appropriate pretreatment (such as alkaline hydrogen peroxide or acid) of lignocellulosic materials can also improve the release of fermentable sugars by mixtures of enzymes that include α-xylosidase. 
     One aspect of the invention is a composition or enzyme mixture comprising an isolated α-xylosidase. In some embodiments, the isolated α-xylosidase is a purified α-xylosidase. The enzyme mixture can include an isolated α-xylosidase with at least one other enzyme (e.g., one or more cellulases). For example, the mixture can include at least one cellulose converting or depolymerizing enzyme, at least one cellulase, and/or at least one other enzyme that can cleave linkages found in the polysaccharides of plant cell walls. Examples of enzymes can, for example, be selected from the group consisting of a cellobiohydrolase, an endoglucanase, a polysaccharide monooxygenase (e.g., cel61, see NCBI accession no. AY094489.1 GI:21694046), an endoxylanase, a β-glucosidase, a β-1,4-glucanase, a β-galactosidase, an α-fucosidase, a β-galactosidase, an endoxylanase, a β-xylosidase, α-arabinosidase, α-glucuronidase, polysaccharide mono-oxygenase, an esterase and combinations thereof. Such a cellulose enzyme mixture or composition can have at least 5%, or at least 10%, or at least 15% cellulase or at least 20%, or at least 25% cellulase, or at least 30% cellulase, or at least 40% cellulase, or at least 50%, or at least 60% cellulase. 
     In some embodiments, the mixture can include at least two, or at least three cellulose depolymerizing enzymes or cellulases. 
     The α-xylosidase can be a secreted enzyme. The α-xylosidase can have substantially no quaternary structure. In one embodiment, the α-xylosidase has a pH optimum of approximately 4.0. In one embodiment, the α-xylosidase has a temperature optimum of approximately 50° C. The α-xylosidase can be obtained or cloned from a fungal, or bacterial species. In one embodiment, the α-xylosidase is derived from a fungal extracellular extract. In one embodiment, the fungal extracellular extract is derived from an  Aspergillus niger  extracellular extract. In one embodiment, the  Aspergillus niger  secreted α-xylosidase is Aspni5|43342 (DOE-JGI database) or has the GenBank accession number DAA35002.1. 
     Another aspect of the invention is a method that includes: 
     a) providing; 
     i) a plant biomass that includes hemicellulose; and 
     ii) an enzyme mixture comprising an isolated α-xylosidase; and 
     b) incubating the biomass with the enzyme mixture to create a degradation product that comprises fermentable sugars. The enzyme mixture can include other enzymes such as cellulases, depolymerizing enzymes, and/or other enzymes that can cleave linkages found in the polysaccharides of plant cell walls. For example, the enzyme mixture can include a cellobiohydrolase, an endoglucanase, a polysaccharide monooxygenase (e.g., cel61, see NCBI accession no. AY094489.1 GI:21694046), an endoxylanase, a β-glucosidase, a β-1,4-glucanase, an α-fucosidase, a β-galactosidase, and combinations thereof. 
     In some embodiments, the method can also include c) identifying the percentage of free fermentable xylose and glucose residues in the degradation product; or c) isolating the free fermentable xylose and glucose residues from the degradation product. Such a method can further comprise treating the plant biomass with alkaline hydrogen peroxide or acid, for example, before incubation with the enzyme mixture. 
     Such a method can release substantial proportions of free fermentable sugars from the plant biomass. For example, such a method can release about 50%, or about 60%, or about 70%, or about 75%, or about 85%, or about 90%, or about 95% of free fermentable sugars contained within the plant biomass. In one embodiment, the degraded hemicellulose material is completely (e.g., 98%-99.9%) degraded by the enzyme mixture into a plurality of free fermentable xylose and glucose residues. 
     The plant biomass can be derived from a number of sources. For example, the plant biomass can be derived from a dicotyledonous plant. In another embodiment, the plant biomass can be derived from a monocotyledon plant. In one embodiment, plant biomass can be derived from grass or wood. In one embodiment, the plant biomass comprises corn stover. 
     The conditions employed for the plant biomass into fermentable sugar can vary. In one embodiment, the plant biomass is first exposed to a pretreatment such as alkaline hydrogen peroxide or sulfuric acid or ammonia. Incubation with the enzyme mixture can be performed at a temperature ranging from approximately 40° to approximately 50° C. In one embodiment, the incubation is performed at a pH ranging from approximately 4 to approximately 5. 
     DEFINITIONS 
     The term “converting enzyme mixture” as used herein, refers to a mixture that contains an isolated α-xylosidase and at least one, and preferably more than one, enzyme having catalytic activity directed towards cleavage of covalent bonds in plant biomass materials. For example, the at least one enzyme may hydrolyze saccharide linkages of an alpha or beta nature, to release free fermentable sugar residues including, but not limited to, glucose, galactose, mannose, fucose, or xylose. 
     The term “lignocellulose” as used herein, refers to any of several closely related substances comprising plant cell walls comprising sugar-based backbone polymers including, but not limited to, cellulose and/or hemicellulose. 
     The term “plant biomass” as used herein, refers to any collection of biological material derived from a plant source. 
     The term “secreted”, “secrete” and/or “secreting” as used herein, refers to the process of segregating, elaborating, and releasing some material (e.g., a protein or enzyme) from a cell or across a cell wall or membrane into the extracellular environment. 
     The term “extracellular” as used herein, refers to any product, compound or process situated or occurring outside a cell. 
     The term “degrade”, “degrading”, or “degraded” as used herein, refers to any process that reduces the complexity of a material (e.g., an organic chemical compound such as a polysaccharide) by splitting off one or more groups or larger components (e.g., free fermentable sugar residues). A material or product that is “degraded” has reduced complexity relative to the original material or product, for example, because polymers in the material or product have been converted (e.g., cleaved) into subunits (e.g., fermentable sugars) and/or oligomers (e.g., oligosaccharides). 
     The term “free, fermentable sugar residues” as used herein, refers to any hexose or pentose sugar moiety that can be metabolized by a biochemical catabolic pathway. For example, one biochemical catabolic pathway produces ethanol as an end product. In some embodiments, the hexose or pentose is underivatized. 
     The term “quaternary structure” as used herein, refers to a protein multi-unit complex that includes three dimensionally folded proteins and/or enzymes. 
     The term “xyloglucan” as used herein, refers to hemicellulose that occurs mainly in the primary cell wall of vascular plants having a backbone of β1→4-linked glucose residues, some of which are substituted with α1→6 linked xylose. About 60-75% (or, in grasses, about 30-40%) of the glucose residues have side-chains attached to position 6, and alpha-linked D-xylopyranosyl is one of the major moieties attached at position 6. The xylose residues are often capped with a galactose residue sometimes followed by a fucose residue. The specific structure of xyloglucan varies among plant families. Other side chains attached to the β-(1→4)-D-glucopyranose backbone include: D-galactopyranosyl-β-(1→2)-D-xylopyranosyl-α-(1→6), L-arabinofuranosyl-(1→2)-D-xylopyranosyl-α-(1→6), and (except in grasses) L-fucopyranosyl-α-(1→2)-D-galactopyranosyl-β-(1→2)-D-xylopyranosyl-α-(1→6). 
     The term “dicot” as used herein, refers to a group of flowering plants known as dicotyledons, whose seed typically has two embryonic leaves or cotyledons. 
     The term “monocot” as used herein, refers to a group of flowering plants known as monocotyledons, whose seed typically has one embryonic leaf or cotyledon. 
     The term “stover” as used herein, refers to the residual leaves, stalks and other above-ground plant materials left in a field after harvest, as well as other plants materials such as weeds and plant-derived waste (e.g., paper, cardboard, etc.). Stover makes up a substantial proportion of a crop (e.g., half or more of a crop such as wheat or maize). Stover may be derived from any plant source including but not limited to, corn, peas, carrots, grasses, recycled paper, recycled cardboard, and the like. 
     The term “derived from” as used herein, refers to the source of a compound or sequence. In one respect, a compound or sequence can be derived from an organism or particular species. In another respect, a compound or sequence may be derived from a larger complex or sequence. 
     The term “protein” as used herein, refers to any of numerous naturally occurring extremely complex substances (as an enzyme or antibody) that consist of amino acid residues joined by peptide bonds, and containing the elements carbon, hydrogen, nitrogen, oxygen, and usually sulfur. A protein is generally larger than a peptide. For example, a protein can comprise more than 100 amino acids. 
     The term “peptide” as used herein, refers to a short polymer of amino acids where various amino acids are linked by amide bonds formed between the amino group of one acid with the carboxyl group of another. Peptides can be obtained by partial hydrolysis of proteins. For example, a peptide can comprise about 10-100 amino acids. 
     The term, “purified” as used herein, refers to any molecule or compound (e.g., a proteinaceous enzyme, such as an α-xylosidase) that has been subjected to treatment (for example, fractionation) to remove various components with which it is naturally associated or with which it is naturally secreted. Such a purified molecule or compound substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the molecule or compound forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition (for example, weight/weight and/or weight/volume). The purified molecule or compound (e.g., an α-xylosidase) can be purified from an  Aspergillus niger  extracellular extract. The term “purified to homogeneity” is used to include a molecule or compound that has been purified to “apparent homogeneity” such that there is single molecule or compound species (for example, based upon SDS-PAGE or HPLC analysis). A purified composition can contain some trace impurities. A purified composition includes the molecule or compound with a carrier. 
     The terms “amino acid sequence,” “protein sequence” and “polypeptide sequence” as used herein, are interchangeable and to refer to a sequence of amino acids. 
     As used herein the term “portion” when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments can range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. 
     The term “derivative” as used herein, refers to any chemical modification of a nucleic acid, sugar, polysaccharide or an amino acid. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. 
     The terms “homology” and “homologous” as used herein in reference to nucleic acid and/or amino acid sequences refer to the degree of identity of the primary structure between two sequences. Such a degree of identity may be directed a portion of each sequence, or to the entire length of the sequence. Two or more nucleic acid or two or more amino acid sequences that are “substantially homologous” may have at least 50% identity, preferably at least 75% identity, more preferably at least 85% identity, most preferably at least 95%, or 100% identity. 
     ABBREVIATIONS 
     AHP, alkaline hydrogen peroxide; Ara, arabinose; BSA, bovine serum albumin; Fuc, fucose; Gal, galactose; Glc, glucose; IgG, immunoglobulin; IP, isoprimeverose; Man, mannose; pNPαX, p-nitrophenyl-α-xyloside; Xyl, xylose; XG, xyloglucan; xyloglucan heptasaccharide, a chain of four glucose residues linked together by beta 1,4 linkages where three of the glucose residues are linked by alpha 1,6 linkages to a xylose (shorthand notation XXXG). 
    
    
     
       DETAILED DESCRIPTION OF THE FIGURES 
       In the figures, “% of maximum” means the Glc or Xyl released as a percentage of the total Glc and Xyl content of the biomass, as determined by the methods cited in Banerjee et al.,  Bioresour. Technol.  101: 9097-9105 (2010). 
         FIG. 1A  illustrates exemplary structures of xyloglucan molecules from dicots and grasses. A secreted α-xylosidase (Ax1A) described herein cleaves the α1,6-linked xylose (Xyl) from the glucan backbone. See, Kato et al., Plant Cell Physiol 23: 351 (1982); M. Pauly and K. Keegstra (2008) Cell-wall carbohydrates and their modification as a resource for biofuels. Plant J. 54:559-568.  FIG. 1B  illustrates release of free glucose and xylose from tamarind xyloglucan by commercial enzyme mixtures as assessed by the percentage of the total glucose and xylose released. MP, Multifect Pectinase; Acc1000, Accellerase 1000; MX, Multifect Xylanase; Acc1000+MX, 50:50 mixture of Accellerase 1000 and Multifect Xylanase; and CTec2+HTec2, 50:50 mixture. CMAX (a product of Dyadic, Inc.) did not release glucose or xylose from tamarind xyloglucan. 
         FIG. 2  is a bar graph illustrating that a secreted α-xylosidase described herein increases glucose (Glc) release from corn stover when mixed with an 11-component synthetic enzyme mixture (11C) at equal total protein loading. The 11C enzyme mixture is described by Banerjee et al.  Bioresource Technology  101:9097-9105 (2010). The numbers in parentheses indicate the mg of protein/gm glucan for each mixture. Corn stover was incubated with the indicated enzyme mixtures for 24 or 48 hr and the released monomeric glucose was measured. The 11-component mixture alone (11C) released 60% of available glucose in 48 hr; a 50:50 mixture of 11C supplemented with secreted α-xylosidase released 65% of the available glucose. Therefore, use of the α-xylosidase allowed 5% more glucose to be obtained with the same total protein loading. Total enzyme concentration was kept constant in all experiments at a total protein loading of 15 mg/g glucan. Numbers in parentheses on the x-axis are the individual loading enzyme concentrations in mg/g glucan for each assay. The numbers above the data bars are the actual glucose values, given as a percentage of the total maximum possible yield. For the composition of the 11-C mixture, see Banerjee et al.,  Bioresour Technol  101:9097-9105 (2010). Control: no enzymes. Abbreviations: 11C: 11-component synthetic enzyme mixture; AXL: α-xylosidase (Aspni5|43342; NCBI accession no. DAA35002.1). 
         FIG. 3  presents exemplary data showing that the α-xylosidase as described herein increases glucose yield from alkaline hydrogen peroxide (AHP) treated corn stover when added to the commercially available Accellerase® 1000 (Acc1000) enzyme mixture. Acc1000 loading was 10 mg/g glucan. The α-xylosidase (Ax1A) increased the yield of glucose from 76% of maximum possible yield to 85%, an absolute increase of 9%. Ax1A: α-xylosidase (Aspni5|43342; GenBank DAA35002.1). 
         FIG. 4  presents exemplary data showing a dose response effect of the α-xylosidase (Ax1A) described herein on glucose yield in combination with a commercially available enzyme mixture composed of 50:50 Cellic® CTec2 and Cellic® HTec2 (“Tek2” in the figure) at different loadings of 0, 0.4, 1 and 2.5 mg/g glucan. The stimulating effect of α-xylosidase is most pronounced at the 2.5 mg/g CTec2:HTec2 loading. 
         FIG. 5  graphically illustrates an expanded scale of the CTec2:HTec2 2.5 mg/g loading data from  FIG. 4 . 
         FIG. 6  graphically illustrates a secreted α-xylosidase (Ax1A) dose response curve for glucose release when combined with various proportions of CTec2 and HTec2. Total CTec2+HTec2 loading in every case was 1 mg/g glucan. As the concentration of α-xylosidase was increased, glucose release was enhanced at almost all proportions of CTec2 and HTec2, including 100:0 (diamond symbols), 50:50 (triangle symbols), and 75:25 (square symbols), but not 25:75 (X symbols). 
         FIG. 7  illustrates enhancement of xylose yields from alkaline hydrogen peroxide-treated corn stover by α-xylosidase (Ax1A) used in combination with a CTec2:HTec2 mixture (75:25) at an enzyme loading of 2.5 mg/g glucan. The data are from the same experiment shown in  FIG. 4  at expanded scale 
         FIG. 8  illustrates a time course of enzymatic hydrolysis of alkaline-hydrogen peroxide-pretreated corn stover with or without supplementation with α-xylosidase (Ax1A), as detected by glucose release from the corn stover. The α-xylosidase loading was 0 or 4 mg/g glucan. CTec2 and HTec2 (in the proportion 75:25) loading was 0.8 or 2 mg/g glucan. Addition of the α-xylosidase (+Ax1A) enhanced glucose yield (solid lines) compared to the glucose release without addition of α-xylosidase (−Ax1A; dashed lines). 
         FIG. 9  illustrates enhancement of xylose (XY) yields from alkaline hydrogen peroxide-treated corn stover in response to addition of α-xylosidase (Ax1A) to a CTec2:HTec2 mixture (enzyme loading concentration of 1 mg/g glucan) at the indicated proportions. 
         FIG. 10A-B  presents exemplary data of a final purification step of α-xylosidase by hydrophobic interaction chromatography.  FIG. 10A  shows the ultraviolet absorption of proteins eluted.  FIG. 10B  correlates α-xylosidase and β-glucosidase (βG) activities with the elution fraction number, where the α-xylosidase and β-glucosidase (βG) were determined using pNPαX and p-nitrophenyl-β-D-glucoside (pNPβG), respectively, as substrates. mAU=milliabsorbance units. 
         FIG. 11A-B  shows proteins separated by SDS-PAGE after isolation and purification of the secreted α-xylosidase enzyme (Ax1A) described herein. The standards and unknowns are from the same gel. The gels were stained with Coomassie Blue.  FIG. 11A : Native α-xylosidase enzyme purified from  Aspergillus niger .  FIG. 11B : Recombinant secreted α-xylosidase enzyme expressed in  Pichia pastoris.    
         FIG. 12A-B  illustrate the pH and temperature optima for α-xylosidase (AXL) enzyme activity.  FIG. 12A  graphically illustrates the pH dependence of secreted α-xylosidase (Ax1A) enzyme activity. Assays were performed with 10 mM pNPαX at 50° C. for 30 min.  FIG. 12B  graphically illustrates the response of α-xylosidase (Ax1A) enzyme activity to temperature, where the enzyme concentration was 88 ng/ml, the concentration of pNPαX was 2.5 mM, and the reaction time was 60 min. 
         FIG. 13  presents exemplary data showing the digestion of xyloglucan heptasaccharide (a chain of four glucose residues linked together by beta 1,4 linkages where three of the glucose residues are linked by alpha 1,6 linkages to a xylose residue (shorthand notation XXXG)) to free xylose by the α-xylosidase (AXL) described herein (dashed line with large filled triangles), by β-glucosidase (dashed, dotted line with small filled triangles), or by a 50:50 combination of the two (diamond symbols). The concentration of the heptasaccharide was 1 mg/ml. Total protein concentration was 30 μg/ml. The results show that either enzyme alone cannot completely degrade xyloglucan heptasaccharide, but a combination of the two can. The xylose released by the α-xylosidase alone is consistent with the α-xylosidase being able to remove one alpha-linked xylose residue but not the other two without the intervening action of β-glucosidase. 
         FIG. 14  shows that α-xylosidase (Ax1A) supplementation of a 75:25 mixture of CTec2 and HTec2 strongly enhances release of free glucose from purified pea xyloglucan. Each reaction contained 10 μg xyloglucan, 50 ng of the CTec2:HTec2 mixture, and 80 ng α-xylosidase in a total volume of 50 μl. The free glucose content in 10 μl of the reaction mixture was measured at each time point by an enzyme-linked assay (Banerjee et al.,  Bioresour. Technol.  101: 9097-9105 (2010); Scott-Craig et al. ( J Biol Chem  286:42848-42854 (2011)). 
         FIG. 15  illustrates glucose release from tamarind xyloglucan as a function of α-xylosidase (Ax1A) concentration. The α-xylosidase was combined with 75:25 CTec2:HTec2 loaded at 2.5 mg/g glucan using a reaction volume of 500 μl. The xyloglucan concentration was 3 mg/ml, giving a maximum possible yield of 1.5 mg glucose/ml. 
         FIG. 16  shows that supplementation of the CTec2:HTec2+α-xylosidase enzyme mixture with β-galactosidase (B) improves yields of glucose from tamarind xyloglucan, compared to α-xylosidase alone (A), and/or a mix of CTec2:HTec2 (C). The tamarind xyloglucan concentration was 3 mg/ml, the CTec2:HTec2 (75:25) loading was 2.5 mg/g glucan, and the α-xylosidase and β-galactosidase loadings were each 8 mg/g glucan. A, α-xylosidase; B, β-galactosidase; C, CTec2:HTec2. 
         FIG. 17  shows that α-xylosidase (Ax1A) supplementation of CTec2:HTec2 improves glucose yields from alkaline hydrogen peroxide-pretreated corn stover. The CTec2:HTec2 ratio was 100:0. The CTec2:HTec2 loading was 0, 0.4, 1.0, or 2.5 mg/g glucan and the incubation time was 48 hr. Similar results were obtained with a 75:25 ratio of CTec2:HTec2 (data not shown). 
         FIG. 18A-18B  illustrate the effect of α-xylosidase supplementation on sugar yields, shown in expanded scale.  FIG. 18A  illustrates glucose release in an expanded scale of the results from  FIG. 17  for 2.5 mg/g glucan CTec2:HTec2.  FIG. 18B  illustrates xylose release from the same experiment shown in expanded scale. 
         FIG. 19A-19B  shows that α-xylosidase (Ax1A) supplementation also enhances the activity of the cellulase enzyme mixture known as Accellerase 1000.  FIG. 19A  illustrates glucose yield at 24 hr and 48 hr hydrolysis times from AHP-pretreated corn stover in response to 5 mg/g glucan Accellerase 1000 and the indicated concentrations of Ax1A.  FIG. 19B  shows xylose yields from the same experiment. 
         FIG. 20  illustrates that α-xylosidase-enhanced sugar release is not just a general protein effect. “CTec2” indicates a 75:25 mixture of CTec2 and HTec2 at a loading of 2.5 mg/g glucan. Neither BSA nor IgG stimulated glucose yields in response to CTec2:HTec2, nor did either protein affect the enhancement by α-xylosidase. The loadings of α-xylosidase (Ax1A), bovine serum albumin (BSA), and bovine immunoglobulin (IgG) were 8 mg/g glucan. Lowercase letters above the data bars indicate significantly different or not from CTec2:HTec2 alone (P&lt;0.05 in Tukey&#39;s multiple comparison test, n=6). 
         FIG. 21  shows that α-xylosidase supplementation does not enhance glucose yield from pea ( Pisum sativum ) biomass. Etiolated (dark-grown) and green (light-grown) peas (all above-ground parts) were not washed before or after AHP pretreatment. “+CTec2” indicates that 15 mg/g glucan 75:25 CTec2:HTec2 was used; “−CTec2” indicates neither CTec2 nor HTec2 was used. Within each group of four data bars, the mg α-xylosidase/g glucan loadings increased from left to right: 0 mg α-xylosidase/g glucan (first bar), 4 mg α-xylosidase/g glucan (second bar), 8 mg α-xylosidase/g glucan (third bar), and 16 mg α-xylosidase/g glucan (fourth bar). 
         FIG. 22A-22B  show that α-xylosidase (Ax1A) supplementation enhances glucose and xylose yields from the herbaceous dicot (forb) lamb&#39;s quarters ( Chenopodium album ).  FIG. 22A  shows glucose yield, while  FIG. 22B  shows xylose yield. The CTec2:HTec2 ratio was 75:25. Hydrolysis time was 48 hr. 
     
    
    
     DETAILED DESCRIPTION 
     This invention is related to the production of biofuels by converting cellulosic materials into fermentable sugars. For example, the release of fermentable sugars from a plant biomass may be enhanced using an extracellular and/or secreted α-xylosidase fungal enzyme. For example, the secreted α-xylosidase fungal enzyme efficiently degrades xyloglucans into xylose and glucose, a compound typically not degraded to xylose and glucose by most commercially available enzyme mixtures. Additionally, chemical and/or heat pretreatment of the plant biomass (e.g., with alkaline hydrogen peroxide), further enhances the release of fermentable sugars from a lignocellulose material by an α-xylosidase and other enzymes. 
     In one embodiment, the present invention contemplates a composition comprising an α-xylosidase and a plurality of microbial enzymes that can depolymerize plant biomass materials. In one embodiment, the α-xylosidase is a secreted α-xylosidase. For example, the α-xylosidase can be derived from a fungal species. Addition of a secreted α-xylosidase to a plurality of microbial enzymes provides a novel enzymatic activity that is not present in current commercial cellulase mixtures, and increases fermentable sugar release from a plant biomass. Commercially, such an increased sugar release lowers the overall cost of biofuel production (expressed as dollars of enzyme needed per liter of fuel). 
     One embodiment of the invention is a method for expressing α-xylosidase in vitro (e.g. in a cell culture vat, bioreactor or fermenter) that involves obtaining a host cell that includes an isolated nucleic encoding an α-xylosidase enzyme and culturing the host cell for a time and under conditions for expression of the α-xylosidase enzyme from the isolated nucleic acid. The α-xylosidase enzyme can be a secreted α-xylosidase. Such a secreted α-xylosidase enzyme can be isolated from cell culture medium without destruction of the host cells. For example, the cells can be removed and recycled, or the cell culture medium can be decanted, filtered or otherwise separated from host cells that are retained in the cell culture apparatus. The host cells can include bacterial, fungal, insect or other cell types. For example, the host cells can be yeast or filamentous fungi cells. Examples of suitable host cells include  Trichoderma reesei  cells,  Sporotrichum thermophile  cells,  Pichia pastoris  cells,  Aspergillus niger  cells, and combinations thereof (see  FIG. 11 ). The isolated nucleic acid can also include a promoter operably linked to a nucleic acid segment that encodes the α-xylosidase enzyme. For example, the promoter can include a native secreted α-xylosidase gene promoter, an inducible promoter, a constitutively active promoter, a developmentally regulated promoter, a tissue-specific promoter, or a combination thereof. In one embodiment, the method includes simultaneous production of a plurality of enzymes, for example, in a cell culture that includes a plurality of host cells (of the same of different species) that express a plurality of enzymes. 
     The results described herein indicate that α-xylosidase can be more effective at the “limit” of glucose and xylose production, i.e., when glucose and xylose yields are highest due to extended hydrolysis time or to high enzyme loadings. For example, the α-xylosidase can catalyze the final step in the release of glucose and xylose from xyloglucan. 
     I. Conventional Plant Biomass Degradation 
     Currently, the production of ethanol and/or other biofuels derived from a lignocellulosic material begins with the conversion of the lignocellulosic material into free, fermentable sugar compounds (e.g., glucose, xylose etc.). Usually, this conversion (also referred to as deconstruction) is performed with a mixture of microbial enzymes. Many of these lignocellulosic-depolymerizing microbial enzymes can be obtained from fungi. An example of a fungal species from which these lignocellulosic-depolymerizing enzymes can be obtained includes, but are not limited to  Trichoderma reesei  and  Sporotrichum thermophile . Pre-made microbial enzyme mixtures, containing more than eighty (80) proteins, are commercially available (i.e., for example, Accellerase 1000 and Spezyme CP) and generally made by expression from  Trichoderma reesei  host cells. However, one technical disadvantage of these commercial mixtures is that their effectiveness is limited to the specific catalytic activity of each individual enzyme. For example, if one wishes to degrade cellulose, the microbial enzyme mixture must contain a β-1,4-glucanase. Similarly, if one wishes to degrade xylan, the microbial enzyme mixture should contain a β-1,4-xylanase. Use of a variety of different enzymes allows release of more fermentable sugars. 
     Commercial enzyme mixtures generally have high levels of cellulases (for example, cellobiohydrolase, endoglucanase, and β-glucosidase), which degrade cellulose. However, these enzyme mixtures are suboptimal for degrading hemicelluloses. 
     Hemicelluloses are structurally more complex than cellulose and can have different monosaccharides. Moreover, different plant species and different parts of the same plant can have different types of hemicelluloses. 
     One of the major types of hemicellulose in the primary walls of herbaceous dicotyledons is xyloglucan. Xyloglucan comprises a backbone of β-1,4-glucose substituted with α-1,6-linked xylose, β-linked galactose, and in some plants, α-linked fucose. Hsieh et al.,  Mol. Plant.  2:943-965 (2009). Another hemicellulose, glucuronoarabinoxylan, is present with xyloglucan in some grasses (e.g., the Poaceae family). Most plants comprise α-linked xylose sugars in polysaccharide xyloglucan complexes. Xyloglucan is comprised of a hemicellulose residing in the primary cell walls of all plants. Furthermore, xyloglucan may or may not be substituted with galactose (Gal) and/or fucose (Fuc). For example, in some grasses, xyloglucan is less substituted, typically lacking galactose or fucose. It has been observed that xyloglucan in some grasses has reduced numbers of xylose, galactose and/or fucose substitutions compared with other plant species. Hayashi T.,  Annu. Rev. Plant Physiol. Plant Mol. Biol.  40:139-168 (1989); see  FIG. 1 . As described herein, xyloglucan is substituted with xylose, a sugar that cannot be released efficiently with any commercially available cellulase enzyme mixtures. Considering that plant cell-derived xyloglucan comprises metabolizable sugars (e.g., fermentable sugars) an efficient mixture for biomass deconstruction of xyloglucan-containing plant biomass should have the full range and proper proportions of enzymes needed for its degradation. For example, these mixtures should contain enzymes capable of efficiently cleaving xylose residues from the xyloglucan backbone structure. 
     Although it is not necessary to understand the mechanism of an invention, it is believed that degrading xyloglucan hemicellulose is advantageous for two reasons: i) hemicelluloses inhibit cellulase degradation of cellulose by blocking cellulase access to cellulose; and ii) hemicelluloses comprise fermentable sugars, including but not limited to, glucose, xylose, galactose, fucose, and mannose. 
     Some commercial enzyme mixtures comprise hemicellulose degrading enzymes, including but not limited to, β-1,4-xylanase, β-xylosidase, α-arabinosidase, mixed-linked glucanase, α-glucuronidase, etc. In contrast, the most common commercial enzyme mixtures (e.g., Spezyme CP, Accellerase® 1000, Multifect Xylanase, Cellic® CTec2, HTec2, CTec3, HTec3, and AlternaFuel® CMAX) do not include an α-xylosidase enzyme that has catalytic activity directed to hydrolyzing α-linked xylose (Xyl) residues from substrates such as isoprimeverose, or xyloglucan ( FIG. 1B  and data not shown). 
     A complete deconstruction of xyloglucan can involve use of multiple enzymes including, but not limited to: i) α-fucosidase to remove a terminal fucose residue; ii) β-galactosidase to remove a penultimate galactose; iii) α-xylosidase to remove an α-1,6-linked xylose residue, preferably a secreted α-xylosidase; and iv) a β-1,4-glucanase and/or a β-glucosidase to depolymerize a glucan backbone. Some β-1,4-glucanases have xyloglucanase activity, i.e. they can hydrolyze β-1,4-glucan linkages in substituted glucans such as xyloglucan. However, other β-1,4-glucanases act only on unsubstituted β-1,4-glucans such as cellulose. Grishutin et al.,  Biochim. Biophys. Acta  1674:268-281 (2004). Neither β-1,4-glucanases nor xyloglucanases can release xylose from xyloglucan. This is a property only of an effective α-xylosidase. 
     Effective enzyme mixtures for biomass degradation and/or deconstruction should have a combined catalytic activity capable of cleaving any saccharide linkage found in plant cell walls to release free, fermentable sugar residues. Many microorganisms that live in lignocellulose-rich environments secrete large numbers and broad ranges of cell wall-active enzymes, including, but not limited to, cellulases, hemicellulases, pectinases, and/or proteases. Most commercially available deconstruction enzyme mixtures contain between approximately twenty-five to one hundred and fifty (25-150) enzymes. Nagendran et al.,  Fung. Genet. Biol.  46: 427-435 (2009); Banerjee et al.,  Bioresour. Technol.  101: 9097-9105 (2010); and Scott-Craig et al.,  J Biol Chem  286:42848-42854 (2011). However, these mixtures are not necessarily ideal with respect to the range of combined catalytic activities or the relative proportions of such catalytic activities. Such suboptimal ranges and proportions of catalytic activity limit the applicability of these commercially available enzyme mixtures. For example, the commercially available enzyme mixtures may work well with certain biomass types that have been subjected to certain pretreatment conditions. But the current commercially available enzyme mixtures are not effective for all types of biomasses. To achieve optimal release of fermentable sugars, diverse types of biomasses subjected to various pretreatment conditions will need an enzyme mixture containing diverse enzymes. 
     Superior and more efficient enzyme mixtures would ensure that the appropriate enzyme catalytic activity is present for any particular biomass being degraded. For example, although all higher plant cell walls contain cellulose, different plant species and even different tissues within a plant can have quite different hemicellulose compositions and proportions. Pauly et al.,  Plant J.  54:559-568 (2008). Hemicelluloses are present within many plant cell wall components including, but not limited to, xyloglucan, glucuronoarabinoxylan, mannan, galactan, arabinan, mixed-linked glucan, and/or glucuronoarabinoxylan. Carpita, N., and McMann, M. (2000), In: B IOCHEMISTRY AND  M OLECULAR  B IOLOGY OF  P LANTS  (Buchanan, B. B., Gruissem, W., and Jones, R. L., eds.) pp. 52-108, American Society of Plant Physiologists, Rockville, Md. Hemicelluloses contain a number of fermentable, or potentially fermentable, monosaccharides including, but not limited to, glucose, xylose, galactose, arabinose, mannose, fucose, rhamnose, and uronic acids. Many of these sugars are also found in pectins and wall proteins such as extensins and arabinogalactan proteins. 
     II. α-Xylosidase Mediated Plant Biomass Degradation 
     Amongst the cell wall active depolymerases, α-xylosidase is not a well understood enzyme because relatively few microbial α-xylosidase enzymes have been described in the literature. α-Xylosidase enzymes are classified in glycosyl hydrolase family 31 (as per the CAZy database), which also includes enzymes with a number of other activities, especially α-glucosidases. Henrissat et al,  Curr. Opin. Struct. Biol.  7:637-644 (1997). α-Xylosidase enzymes have been identified in various biological sources including, but not limited to, fungi, bacteria, and/or plants. Notably, distinguishing α-xylosidase enzymes from α-glucosidases based solely on nucleic acid and/or amino acid sequence information is not possible, so comparative biochemical data should be used for this purpose. Thus, an enzyme should be purified or cloned to permit testing and characterization of its enzymatic activity. 
     In most bacteria and fungi that can metabolize xyloglucan, extracellular enzymes first break the xyloglucan down to the disaccharide isoprimeverose, the isoprimeverose is imported into the cytoplasm, and then the isoprimeverose is broken down into free xylose and glucose using a cytoplasmic α-xylosidase. For example, the bacterium  Lactobacillus pentosus  has an isoprimeverose (IP) utilization operon, which includes an isoprimeverose transporter and a cytoplasmic α-xylosidase. Chaillou et al.,  J Bacterial.  180:2312-2320 (1998). Other bacteria have been reported to have α-xylosidase enzymes encoded in their genomes, for example: i)  Escherichia coli  (yicI) (Lovering et al.,  J Biol Chem  280:2105-2115 (2005)), ii)  Sulfolobus solfataricus  (xylS) (Moracci et al.,  J Biol Chem  275:22082-22089 (2000); and iii)  Cellvibrio japonicus  (xyl31A); Larsbrink et al.,  Biochem J  436:567-580 (2011); Okuyama et al., Protein Expr. Purif. 37:170-179 (2004). The prokaryotic cytosolic α-xylosidase from the archaean  Sulfolobus solfataricus  has been characterized, and has a preferred temperature of greater than 80° C., with low activity at 50° C. Consequently, one would not expect the α-xylosidase enzyme from  Sulfolobus solfataricus  (xylS) to improve the efficiency of commercially available lignocellulose depolymerizing enzyme mixtures from, for example,  Trichoderma  and/or  Aspergillus , which optimally degrade biomass between approximately 40-50° C. 
     In regard to fungi, the only α-xylosidase enzymes that have been studied are cytoplasmic, from  Aspergillus flavus, Aspergillus niger , and  Penicillium wortmanii , The α-xylosidases characterized from  A. niger  are cytoplasmic, not secreted, and therefore significantly different from the preferred α-xylosidase described herein. They also have quaternary structure and therefore would not be preferred for industrial applications, e.g., biomass deconstruction. Furthermore, the genes encoding any fungal α-xylosidase have not previously been unequivocally identified and/or characterized. Matsuo et al.,  Biosci. Biotechnol. Biochem.  60:341-343 (1996); Matsushita et al.,  Agric. Biol. Chem.  51:2015-2016 (1987); and Yoshikawa et al.,  Biosci. Biotechnol. Biochem.  58:1392-1398 (1994). For example, a gene referred to as AN7505, which purportedly encodes an α-xylosidase from  A. nidulans , was identified by expression in  Pichia pastoris . The function of AN7505 was not well characterized because it was tested only against the synthetic α-xylosidase substrate pNPαX, and not against a more complex and demanding substrate such as that found in lignocellulosic biomass. Bauer et al.,  Proc. Natl. Acad. Sci. U.S.A.  103:11417-11422 (2006). Substrates used to characterize α-xylosidase enzyme activity should include activity comparison of the substrates p-nitrophenyl-α-xyloside (pNPαX), isoprimeverose (IP), and xyloglucan oligosaccharides and polysaccharides. Furthermore AN7505 lacks a signal peptide and is therefore probably a cytoplasmic enzyme, and thus not suited to industrial biotechnology applications. 
     A. α-Xylosidase Enzymes 
     α-Xylosidase enzymes in plants may be involved in mobilization of seed storage xyloglucan and/or remodeling of cell wall xyloglucan. Nakai et al.,  J Biochem.  142:491-500 (2007); O&#39;Neill et al.,  J Biol. Chem.  264:20430-20437 (1989); and Sampedro et al.,  Plant Physiol.  126:910-920 (2001). These α-xylosidases are not of fungal origin, and would be less suited to industrial-scale conversion of xyloglucan to free glucose and xylose. One salient feature of the microbial α-xylosidase enzymes studied to date is that most (probably all) are intracellular (e.g., cytoplasmic) enzymes and few, if any, have been reported to be secreted free into the medium. Matsushita et al., Agric. Biol. Chem. 51:2015-2016 (1987). Further, it has been reported that Xyl31A ( C. japonicus ) is partially cytoplasmic and partially anchored to the outer cell wall. Larsbrink et al.,  Biochem. J.  436:567-580 (2011). The intracellular location of XylS of  S. solfataricus  has not been reported, but clustering of its encoding gene with a gene for a disaccharide transporter suggests that it is cytoplasmic, like the α-xylosidase of  L. pentosus . Moracci et al.,  J. Biol. Chem.  275:22082-22089 (2000). Consistent with a cytoplasmic location for most fungal α-xylosidase enzymes, the majority of the fungal proteins in GenBank™ that are annotated as belonging to GH31 lack predicted signal peptides. 
     Because intracellular fungal α-xylosidases are reported to be labile, comprise a quaternary structure, and lack a signal sequence, it is believed that intracellular fungi α-xylosidases are insufficiently robust to support industrial fermentation applications. Yoshikawa et al.  Biosci Biotechnol Biochem  57:1275 (1993); and Yoshikawa et al.  Biosci Biotechnol Biochem  58:1392 (1994). 
     B. Secreted Extracellular α-Xylosidase Enzymes 
     Unlike intracellular cytosolic α-xylosidases, the secreted α-xylosidase enzymes are structurally configured to survive in harsh and unstable extracellular environments and are therefore generally preferred over cytoplasmic proteins for the compositions and methods provided herein. In one embodiment, a composition is provided comprising a secreted, extracellular α-xylosidase. In one embodiment, the secreted α-xylosidase enzyme is derived from a fungus. In one embodiment, the fungus is  A. niger.    
     Extracellular α-xylosidases can be active at mesophilic temperatures (about 50° C.) and have an optimum pH close to that of other fungal enzymes (about pH 4.8). Furthermore, extracellular α-xylosidases can degrade isoprimeverose and xyloglucan oligosaccharides that are native components of plant cell walls. As a result, an α-xylosidase that is normally secreted as an extracellular fungal enzyme can be more robust than intracellular α-xylosidase enzymes. For an industrial process such as lignocellulose breakdown, which takes places in a potentially harsh environment, an extracellular enzyme is preferable. However, secreted fungal α-xylosidases are believed to be rare and have not previously been documented in the literature. 
     Even though some plants (i.e., for example, rice, nasturtium, and/or  Arabidopsis ) have secreted α-xylosidases that degrade storage xyloglucan and/or remodel their cell walls during growth, α-xylosidases from such plants would not be a preferred source for biomass degradation because it is more likely that a fungal α-xylosidase would be compatible with other fungal enzymes for biomass degradation applications. Crombie et al.,  Planta  214:406-413 (2002); and Nakai et al.  J Biochem  142:491-500 (2007). 
     1. Identification and Purification 
     The data presented herein evaluate several fungi for secreted α-xylosidase activity on a variety of substrates. These fungi were  Cochliobolus carbonum, Fusarium graminearum, Trichoderma reesei, Aspergillus niger , and  Phanerochaete chrysosporium . The fungi were cultured on ground tamarind seed (which contains high levels of xyloglucan), corn stover ( Zea mays ), pea cell walls ( Pisum sativum ), and carrot cell walls ( Daucus carota ) that were either supplemented or not supplemented with lactose or xylose for 5-14 days. No activity against pNPαX was seen in any of the resulting culture filtrates indicating that α-xylosidase was not being secreted under these growth conditions. The following commercial enzyme products were also examined: Accellerase 1000, Accellerase XY, Multifect Xylanase, Multifect Pectinase, Novozyme 188, CTec2, and HTec2. α-Xylosidase activity against pNPαX was not seen in any of the commercially available enzyme mixtures tested except Multifect Pectinase, which had a specific activity of 0.197 μmol/min/mg. Consistent with the presence of α-xylosidase activity in this preparation, and only in this preparation, degradation of tamarind xyloglucan to free xylose and glucose was observed.  FIG. 1B . Among all commercial enzyme mixtures tested, Multifect Pectinase was also the only one that exhibited activity against isoprimeverose. However, Multifect Pectinase is no longer available in the market, and it was a complex mixture of previously unidentified proteins. It was used in the food industry (e.g., for processing fruits) and no evidence is available that it has been used for digestion of cellulose-containing plant biomasses. Testing shows that Multifect Pectinase contained 4% or less of the protein as cellulase. Commercially available enzyme mixtures are typically made by growing fungal and/or bacterial cells and collecting the secreted enzymes. Thus, commercially available enzyme mixtures contain hundreds of enzymes. The amount and identity of the enzymes in the mixture is typically unknown. 
     To evaluate potential sources of α-xylosidases, mixtures were purified by High Performance Liquid Chromatography (HPLC) using three high resolution purification stages. A low level of β-glucosidase (βG) activity was consistently associated with the peak of α-xylosidase activity.  FIG. 10 . The secreted α-xylosidase activity peak did not contain any α-glucosidase or β-xylosidase activity as measured using pNPαG and p-nitrophenyl-β-D-xyloside, respectively. Other data indicates that the β-glucosidase activity was probably due to co-purification of a separate enzyme (infra). Their co-elution through multiple purification steps suggests that the two enzymes might form a complex in vivo. Although the secreted proteins of aerobic filamentous fungi are generally considered to be “noncomplexed,” evidence for the formation of complexes between the secreted enzymes of a filamentous fungus has been reported recently. Gonzalez-Vogel et al.,  Appl. Microbial. Biotechnol.  89:145-155 (2011). 
     The molecular weight of the secreted α-xylosidase enzyme by SDS-PAGE was about 85 kDa.  FIG. 11A . This secreted α-xylosidase enzyme has been identified as Aspni5|43342 (a numerical identification from the Department of Energy Joint Genome Institute) by proteomics. The dominant band was excised and subjected to tryptic digestion and mass spectrometric proteomics based on the whole predicted proteome of  A. niger  ATCC 1015 as the query database. Eight unique peptides amounting to 16% coverage of the Aspni5|43342 amino acid sequence were detected at about 95% probability. The only other protein detected was Aspni5|50997 which is an β-glucosidase in GH family 3. This might account for the residual β-glucosidase (βG) activity co-eluting with secreted α-xylosidase (supra), a conclusion that was supported by heterologous expression data (infra). 
     Unfractionated Multifect Pectinase enzyme mixture was also analyzed by mass spectrometric proteomics. At high confidence (about 95% probability), 132 proteins were identified (Table 4). More than 90% of the proteins have predicted signal peptides. Both Aspni5|43342 (secreted α-xylosidase) and Aspni5|50997 (βG) were detected (Table 4). However, Aspni5|56782 was the most abundant β-glucosidase (βG) in Multifect Pectinase (Table 4). In the JGI database, Aspni5|43342 is annotated as “Glycoside hydrolase family 31”. Before the invention, the precise biochemical function of Aspni5|43342 was not known. 
     Aspni5|43342 (identified in various databases as XP_001393647, An09g03300, CAK40270, jgi|Aspni5|43342, fgenesh1_pg.C_scaffold_11000279) is a predicted protein in GH family 31, a family which includes predominantly α-glucosidases and known or putative α-xylosidases. Such putative α-xylosidases may not actually have any α-xylosidase activity, and/or may not have adequate α-xylosidase activity. Unless significant sequence identity is present, testing is needed to definitively establish whether a protein has α-xylosidases activity. 
     The cytosolic protein AN7505 (Genbank DQ490509.1) of  A. nidulans  has minimal amino acid sequence identity with the extracellular Aspni5|43342 enzyme of  A. niger  (also referred to herein as Ax1A). Yuan et al. showed that the gene for Aspni5|43342 is induced by growth on xylose and speculated that Aspni5|43342 may be a secreted α-xylosidase because of its weak homology to AN7505. Yuan et al.,  Mol. Genet. Genomics  279:545-561 (2008). However, Yuan et al. presented no biochemical or enzymatic data to support such a conclusion. For example, a 25% amino acid identity between AN7505 and Ax1A as described herein is weak. Furthermore xylose-induction of α-xylosidase expression is contrary to accepted understandings of biochemical feedback mechanisms. Typically, expression of an enzyme is repressed, not induced, by the products of the enzyme (in this case xylose). This makes biological sense because when free xylose is present, the fungus does not need to make enzymes to produce xylose. This is the biological logic of why, for example, glucose represses the expression of cellulase genes. 
     Consequently, the data presented herein provide the first experimental evidence that isolated and purified Aspni5|43342 (Ax1A) is, in fact, a true secreted α-xylosidase that has its primary function in the extracellular environment. 
     The data presented herein identifies an extracellular (i.e., secreted) α-xylosidase with a predicted signal sequence extracted from a filamentous fungus (i.e., for example,  Aspergillus niger ; Aspni5|43342, XP_001393647, GI: 145242002, shown below as SEQ ID NO:1). 
     
       
         
           
               
               
            
               
                 1 
                 MYFSSFLALG ALVQAAAATY FAPNSTGLRI QHGFETILIQ 
               
               
                   
               
               
                 41 
                 PFGYDGFRVR AWPFRPPSGN EISFIYDPPI EGYEDTAHGM 
               
               
                   
               
               
                 81 
                 SYDTATTGTE PRTLRNGNII LRTTGWGGTT AGYRLSFYRV 
               
               
                   
               
               
                 121 
                 NDDGSETLLT NEYAPLKSLN PRYYYWPGPG AEFSAEFSFS 
               
               
                   
               
               
                 161 
                 ATPDEQIYGT GTQQDHMINK KGSVIDMVNF NSYIPTPVFM 
               
               
                   
               
               
                 201 
                 SNKGYAFIWN MPAEGRMEFG TLRTRFTAAS TTLVDYVIVA 
               
               
                   
               
               
                 241 
                 AQPGDYDTLQ QRISALTGRA PAPPDFSLGY IQSKLRYENQ 
               
               
                   
               
               
                 281 
                 TEVELLAQNF HDRNIPVSMI VIDYQSWAHQ GDWALDPRLW 
               
               
                   
               
               
                 321 
                 PNVAQMSARV KNLTGAEMMA SLWPSVADDS VNYAALQANG 
               
               
                   
               
               
                 361 
                 LLSATRDGPG TTDSWNGSYI RNYDSTNPSA RKFLWSMLKK 
               
               
                   
               
               
                 401 
                 NYYDKGIKNF WIDQADGGAL GEAYENNGQS TYIESIPFTL 
               
               
                   
               
               
                 441 
                 PNVNYAAGTQ LSVGKLYPWA HQQAIEEGFR NATDTKEGSA 
               
               
                   
               
               
                 481 
                 CDHVSLSRSG YIGSQRFCSM IWSGDTTSVW DTLAVQVASG 
               
               
                   
               
               
                 521 
                 LSAAATGWGW WTVDAGGFEV DSTVWWSGNI DTPEYRELYV 
               
               
                   
               
               
                 561 
                 RWLAWTTFLP FMRTHGSRTC YFQDAYTCAN EPWSYGASNT 
               
               
                   
               
               
                 601 
                 PIIVSYIHLR YQLGAYLKSI FNQFHLTGRS IMRPLYMDFE 
               
               
                   
               
               
                 641 
                 KTDPKISQLV SSNSNYTTQQ YMFGPRLLVS PVTLPNVTEW 
               
               
                   
               
               
                 681 
                 PVYLPQTGQN NTKPWTYWWT NETYAGGQVV KVPAPLQHIP 
               
               
                   
               
               
                 721 
                 VFHLGSREEL LSGNVF 
               
            
           
         
       
     
     A cDNA for the SEQ ID NO:1 protein is available from the NCBI database (www.ncbi.nlm.nih.gov) as accession number XM_001393610.1, GI:145242001, and provided below as SEQ ID NO:2. 
     
       
         
           
               
               
            
               
                 1 
                 ATGTACTTCT CTTCCTTCTT GGCCCTAGGG GCCTTGGTTC 
               
               
                   
               
               
                 41 
                 AGGCTGCAGC AGCAACCTAT TTTGCCCCCA ACTCTACCGG 
               
               
                   
               
               
                 81 
                 TCTTCGTATC CAGCATGGTT TTGAGACGAT TCTTATCCAG 
               
               
                   
               
               
                 121 
                 CCGTTTGGGT ACGACGGATT CCGTGTGCGC GCATGGCCCT 
               
               
                   
               
               
                 161 
                 TCCGTCCGCC TTCGGGTAAC GAGATCAGCT TCATCTACGA 
               
               
                   
               
               
                 201 
                 TCCCCCGATC GAAGGCTATG AGGACACTGC GCATGGCATG 
               
               
                   
               
               
                 241 
                 AGCTATGACA CCGCAACCAC CGGCACGGAG CCTCGCACCT 
               
               
                   
               
               
                 281 
                 TGCGCAACGG CAATATCATC CTGCGCACCA CCGGCTGGGG 
               
               
                   
               
               
                 321 
                 TGGTACCACA GCCGGATACC GACTGTCCTT TTATCGCGTC 
               
               
                   
               
               
                 361 
                 AATGACGATG GAAGTGAGAC CCTTCTCACA AACGAATATG 
               
               
                   
               
               
                 401 
                 CTCCGCTGAA GTCTCTCAAC CCCCGGTACT ATTACTGGCC 
               
               
                   
               
               
                 441 
                 GGGACCTGGG GCCGAATTCT CAGCTGAGTT CTCTTTCAGT 
               
               
                   
               
               
                 481 
                 GCGCAGCCGG ATGAGCAGAT CTATGGTACG GGCACGCAAC 
               
               
                   
               
               
                 521 
                 AGGATCATAT GATCAACAAG AAGGGCTCCG TAATTGACAT 
               
               
                   
               
               
                 561 
                 GGTCAACTTC AACTCCTACA TCCCTACCCC GGTCTTCATG 
               
               
                   
               
               
                 601 
                 AGCAATAAAG GCTATGCCTT CATCTGGAAC ATGCCAGCTG 
               
               
                   
               
               
                 641 
                 AGGGGCGTAT GGAATTTGGC ACCCTCCGGA CCAGATTCAC 
               
               
                   
               
               
                 681 
                 CGCCGCGTCC ACGACGCTTG TTGACTATGT AATCGTCGCC 
               
               
                   
               
               
                 721 
                 GCGCAGCCGG GCGACTACGA CACCTTGCAG CAGCGGATTT 
               
               
                   
               
               
                 761 
                 CGGCCCTCAC AGGACGGGCC CCGGCCCCGC CTGACTTCTC 
               
               
                   
               
               
                 801 
                 GCTTGGATAC ATCCAGTCCA AGCTACGATA TGAAAACCAA 
               
               
                   
               
               
                 841 
                 ACGGAGGTGG AGCTGCTGGC TCAAAACTTC CATGACCGAA 
               
               
                   
               
               
                 881 
                 ACATCCCGGT GTCCATGATC GTTATTGACT ACCAGTCCTG 
               
               
                   
               
               
                 921 
                 GGCTCACCAG GGTGATTGGG CGCTCGATCC TCGCCTGTGG 
               
               
                   
               
               
                 961 
                 CCCAATGTTG CGCAGATGTC GGCGCGGGTC AAGAACCTCA 
               
               
                   
               
               
                 1001 
                 CCGGCGCCGA AATGATGGCA TCGCTATGGC CCAGTGTTGC 
               
               
                   
               
               
                 1041 
                 CGACGACAGC GTCAATTACG CAGCCCTGCA GGCGAACGGC 
               
               
                   
               
               
                 1081 
                 CTTCTCTCGG CCACGCGCGA TGGACCTGGT ACCACTGACT 
               
               
                   
               
               
                 1121 
                 CCTGGAACGG ATCATACATC CGGAACTATG ACTCCACCAA 
               
               
                   
               
               
                 1161 
                 CCCCTCGGCG CGGAAGTTCC TCTGGAGCAT GCTGAAGAAG 
               
               
                   
               
               
                 1201 
                 AACTACTACG ACAAGGGTAT CAAAAACTTT TGGATTGACC 
               
               
                   
               
               
                 1241 
                 AAGCCGATGG CGGAGCGCTG GGTGAGGCGT ATGAGAACAA 
               
               
                   
               
               
                 1281 
                 CGGACAGAGC ACGTATATTG AGTCCATCCC GTTCACCCTG 
               
               
                   
               
               
                 1321 
                 CCAAACGTGA ACTATGCCGC TGGTACGCAG CTCAGCGTGG 
               
               
                   
               
               
                 1361 
                 GTAAGCTGTA CCCCTGGGCG CATCAGCAGG CAATTGAGGA 
               
               
                   
               
               
                 1401 
                 GGGGTTCCGC AATGCAACAG ATACCAAGGA AGGGAGCGCA 
               
               
                   
               
               
                 1441 
                 TGCGATCATG TCTCCCTGAG TCGGTCTGGA TACATCGGAT 
               
               
                   
               
               
                 1481 
                 CCCAGCGGTT CTGCAGCATG ATCTGGTCGG GAGACACTAC 
               
               
                   
               
               
                 1521 
                 ATCCGTTTGG GACACCCTGG CAGTGCAAGT AGCCAGTGGA 
               
               
                   
               
               
                 1561 
                 CTGTCCGCCG CAGCAACAGG CTGGGGTTGG TGGACGGTCG 
               
               
                   
               
               
                 1601 
                 ATGCCGGTGG CTTCGAAGTC GACTCGACTG TTTGGTGGAG 
               
               
                   
               
               
                 1641 
                 TGGAAACATT GACACGCCTG AATACCGGGA GTTGTATGTG 
               
               
                   
               
               
                 1681 
                 CGCTGGCTGG CTTGGACGAC TTTCCTGCCA TTCATGCGCA 
               
               
                   
               
               
                 1721 
                 CTCACGGTAG CCGGACCTGC TATTTCCAGG ACGCCTACAC 
               
               
                   
               
               
                 1761 
                 CTGTGCCAAT GAGCCGTGGT CCTATGGTGC AAGCAACACA 
               
               
                   
               
               
                 1801 
                 CCCATCATTG TCTCGTACAT TCATCTGCGC TACCAGCTGG 
               
               
                   
               
               
                 1841 
                 GTGCTTACCT GAAGTCCATC TTCAACCAGT TCCACCTCAC 
               
               
                   
               
               
                 1881 
                 AGGCCGGAGC ATCATGCGCC CATTGTATAT GGATTTCGAG 
               
               
                   
               
               
                 1921 
                 AAGACAGACC CGAAGATCTC CCAGCTGGTG TCGTCGAACA 
               
               
                   
               
               
                 1961 
                 GCAACTACAC GACGCAACAG TACATGTTTG GCCCACGTCT 
               
               
                   
               
               
                 2001 
                 CCTGGTCTCG CCAGTGACCT TGCCGAACGT GACTGAGTGG 
               
               
                   
               
               
                 2041 
                 CCCGTGTATC TGCCGCAGAC GGGACAGAAC AACACCAAGC 
               
               
                   
               
               
                 2081 
                 CTTGGACATA CTGGTGGACG AATGAAACGT ATGCCGGAGG 
               
               
                   
               
               
                 2121 
                 ACAGGTCGTC AAGGTGCCTG CCCCCTTGCA ACATATCCCC 
               
               
                   
               
               
                 2161 
                 GTGTTTCATC TGGGATCGCG CGAAGAGCTT CTCTCGGGTA 
               
               
                   
               
               
                 2201 
                 ATGTTTTCTA G 
               
            
           
         
       
     
     Ax1A fungal orthologs were identified by BLASTP against the GenBank™ nonredundant database. Many of these orthologs are annotated as belonging to GH family 31 and as having β-glucosidase and/or α-xylosidase activity. However, there is no supporting biochemical evidence for any of the α-xylosidase activity annotations in GenBank except perhaps for AN7505 ( A. nidulans ). Bauer et al.,  Proc. Natl. Acad. Sci. U.S.A.  10311417-11422 (2006). However, AN7505 has no predicted signal peptide, and there is no evidence that the native protein is secreted from its native host. AN7505 has been tested on only a single, artificial substrate and never shown to be active on natural α-xylosides such as tamarind xyloglucan or isoprimeverose, which are the substrates of industrial, practical relevance. AN7505 is only weakly related (25% sequence identity) to Ax1A of  A. niger.    
     The top BLASTP hits for the Ax1A of  A. niger  (e.g., E-values=0.0 and percent identities ranging from 52 to 81%) were from  Aspergillus  species,  Neosartorya fischeri , and  Schizophyllum commune  (XP_003031084) and  Serpula lachrymans  (EG001163). 
     The  Schizophyllum commune  (XP_003031084.1, GI:302682806) amino acid sequence is shown below as SEQ ID NO:3. 
                        1   MLLRSLAALC AALACANLAL AQGSETNSTG IKLQNGFERV               41   FIQPFGENGF RVRTSLMRDP TGNEWSGLID PPLEGPGGNA               61   GLTYDTLLPY HGNATIQNGN ILATVDLGVL SFFRLEPNGS               121   TTLLTGEFTD EKAIPARYYT RNFLSDSFAV DLAFSAEKDE               161   QFYGTGQQAC CKDHSVNKKG QVVDLFNFNS NVALPVYMSS               201   KGYLQFFNMP SQGRIEFSPL RTRFHATETT VVDYYITTAQ               241   PGDYDTLQKQ FTSVTGRQPT PPDFLLGYQH SKLRYFEQQQ               281   VLDVAQRFHD EQINVSLLVV DFFAWKYQGD WSFNPEYWPD               321   PEGMAAKVKE LTGAEMMASL WPSVEDNSEN YAALQEQGLL               361   ATTRDGTGVT DSFAGAYTRL IDSTNPAARE FLWKRLNDSY               401   FSKGIYNFWI DQADGGTLGE AFENNGQTIQ NIPYSRAFTQ               441   YYIGTQEGAG KMYPWFHEQA VDEGHRNLTN TARDDPACPY               481   MSLTRSTWVG GQRFCTYLWS GDTRSEWATL SQQVTAGASV               521   AASGISSWTL DIGGFAGLNV DQEEDRELFV RWFGFGTFLP               561   YVSTYTVAGE REPWSFGDDN FVVLKKYISL RYQLVPYVKK               601   LFVDLQASGK TIMRALYYDF SLSDPAVVEG TRTNDPSIVH               641   EYMLGPRLLV APVWATNVTS WEVYLPKLPE AYVDEGWEWT               681   HWWTDEAYGA GGEKVNVSAQ LDEIPVFYLG SKDDIFSGNV            
A nucleotide sequence for the SEQ ID NO:3 protein is available as accession number XM_003031038.1 (GI:302682805), provided below as SEQ ID NO:4.
 
     
       
         
           
               
               
            
               
                 1 
                 ATGCTACTAA GATCACTTGC CGCCCTATGT GCGGCGCTTG 
               
               
                   
               
               
                 41 
                 CTTGCGCGAA CCTTGCCCTC GCGCAAGGTT CCGAGACCAA 
               
               
                   
               
               
                 61 
                 CTCCACGGGC ATCAAACTTC AGAACGGCTT CGAACGCGTC 
               
               
                   
               
               
                 121 
                 TTCATTCAAC CCTTTGGCGA GAATGGCTTC CGCGTCCGGA 
               
               
                   
               
               
                 161 
                 CCAGCCTCAT GCGCGATCCC ACCGGGAACG AATGGAGCGG 
               
               
                   
               
               
                 201 
                 CCTTATCGAC CCGCCCCTCG AAGGCCCCGG AGGCAATGCG 
               
               
                   
               
               
                 241 
                 GGACTCACCT ACGACACCCT CCTCCCCTAC CACGGCAACG 
               
               
                   
               
               
                 281 
                 CGACTATCCA GAACGGCAAC ATTCTCGCCA CCGTAGACCT 
               
               
                   
               
               
                 321 
                 CGGCGTTCTC TCCTTCTTCC GCCTCGAGCC TAACGGTAGC 
               
               
                   
               
               
                 361 
                 ACCACGCTTC TCACCGGCGA GTTTACCGAC GAGAAGGCGA 
               
               
                   
               
               
                 401 
                 TCCCGGCGCG ATACTACACG CGCAACTTCC TCTCCGATAG 
               
               
                   
               
               
                 441 
                 CTTTGCCGTC GATCTCGCGT TCTCGGCGGA GAAGGACGAG 
               
               
                   
               
               
                 481 
                 CAGTTCTATG GCACGGGGCA GCAGGCGTGT TGCAAGGACC 
               
               
                   
               
               
                 521 
                 ACTCGGTCAA TAAGAAGGGG CAGGTGGTGG ACTTGTTCAA 
               
               
                   
               
               
                 561 
                 CTTCAATAGC AATGTGGCAC TTCCGGTGTA TATGTCGAGC 
               
               
                   
               
               
                 601 
                 AAGGGGTACC TGCAGTTCTT CAATATGCCT AGTCAAGGGA 
               
               
                   
               
               
                 641 
                 GGATAGAGTT CAGCCCATTG AGGACTCGTT TCCATGCCAC 
               
               
                   
               
               
                 681 
                 GGAAACGACC GTCGTGGATT ACTATATCAC GACCGCACAA 
               
               
                   
               
               
                 721 
                 CCCGGCGACT ATGATACCCT GCAGAAACAG TTCACCTCCG 
               
               
                   
               
               
                 761 
                 TCACCGGGCG TCAGCCTACG CCGCCCGACT TCCTTCTCGG 
               
               
                   
               
               
                 801 
                 CTACCAGCAC TCCAAACTGC GGTACTTTGA GCAGCAACAA 
               
               
                   
               
               
                 841 
                 GTCCTCGACG TCGCGCAGCG CTTCCATGAT GAACAGATCA 
               
               
                   
               
               
                 881 
                 ACGTCTCGCT GCTGGTCGTA GACTTCTTTG CTTGGAAGTA 
               
               
                   
               
               
                 921 
                 CCAGGGTGAC TGGTCTTTCA ACCCAGAGTA TTGGCCCGAC 
               
               
                   
               
               
                 961 
                 CCCGAGGGCA TGGCCGCGAA AGTCAAGGAG CTCACTGGCG 
               
               
                   
               
               
                 1001 
                 CCGAGATGAT GGCCTCGCTC TGGCCCAGCG TCGAAGATAA 
               
               
                   
               
               
                 1041 
                 CTCCGAGAAC TACGCAGCGC TGCAGGAGCA GGGTCTGTTG 
               
               
                   
               
               
                 1081 
                 GCGACGACGC GTGATGGCAC GGGCGTGACG GACTCATTTG 
               
               
                   
               
               
                 1121 
                 CGGGGGCGTA TACGAGGTTG ATCGACTCGA CGAATCCGGC 
               
               
                   
               
               
                 1161 
                 AGCGCGCGAG TTTTTGTGGA AGCGGCTGAA TGATAGTTAC 
               
               
                   
               
               
                 1201 
                 TTCTCTAAGG GTATATACAA CTTCTGGATC GATCAGGCAG 
               
               
                   
               
               
                 1241 
                 ACGGTGGAAC CCTCGGAGAG GCTTTCGAGA ACAACGGTCA 
               
               
                   
               
               
                 1281 
                 AACCATCCAA AACATCCCCT ACAGCCGCGC CTTCACCCAA 
               
               
                   
               
               
                 1321 
                 TACTACATCG GCACGCAGGA AGGCGCCGGC AAGATGTACC 
               
               
                   
               
               
                 1361 
                 CCTGGTTCCA CGAACAAGCC GTCGACGAGG GCCACCGCAA 
               
               
                   
               
               
                 1401 
                 CCTCACCAAC ACCGCGCGCG ACGACCCCGC GTGCCCCTAC 
               
               
                   
               
               
                 1441 
                 ATGTCCCTCA CGCGCAGCAC GTGGGTCGGC GGGCAGCGCT 
               
               
                   
               
               
                 1481 
                 TCTGCACGTA CCTCTGGTCG GGCGACACGC GCTCGGAGTG 
               
               
                   
               
               
                 1521 
                 GGCGACGCTG TCGCAGCAGG TGACGGCGGG CGCGAGCGTC 
               
               
                   
               
               
                 1561 
                 GCGGCATCGG GCATCTCGTC GTGGACGCTC GATATTGGCG 
               
               
                   
               
               
                 1601 
                 GGTTTGCGGG GTTGAATGTC GATCAGGAGG AGGATAGGGA 
               
               
                   
               
               
                 1641 
                 GTTGTTTGTG CGGTGGTTTG GGTTTGGGAC GTTTTTGCCG 
               
               
                   
               
               
                 1681 
                 TATGTGAGTA CATACACGGT GGCGGGAGAG AGGGAGCCCT 
               
               
                   
               
               
                 1721 
                 GGTCCTTCGG AGATGACAAC TTCGTTGTTT TGAAGAAGTA 
               
               
                   
               
               
                 1761 
                 CATCTCTCTG CGCTACCAGC TCGTCCCCTA CGTCAAGAAG 
               
               
                   
               
               
                 1801 
                 CTCTTCGTCG ACCTCCAGGC CTCGGGCAAG ACGATCATGC 
               
               
                   
               
               
                 1841 
                 GCGCGCTTTA CTACGACTTC TCGCTCTCGG ACCCAGCAGT 
               
               
                   
               
               
                 1861 
                 AGTCGAGGGC ACGCGCACCA ACGACCCCGC GATCGTCCAC 
               
               
                   
               
               
                 1921 
                 GAGTACATGC TGGGCCCGCG GCTGCTTGTT GCGCCGGTGT 
               
               
                   
               
               
                 1961 
                 GGGCGACAAA CGTGACGAGC TGGGAGGTGT ATCTTCCGAA 
               
               
                   
               
               
                 2001 
                 GTTGCCGGAG GCTTATGTGG ATGAGGGTTG GGAGTGGACG 
               
               
                   
               
               
                 2041 
                 CATTGGTGGA CGGACGAGGC TTACGGCGCC GGGGGCGAGA 
               
               
                   
               
               
                 2081 
                 AGGTGAACGT AAGCGCGCAG CTGGACGAGA TTCCTGTGTT 
               
               
                   
               
               
                 2121 
                 CTATCTCGGG TCCAAGGACG ATATCTTCTC AGGCAATGTT 
               
               
                   
               
               
                 2161 
                 TGA 
               
            
           
         
       
     
     Among species of  Aspergillus, Ax 1A orthologs with strong E-values and percent amino acid identity are present in  A. flavus, Aspergillus oryzae, Aspergillus terreus, Aspergillus aculeatus , and  Aspergillus carbonarius . Proteins with strong identity to Ax1A were not observed in  A. fumigatus, A. clavatus , or  A. nidulans  (Aspergillus Comparative Database (Broad Institute) and DOE Joint Genome Institute). All of the Ax1A orthologs in  Aspergillus  have strongly predicted signal peptides, like Ax1A as described herein. 
     Reannotation of protein XP_002378848 from  A. flavus  by reassigning the ATG start codon indicates that it probably also has a signal peptide. The sequence for this  A. flavus  protein is shown below as SEQ ID NO:5. 
                        1   MLILALGAVK FAGVGHHIPW LMVKDPASLR IWAKYLLALS               41   FLYLGSVNLP KFSILLLYHR LFPTKKMGAI IKLMMVVLCV               81   ITISTIVGAS LVCRPFSANW DGPIPGNCGN KKVLYIWASF               121   PNIVTDVILL LLPMPVLWSL NVSPRLKVGL TITFAVGSIG               161   LVTSVMRFQI FFRNNAFLDG TWVAVELIIW TQVETGVYLI               201   SACLPTYRPL IEHGFNPKML SKMYRWLVAL TVCATQLVQA               241   TPIQTRESDY FLPNSTGFRM QHGFETILVQ PFGFDGFRVR               281   AWPFRPPTGH EISFIYDPPL EGFENGQAHG LTFDTAFNGN               321   HTVAIRNGNT IVRTSGWGGN PGGYRLAFYR IEQDGSESLL               361   TNEYAPLKSI NPRYYSWNGP GSEFSAEFSF STDPDEQFYG               401   TGTQQDHLVN KKGTVIDLIN FNTHIPTPVF MSNKGYAFIW               441   NMPAQGRMEF GQLRTKLTAE STTVVDYVIV ATTPGDYDTL               481   QKRLSALTGR APTPPDFSLG YIQSKLRYEN QTELELLAQK               521   FKDNNVPVGM FVIDYQSWRN QGDWGLDPAL WPDVAAMAKK               561   VKDLTGAEIM ASLWPSVSDA SDNYLELQAN GYLSATRDGP               601   GTTDSWNGSY IRNVDSTNPG ARKFIWSTLK RNYYDKGIKN               641   FWIDQADGGA LGEAYENNGQ STYIQSVPFA LPNVLYAAGT               681   QQSAGKYYPW AHQLAIEEGF RNVTDSKEGE ACEHISLSRS               721   GYIGSQRFCS MIWSGDTTSA WETLGLQVAS GLSAAATGWG               761   WWTMDAGGFQ PDPTVPWSSN IDTPEYRELY VRWLQWATFV               801   PFMRTHGQRV CDNQDAYTCN NEPWSYGEKN TPIILSYIHL               841   RYQLASYLRA LFDQFHKTGR MIMRPLYMDF EKTDPKVSQW               881   TQANNNVTTQ QYMFGPRLLV SPITTPNVTE WSVYLPQTGQ               921   NGTKPWTYWW TNQTYAGGQT VTVPAPVEHI PVFHLGKRED               961   ILSGNVF            
A nucleotide sequence for the SEQ ID NO:5 protein is available as XM_002378807.1 (GI:238495223), provided below as SEQ ID NO:6.
 
     
       
         
           
               
               
            
               
                 1 
                 ATGCTAATTC TTGCTTTAGG TGCTGTAAAG TTCGCTGGCG 
               
               
                   
               
               
                 41 
                 TGGGACACCA CATCCCATGG TTAATGGTGA AAGACCCTGC 
               
               
                   
               
               
                 81 
                 CAGTCTAAGA ATTTGGGCGA AATATCTCCT GGCTTTGTCA 
               
               
                   
               
               
                 121 
                 TTTCTCTATT TGGGAAGTGT TAATCTTCCA AAGTTCTCTA 
               
               
                   
               
               
                 161 
                 TCCTATTACT GTACCATAGG CTCTTCCCCA CAAAGAAAAT 
               
               
                   
               
               
                 201 
                 GGGCGCGATC ATCAAATTGA TGATGGTGGT CCTGTGTGTC 
               
               
                   
               
               
                 241 
                 ATCACGATAT CTACGATCGT TGGCGCGAGT CTCGTCTGCC 
               
               
                   
               
               
                 281 
                 GACCGTTCTC CGCTAACTGG GACGGTCCTA TCCCTGGCAA 
               
               
                   
               
               
                 321 
                 CTGTGGTAAC AAGAAAGTTC TTTACATCTG GGCCAGTTTT 
               
               
                   
               
               
                 361 
                 CCTAACATTG TGACCGATGT AATTCTACTG CTCCTTCCAA 
               
               
                   
               
               
                 401 
                 TGCCAGTGCT GTGGTCACTT AATGTCAGTC CACGACTGAA 
               
               
                   
               
               
                 441 
                 GGTAGGACTG ACAATCACAT TCGCAGTAGG GAGCATAGGC 
               
               
                   
               
               
                 481 
                 TTAGTCACTT CCGTTATGCG CTTCCAGATC TTTTTTCGAA 
               
               
                   
               
               
                 521 
                 ACAACGCCTT CCTCGATGGG ACCTGGGTAG CGGTTGAGCT 
               
               
                   
               
               
                 561 
                 GATTATATGG ACCCAAGTCG AGACCGGGGT TTACCTGATA 
               
               
                   
               
               
                 601 
                 TCTGCCTGCC TGCCCACATA TAGACCACTT ATCGAACACG 
               
               
                   
               
               
                 641 
                 GCTTCAATCC CAAGATGTTG AGCAAAATGT ATCGCTGGCT 
               
               
                   
               
               
                 681 
                 GGTGGCCCTA ACAGTCTGCG CCACACAGCT GGTGCAGGCG 
               
               
                   
               
               
                 721 
                 ACCCCAATCC AGACGCGGGA GTCGGACTAC TTCCTGCCCA 
               
               
                   
               
               
                 761 
                 ACTCGACTGG ATTTCGCATG CAGCATGGCT TCGAGACTAT 
               
               
                   
               
               
                 801 
                 TCTGGTACAG CCCTTTGGCT TCGATGGGTT CCGTGTGCGC 
               
               
                   
               
               
                 841 
                 GCCTGGCCCT TCCGGCCGCC TACGGGCCAT GAGATCAGCT 
               
               
                   
               
               
                 881 
                 TCATCTACGA TCCACCATTG GAAGGATTCG AGAATGGACA 
               
               
                   
               
               
                 921 
                 AGCGCATGGA CTAACCTTTG ACACGGCATT TAATGGCAAT 
               
               
                   
               
               
                 961 
                 CACACTGTTG CTATCCGCAA TGGAAACACT ATCGTGCGCA 
               
               
                   
               
               
                 1001 
                 CCTCTGGCTG GGGTGGAAAT CCCGGAGGAT ATCGGCTGGC 
               
               
                   
               
               
                 1041 
                 ATTCTACCGC ATCGAGCAAG ATGGTTCTGA GTCACTGTTA 
               
               
                   
               
               
                 1081 
                 ACAAACGAGT ATGCGCCACT CAAATCGATC AATCCACGAT 
               
               
                   
               
               
                 1121 
                 ACTACTCGTG GAACGGCCCG GGAAGCGAAT TTTCTGCCGA 
               
               
                   
               
               
                 1161 
                 GTTTTCATTC AGTACGGACC CCGACGAGCA GTTCTATGGC 
               
               
                   
               
               
                 1201 
                 ACGGGTACGC AACAGGACCA TCTTGTCAAC AAGAAAGGAA 
               
               
                   
               
               
                 1241 
                 CGGTCATTGA CTTGATCAAC TTCAATACCC ACATCCCCAC 
               
               
                   
               
               
                 1281 
                 ACCTGTGTTC ATGAGCAACA AGGGCTACGC CTTCATCTGG 
               
               
                   
               
               
                 1321 
                 AATATGCCAG CTCAGGGTCG CATGGAATTT GGACAGCTAC 
               
               
                   
               
               
                 1361 
                 GCACCAAGCT CACCGCGGAG TCCACCACGG TCGTCGACTA 
               
               
                   
               
               
                 1401 
                 TGTCATTGTG GCCACGACAC CAGGCGACTA CGACACATTG 
               
               
                   
               
               
                 1441 
                 CAGAAACGTC TATCCGCCCT GACGGGTAGA GCACCCACTC 
               
               
                   
               
               
                 1481 
                 CGCCTGACTT CTCACTCGGA TACATCCAGT CTAAGCTCCG 
               
               
                   
               
               
                 1521 
                 CTATGAGAAC CAGACTGAAC TAGAACTCCT GGCGAAGAAG 
               
               
                   
               
               
                 1561 
                 TTCAAGGACA ACAACGTCCC CGTTGGAATG TTCGTCATCG 
               
               
                   
               
               
                 1601 
                 ACTACCAATC CTGGCGGAAT CAAGGCGACT GGGGTCTTGA 
               
               
                   
               
               
                 1641 
                 CCCAGCGCTA TGGCCGGACG TAGCAGCAAT GGCGAAGAAG 
               
               
                   
               
               
                 1681 
                 GTAAAGGATC TCACCGGAGC AGAGATCATG GCATCTCTCT 
               
               
                   
               
               
                 1721 
                 GGCCCAGTGT ATCGGATGCG AGCGACAACT ACTTGGAGCT 
               
               
                   
               
               
                 1761 
                 TCAAGCCAAC GGATACCTAT CTGCGACTCG CGACGGACCC 
               
               
                   
               
               
                 1801 
                 GGAACCACCG ATTCATGGAA CGGCTCGTAC ATCCGCAACG 
               
               
                   
               
               
                 1841 
                 TGGACTCTAC GAACCCAGGC GCACGGAAAT TCATCTGGTC 
               
               
                   
               
               
                 1881 
                 GACCTTGAAG CGCAACTACT ACGACAAGGG AATCAAGAAC 
               
               
                   
               
               
                 1921 
                 TTCTGGATCG ACCAAGCTGA CGGTGGTGCC CTGGGCGAAG 
               
               
                   
               
               
                 1961 
                 CCTACGAAAA CAACGGTCAA AGCACCTACA TTCAGTCTGT 
               
               
                   
               
               
                 2001 
                 CCCCTTCGCC CTACCCAACG TCCTCTACGC AGCTGGCACC 
               
               
                   
               
               
                 2041 
                 CAACAGAGCG CCGGAAAATA TTACCCCTGG GCCCACCAGC 
               
               
                   
               
               
                 2081 
                 TGGCAATCGA AGAGGGCTTC CGCAACGTCA CCGACAGCAA 
               
               
                   
               
               
                 2121 
                 GGAAGGCGAA GCCTGCGAGC ACATCTCGCT CAGTCGGTCT 
               
               
                   
               
               
                 2161 
                 GGCTACATCG GATCTCAACG ATTCTGCAGC ATGATCTGGT 
               
               
                   
               
               
                 2201 
                 CAGGAGACAC CACCTCCGCC TGGGAAACAC TAGGCCTCCA 
               
               
                   
               
               
                 2241 
                 AGTTGCTAGT GGAACCACCG CCGCCGCAAC AGGATGGGGC 
               
               
                   
               
               
                 2281 
                 TGGTGGACTA TGGACGCAGG CGGTTTCCAA CCTGACCCGA 
               
               
                   
               
               
                 2321 
                 CAGTACCATG GAGCTCTAAC ATCGACACAC CGGAGTACCG 
               
               
                   
               
               
                 2361 
                 CGAGTTGTAC GTGCGCTGGC TGCAGTGGGC TACATTCGTC 
               
               
                   
               
               
                 2401 
                 CCCTTCATGC GTACACACGG TCAGCGAGTC TGCGACAACC 
               
               
                   
               
               
                 2441 
                 AGGACGCATA CACATGTAAC AACGAGCCGT GGTCGTATGG 
               
               
                   
               
               
                 2481 
                 CGAGAAGAAC ACCCCCATTA TCCTCTCGTA CATTCACCTC 
               
               
                   
               
               
                 2521 
                 CGATACCAAT TGGCCTCGTA TCTGCGTGCC CTCTTCGACC 
               
               
                   
               
               
                 2561 
                 AGTTCCACAA GACCGGTCGC ATGATCATGC GTCCCTTGTA 
               
               
                   
               
               
                 2601 
                 TATGGATTTC GAGAAGACTG ATCCGAAAGT TTCACAGTGG 
               
               
                   
               
               
                 2641 
                 ACGCAGGCCA ACAACAATGT GACAACGCAG CAGTACATGT 
               
               
                   
               
               
                 2681 
                 TCGGCCCGAG ATTGCTGGTA TCACCTATTA CCACGCCGAA 
               
               
                   
               
               
                 2721 
                 TGTCACCGAA TGGTCGGTAT ATCTGCCGCA GACGGGCCAG 
               
               
                   
               
               
                 2761 
                 AATGGGACGA AGCCTTGGAC GTACTGGTGG ACTAATCAGA 
               
               
                   
               
               
                 2801 
                 CATATGCTGG TGGTCAGACG GTTACTGTGC CGGCGCCTGT 
               
               
                   
               
               
                 2841 
                 GGAGCATATT CCTGTGTTCC ATCTTGGGAA GAGAGAGGAT 
               
               
                   
               
               
                 2881 
                 ATTCTCAGTG GTAATGTCTT CTAG 
               
            
           
         
       
     
     An α-xylosidase from  Aspergillus kawachii  strain IFO 4308 has NCBI accession number GAA91593.1, and has 97% sequence identity to the α-xylosidase described herein with SEQ ID NO:1. This α-xylosidase from  Aspergillus kawachii  strain IFO 4308 has SEQ ID NO:7. 
     
       
         
           
               
               
            
               
                 1 
                 MYFSSFLALG ALIQAAAATY LAPNSTGLRI QHGFETILIQ 
               
               
                   
               
               
                 41 
                 PFGYDGFRVR AWPFRPPSGN EISFIYDPPI EGYEDTAHGM 
               
               
                   
               
               
                 61 
                 SYDTATTGTE PRTLRNGNII LRTTGWGGTT AGYRLSFYRV 
               
               
                   
               
               
                 121 
                 NDDGSETLLT NEYAPLKSLN PRYYSWPGPG AEFSAEFSFS 
               
               
                   
               
               
                 161 
                 ATPDEQIYGT GTQQDHMINK KGSVIDLVNF NTHIPTPVFM 
               
               
                   
               
               
                 201 
                 SNKGYAFIWN MPAEGRMEFG SLRTRFTAAS TTLVDYVIVA 
               
               
                   
               
               
                 241 
                 AQPGDYDTLQ QRISALTGRA PTPPDFSLGY IQSKLRYENQ 
               
               
                   
               
               
                 281 
                 TEVELLAQNF HDRDIPVSMI VIDYQSWAHQ GDWALDPRLW 
               
               
                   
               
               
                 321 
                 PNVAQMSATV KNLTGAEMMA SLWPSVADDS VNYAALQANG 
               
               
                   
               
               
                 361 
                 LLSATRDGPG TTDSWNGSYI RNYDSTNPSA RKFLWSMLKK 
               
               
                   
               
               
                 401 
                 NYYDKGIKNF WIDQADGGAL GEAYENNGQS TYIQSIPYTL 
               
               
                   
               
               
                 441 
                 PNVNYAAGTQ LGVGKLYPWA HQQAIEEGFR NATDTKEGSA 
               
               
                   
               
               
                 481 
                 CDHVSLSRSG YIGSQRFCSM IWSGDTTSVW DTLAVQVASG 
               
               
                   
               
               
                 521 
                 LSAAATGWGW WTVDAGGFEV DSTVWWSGNI DTPEFRELYV 
               
               
                   
               
               
                 561 
                 RWLACTTFLP FMRTHGSRAC YYQDAYTCAN EPWSYGASNT 
               
               
                   
               
               
                 601 
                 PIIVSYIHLR YQLGAYLKSI FNQFHLTGRS IMRPLYMDFE 
               
               
                   
               
               
                 641 
                 KTDPKISQLV SSNSNYTTQQ YMFGPRLLVS PVTLPNVTEW 
               
               
                   
               
               
                 681 
                 PVYLPQTGDN STKPWTYWWT NETYAGGQVV KVPAPVQHIP 
               
               
                   
               
               
                 721 
                 VFHLGSREEL LSGDVF 
               
            
           
         
       
     
     An α-xylosidase from  Aspergillus terreus  strain NIH2624 has NCBI accession number XP_001217011.1, and has 81% sequence identity to the α-xylosidase described herein with SEQ ID NO:1. This α-xylosidase from  Aspergillus terreus  strain NIH2624 has SEQ ID NO:8. 
     
       
         
           
               
               
            
               
                 1 
                 MYRWLVALAA CAGQLALANP VHPRDTDYFK PNSTGFRMRH 
               
               
                   
               
               
                 41 
                 GFETVLVQPF GYDGFRVRAW PFRPPTGQEL SFVYDPPLEG 
               
               
                   
               
               
                 81 
                 FEDGQAHGMD YDTAFTGNES LAIRNGNMIV RTTGWGGNPG 
               
               
                   
               
               
                 121 
                 GYRLAFYRVE EDGSETLLTN EYAPLKSVNP RYYSWNGPGA 
               
               
                   
               
               
                 161 
                 EFSAEFTFST TPDEQFYGTG TQQDHLVNKK GTVIDLINFN 
               
               
                   
               
               
                 201 
                 THIPTPVFMS NKGYGFVWNM ASEGRMEFGQ LRNKFTAASA 
               
               
                   
               
               
                 241 
                 TLVDYVIVAS PAGDYDTLQQ RLSALTGRAP TPPDFALGYI 
               
               
                   
               
               
                 281 
                 QSKLRYENQT EVELLAQNFK DHNIPVGMIV IDYQSWADQG 
               
               
                   
               
               
                 321 
                 DWALDPRLWP DVAAMARKVK ELTGAEMMAS LWPSVSDDSV 
               
               
                   
               
               
                 361 
                 NYEALQMNGW LTATRDGPGT TDSWNGSYIR NIDSTNPDAR 
               
               
                   
               
               
                 401 
                 RFLWDTLKRN YYDKGIRNFW IDQADGGALG EAYENNGQSL 
               
               
                   
               
               
                 441 
                 YIQSIPYALP NVLYAAGTQL GVGKMYPWTH QMAIDEGFRN 
               
               
                   
               
               
                 481 
                 ATDSKPGSAC EHISLSRSGY IGSQRFCSMI WSGDITSVWE 
               
               
                   
               
               
                 521 
                 TLGLQVASGL SAAATGWGWW TVDAGGFQPD PTVPWSANID 
               
               
                   
               
               
                 561 
                 TPEYRELYVR WLQWTTFLPF MRTHGSRECD SQNAYTCNNE 
               
               
                   
               
               
                 601 
                 PWSYGEENTP VIVSYIHLRY QLGAYLRAIF KKFHETGRSI 
               
               
                   
               
               
                 641 
                 MRPLYMDFEK TDPRIRTMTQ ANTNVTTQQY MFGPRLLVSP 
               
               
                   
               
               
                 681 
                 VTTPNTTEWP VYLPQTGQNG TKPWTYWWTN ETYAGGQTVK 
               
               
                   
               
               
                 721 
                 VPAPVEHIPV FHLGTREEIL SGDVF 
               
            
           
         
       
     
     An α-xylosidase from  Neosartorya fischeri  NRRL 181 has NCBI accession number EAW23703.1, and has 79% sequence identity to the α-xylosidase described herein with SEQ ID NO:1. This α-xylosidase from  Neosartorya fischeri  strain NRRL 181 has SEQ ID NO:9. 
     
       
         
           
               
               
            
               
                 1 
                 MVSIKRWLLG LCAVSTVWAN PIQTREADYV MPNSTGFRMQ 
               
               
                   
               
               
                 41 
                 HGFETVLVQP FGYDGFRVRA WPYRPPTGNE VSFIYDPPLE 
               
               
                   
               
               
                 81 
                 GFEDGMAHGL GFDTAFNGNR TVAIRNGKIV VRTSGWGGNP 
               
               
                   
               
               
                 121 
                 GGYRLAFYRV EKDGSETLLT NEYAPLKSVN PRYYFWRGPG 
               
               
                   
               
               
                 161 
                 SEFSAEFSFS STPDEQIYGT GTQQDHMVNK KGSVIDLINF 
               
               
                   
               
               
                 201 
                 NTHIPTPVIV SNKGYGFVWN MASEGRMELG ALRTKFTAES 
               
               
                   
               
               
                 241 
                 ATVVDYAIVA AEQGDYDTLQ RRLSALTGRA PTPPEASLGY 
               
               
                   
               
               
                 281 
                 IQSKLRYENQ TEVELLAQQF KDHNIPVSMI VIDYQSWAHQ 
               
               
                   
               
               
                 301 
                 GDWALDPRLW PDVASMAKKV KDLTGAEMMA SLWPSVADNS 
               
               
                   
               
               
                 361 
                 ENYLELIANG LLSATRSGPG TTDSWNGSYI RNIDSTNPAA 
               
               
                   
               
               
                 401 
                 RAFLWKTLKR NYYDKGIKNF WIDQADGGAL GEAYENNGQS 
               
               
                   
               
               
                 441 
                 SYIESIPFSL PNVLYAAGTQ LSAGKLYPWA HQQAIEEGYR 
               
               
                   
               
               
                 481 
                 NATGTKMGEA CDHISLSRSG YIGSQRFCSM IWSGDTTSVW 
               
               
                   
               
               
                 521 
                 DTLAVQVASG LSAAATGWGW WTMDAGGFQA DPTVPWSSNI 
               
               
                   
               
               
                 541 
                 DTPEYRELYV RWFQWAAFLP FMRTHGSRKC NVQNAYTCNN 
               
               
                   
               
               
                 601 
                 EPWSYGEENT PIIVSYIQLR YQLKAYLQAV FEQFHHTGRA 
               
               
                   
               
               
                 641 
                 LMRPLYMDFE RTDPQIAKMT RENVNATTQQ YMLGPRLLVT 
               
               
                   
               
               
                 681 
                 PVTLPNATEW EVYLPLTAQN ETKPWTYWWT NETYAGGQTV 
               
               
                   
               
               
                 721 
                 TVPAPIEHIP LFYLGKREDI LSGSVF 
               
            
           
         
       
     
     An α-xylosidase from  Aspergillus flavus  NRRL3357 has NCBI accession number XP_002378848.1, and has 79% sequence identity to the α-xylosidase described herein with SEQ ID NO:1. This α-xylosidase from  Aspergillus flavus  strain NRRL3357 has SEQ ID NO:10. 
     
       
         
           
               
               
            
               
                 1 
                 MLILALGAVK FAGVGHHIPW LMVKDPASLR IWAKYLLALS 
               
               
                   
               
               
                 41 
                 FLYLGSVNLP KFSILLLYHR LFPTKKMGAI IKLMMVVLCV 
               
               
                   
               
               
                 81 
                 ITISTIVGAS LVCRPFSANW DGPIPGNCGN KKVLYIWASF 
               
               
                   
               
               
                 121 
                 PNIVTDVILL LLPMPVLWSL NVSPRLKVGL TITFAVGSIG 
               
               
                   
               
               
                 161 
                 LVTSVMRFQI FFRNNAFLDG TWVAVELIIW TQVETGVYLI 
               
               
                   
               
               
                 201 
                 SACLPTYRPL IEHGFNPKML SKMYRWLVAL TVCATQLVQA 
               
               
                   
               
               
                 241 
                 TPIQTRESDY FLPNSTGFRM QHGFETILVQ PFGFDGFRVR 
               
               
                   
               
               
                 281 
                 AWPFRPPTGH EISFIYDPPL EGFENGQAHG LTFDTAFNGN 
               
               
                   
               
               
                 321 
                 HTVAIRNGNT IVRTSGWGGN PGGYRLAFYR IEQDGSESLL 
               
               
                   
               
               
                 361 
                 TNEYAPLKSI NPRYYSWNGP GSEFSAEFSF STDPDEQFYG 
               
               
                   
               
               
                 401 
                 TGTQQDHLVN KKGTVIDLIN FNTHIPTPVF MSNKGYAFIW 
               
               
                   
               
               
                 441 
                 NMPAQGRMEF GQLRTKLTAE STTVVDYVIV ATTPGDYDTL 
               
               
                   
               
               
                 481 
                 QKRLSALTGR APTPPDFSLG YIQSKLRYEN QTELELLAQK 
               
               
                   
               
               
                 521 
                 FKDNNVPVGM FVIDYQSWRN QGDWGLDPAL WPDVAAMAKK 
               
               
                   
               
               
                 561 
                 VKDLTGAEIM ASLWPSVSDA SDNYLELQAN GYLSATRDGP 
               
               
                   
               
               
                 601 
                 GTTDSWNGSY IRNVDSTNPG ARKFIWSTLK RNYYDKGIKN 
               
               
                   
               
               
                 641 
                 FWIDQADGGA LGEAYENNGQ STYIQSVPFA LPNVLYAAGT 
               
               
                   
               
               
                 681 
                 QQSAGKYYPW AHQLAIEEGF RNVTDSKEGE ACEHISLSRS 
               
               
                   
               
               
                 721 
                 GYIGSQRFCS MIWSGDTTSA WETLGLQVAS GLSAAATGWG 
               
               
                   
               
               
                 761 
                 WWTMDAGGFQ PDPTVPWSSN IDTPEYRELY VRWLQWATFV 
               
               
                   
               
               
                 781 
                 PFMRTHGQRV CDNQDAYTCN NEPWSYGEKN TPIILSYIHL 
               
               
                   
               
               
                 841 
                 RYQLASYLRA LFDQFHKTGR MIMRPLYMDF EKTDPKVSQW 
               
               
                   
               
               
                 881 
                 TQANNNVTTQ QYMFGPRLLV SPITTPNVTE WSVYLPQTGQ 
               
               
                   
               
               
                 921 
                 NGTKPWTYWW TNQTYAGGQT VTVPAPVEHI PVFHLGKRED 
               
               
                   
               
               
                 961 
                 ILSGNVF 
               
            
           
         
       
     
     An α-xylosidase from  Aspergillus oryzae  has NCBI accession number XP_001823456.1, and has 78% sequence identity to the α-xylosidase described herein with SEQ ID NO:1. This α-xylosidase from  Aspergillus oryzae  has SEQ ID NO:11. 
     
       
         
           
               
               
            
               
                 1 
                 MLSKMYRWLV ALTVCATQLV QATPIQTRES DYFLPNSTGF 
               
               
                   
               
               
                 41 
                 RMQHGFETIL VQPFGFDGFR VRAWPFRPPT GHEISFIYDP 
               
               
                   
               
               
                 81 
                 PLEGFENGQA HGLTFDTAFN GNHTVAIRNG NTIVRTSGWG 
               
               
                   
               
               
                 121 
                 GNPGGYRLAF YRIEQDGSES LLTNEYAPLK SINPRYYSWN 
               
               
                   
               
               
                 161 
                 GPGSEFSAEF SFSTTPDEQF YGTGTQQDHL VNKKGTVIDL 
               
               
                   
               
               
                 201 
                 INFNTHIPTP VFMSNKGYAF IWNMPAQGRM EFGQLRTKLT 
               
               
                   
               
               
                 241 
                 AESTTVVDYV IVATTPGDYD TLQKRLSALT GRAPTPPDFS 
               
               
                   
               
               
                 281 
                 LGYIQSKLRY ENQTELELLA QKFKDNNVPV GMIVIDYQSW 
               
               
                   
               
               
                 321 
                 RNQGDWGLDP ALWPDVAAMA KKVKDLTGAE IMASLWPSVS 
               
               
                   
               
               
                 361 
                 DASDNYLELQ ANGYLSATRD GPGTTDSWNG SYIRNVDSTN 
               
               
                   
               
               
                 401 
                 PGARKFIWST LKRNYYEKGI KNFWIDQADG GALGEAYENN 
               
               
                   
               
               
                 441 
                 GQSTYIQSVP FALPNVLYAA GTQQSAGKYY PWAHQLAIEE 
               
               
                   
               
               
                 481 
                 GFRNVTDSKE GEACEHISLS RSGYIGSQRF CSMIWSGDTT 
               
               
                   
               
               
                 521 
                 SAWETLGLQI ASRLSAAATG WGWWTMDAGG FQPDPTVPWS 
               
               
                   
               
               
                 561 
                 SNIDTPEYRE LYVRWLQWAT FVPFMRTHGQ RVCDNQDAYT 
               
               
                   
               
               
                 601 
                 CNNEPWSYGE KNTPIILSYI HLRYQLASYL RALFDQFHKT 
               
               
                   
               
               
                 641 
                 GRMIMRPLYM DFEKTDPKVS QWTQANNNVT TQQYMFGPRL 
               
               
                   
               
               
                 681 
                 LVSPITTPNV TEWSVYLPQT GQNGTKPWTY WWTNQTYAGG 
               
               
                   
               
               
                 721 
                 QTVTVPAPVE HIPVFHLGKR EDILSGNVF 
               
            
           
         
       
     
     An α-xylosidase from  Macrophomina phaseolina  strain MS6 has NCBI accession number EKG20540.1, and has 70% sequence identity to the α-xylosidase described herein with SEQ ID NO:1. This α-xylosidase from  Macrophomina phaseolina  strain MS6 has a signal sequence and amino acid sequence SEQ ID NO:12. 
     
       
         
           
               
               
            
               
                 1 
                 MHLLYSLVSL PLLTVSAQNI TSEYFAPNST GFRMTHGFET 
               
               
                   
               
               
                 41 
                 ILVQPYGYDG FRVRAWPFRP PNGNEISFLY DPPLEGPENG 
               
               
                   
               
               
                 81 
                 EARAMSYDFT TNGNQSAIIR NGNTVVKTYG LEGAHYRLAF 
               
               
                   
               
               
                 121 
                 YRIEPDGTET LLTNEFNPVK ALNPRYYSWT STGYEFSASF 
               
               
                   
               
               
                 161 
                 SFTTTPDEQI FGTGTQQDFL LNKKGSVIDM INFNSYIPTP 
               
               
                   
               
               
                 201 
                 VFMSSKGYGF VWNSAAQGRM EFGPRRNKFT SDSTTLVDYA 
               
               
                   
               
               
                 241 
                 IVSAPEGDYD SLQQKLTAIT GRAPTPPDFS LGYLHSKLRY 
               
               
                   
               
               
                 281 
                 ENQTEVVLLA QGFRDRNIPV SMIVIDYESW AQNGDWGLDP 
               
               
                   
               
               
                 321 
                 ALWPDVASMA AQVKNLTGAE MMASLWPAVE DDSLNYAEMQ 
               
               
                   
               
               
                 361 
                 QLGLLAATMS GPGTTDSWNG SYIRNYDSTN PRAREFLWNT 
               
               
                   
               
               
                 401 
                 LKRNYYDKGI KNFWIDQADG GALGEAWENN GQTAYVQSIP 
               
               
                   
               
               
                 441 
                 YPLPQVLYHA GTQASVGKLY PWAHQQAIEE GTRNATGTEQ 
               
               
                   
               
               
                 481 
                 GTACDYISLS RSGYIGSQRF CSMIWSGDTE ASWEVLGNQI 
               
               
                   
               
               
                 521 
                 PNALSAAATG WSWYTVDAGG FQPDPAIEWS NNIDRPEYRE 
               
               
                   
               
               
                 561 
                 LYVRWLQWTT FLPFMRNHGS RACDVQHAFT CDNEPWTYGA 
               
               
                   
               
               
                 601 
                 QNTPTIVSYI NLRYRLAPYV RALFEQLSRT GRQILRPLFM 
               
               
                   
               
               
                 641 
                 DFGKSDANVV AWTRENKNIT TQQYMFGPRL LVAPVVLPNV 
               
               
                   
               
               
                 681 
                 TTWPVYLPKT AGEGSGQRPW TYWWTNETFA GGQTVNVSAP 
               
               
                   
               
               
                 721 
                 VEHIPLFYLG DRDDIFSGNV F 
               
            
           
         
       
     
     An α-xylosidase from  Serpula lacrymans  var.  lacrymans  S7.3 has NCBI accession number EKG20540.1, and has 52% sequence identity to the α-xylosidase described herein with SEQ ID NO:1. This α-xylosidase from  Serpula lacrymans  var. lacrymans S7.3 has a signal sequence if it is reannotated to remove the first 20 amino acids, and has amino acid sequence SEQ ID NO:13. 
     
       
         
           
               
               
            
               
                 1 
                 MPYKPSRNIV RLCVPSRTCK MLGILSIVAV ITTAYAANTS 
               
               
                   
               
               
                 41 
                 IPSSTGIKLQ NGFERVYIQP FGNNGIRVRA SLLRDPTGNE 
               
               
                   
               
               
                 61 
                 LSALLDPPLE GPGGNQGLAY DQLVGFQGNA NLTNGNIAAE 
               
               
                   
               
               
                 121 
                 IATGYLSFYR IESNGSRTLL TSEFTDDKAL YPRYYIQEYK 
               
               
                   
               
               
                 161 
                 SPSFSAEFSF TAEPDEQIYG VGQQACCKDN SVNKKGQSID 
               
               
                   
               
               
                 201 
                 LINFNSFVPL PVYMSNKGYL QFFNMPSQGR MEFSPIRTRF 
               
               
                   
               
               
                 241 
                 VSSEATVVDY WITTAEPGDY DTLQEQYTAV TGRQPTPPTF 
               
               
                   
               
               
                 281 
                 THGYQQSKLR YFNQTQVEDL AQEFHDRQIN VSLIVIDFFN 
               
               
                   
               
               
                 321 
                 WKYQGDWSFD PEYWPDPAAM TAKVKELTGA EMMVSLWPSV 
               
               
                   
               
               
                 361 
                 EDLSVNYLTL QEQGLLATTR DGTGISDSFA GVYTRLIDST 
               
               
                   
               
               
                 401 
                 NPASREFLWK RLNESYFSNG IHNFWIDQAD GGTLGEAFEN 
               
               
                   
               
               
                 441 
                 NGQTIETIPY ARAFSQYFIG TQEGAGKMYP WLHQQAINEG 
               
               
                   
               
               
                 481 
                 LHNLTDTPAT ATSCEYMSLT RSTFAGGQRY CSYLWSGDTM 
               
               
                   
               
               
                 521 
                 AEFPVLLQQI TSAVSVAASG ISSWTLDLGG FTGLDIDTAY 
               
               
                   
               
               
                 561 
                 GKELYVRWFA MGVFLPYMRT HGDRICDIPP PTTPSNANYC 
               
               
                   
               
               
                 601 
                 PNEPWSYGEE NYPILKMYIE LRYKLVPYVT QLFAMLQNNG 
               
               
                   
               
               
                 641 
                 RTIMRALYFD FSLSDPFVAS ATAANDPLVS HQFMFGPRIL 
               
               
                   
               
               
                 681 
                 VSPVGVQNAT SKEVYLPRLT QAMLDQNYTW THWWTNTSYG 
               
               
                   
               
               
                 721 
                 QGGASVNVSA PLDQIPVFYL GSMADILSGN I 
               
            
           
         
       
     
     An α-xylosidase from  Agaricus bisporus  has NCBI accession number EKM78298.1, and has 49% sequence identity to the α-xylosidase described herein with SEQ ID NO:1. This α-xylosidase from  Agaricus bisporus  has a signal sequence, and has amino acid sequence SEQ ID NO:14. 
     
       
         
           
               
               
            
               
                 1 
                 MVLQSLILCY LVLPISLSLA ADYFNPNATG IKLQNGFERI 
               
               
                   
               
               
                 41 
                 HIQPFGNHGF RVRASLLRDP TGREPSALID PPLEGPSSKG 
               
               
                   
               
               
                 81 
                 LEHSITIPFR GNATVRNGNL VVDVSFGVTS FSRVEPNGTL 
               
               
                   
               
               
                 121 
                 TLLTSEYADT KVLPARYYVQ DIHGQSFQAQ FGFSADPDEM 
               
               
                   
               
               
                 161 
                 FFGTGQHACC KDHTVNKKGQ IVDLINYNSH VTLPIWMSNK 
               
               
                   
               
               
                 201 
                 GYLMFFNYPG QGRIEFDRLR TRFVADEATV VDYWITTAPP 
               
               
                   
               
               
                 241 
                 EDYDALQQQF TGVTGRQPTP PDFSLGFQQS KLRYYNQTQI 
               
               
                   
               
               
                 281 
                 IDLAQRFHDE QVPISLIVID FFAWKFQGDW SLDVDVWPDP 
               
               
                   
               
               
                 321 
                 TAMAAEVKRL TGAELMVSLW PSVEDLSENY LTLQEEGLLA 
               
               
                   
               
               
                 361 
                 ITRDGTGIQD SFEGVYTRLI DSTNPDAREF LWKRLNDSYF 
               
               
                   
               
               
                 401 
                 SKGIHNFWID QADGGTLGEP FENNGQSISS IPYSRSFTQY 
               
               
                   
               
               
                 441 
                 FLGSQEGFGK MYPWLHQQAI QEGFQNLTGT DSSQESCEYM 
               
               
                   
               
               
                 481 
                 SLTRSTFIGG QRFCSYLWSG DTDSKFDVLL QQITAGVSVA 
               
               
                   
               
               
                 521 
                 ASGISSWTLD IGGFAGLDID TDEGKELFVR WFSMGVFLPY 
               
               
                   
               
               
                 561 
                 TRVHGTRSCN IPRTSTLPHA NPCPNEPWSY GEDNFVILKK 
               
               
                   
               
               
                 601 
                 YIALRYQLIP YVKTLFQMLH TSGKVILRPL YFDFSKSDEF 
               
               
                   
               
               
                 641 
                 VRKGTKTNDP VVVHQFMFGP RLLVAPVGEF GVKTWDVYLP 
               
               
                   
               
               
                 681 
                 KLDTQTWKHW QVTTNQIPRW TDHDFGKGGM SITIDAPLDQ 
               
               
                   
               
               
                 721 
                 IPVFYLGDKD DILNGNI 
               
            
           
         
       
     
     An α-xylosidase from  Penicillium chrysogenum  has NCBI accession number XP_002566456.1, and has 35% sequence identity to the α-xylosidase described herein with SEQ ID NO:1. This α-xylosidase from  Penicillium chrysogenum  has no predicted signal sequence, and has amino acid sequence SEQ ID NO:15. 
     
       
         
           
               
               
            
               
                 1 
                 MLYAEDDKLV FRFDDHILWV QPWGENAFRV RATKQASIPT 
               
               
                   
               
               
                 41 
                 EDWALPSKPS SPSPSIEISA DQEATITNGK IKATVSRRGK 
               
               
                   
               
               
                 81 
                 IIIYDSKGNK LLEEYARHRQ DPMDPKCSAL TVEARELRPI 
               
               
                   
               
               
                 121 
                 LGGDYHLTMR FESLDHKEKI FGMGQYQQPY LNLKGADLEL 
               
               
                   
               
               
                 161 
                 AHRNSQASVP FAVSSLGYGF LWNNPGIGRA VLGTNVMSFE 
               
               
                   
               
               
                 201 
                 AYSTKALDYW VVAGDTPAEI EEAYAKVTGY VPMMPEYGLG 
               
               
                   
               
               
                 241 
                 FWQCKLRYTN QEQLLNIARE YRRREVPLDL IVIDFFHWKH 
               
               
                   
               
               
                 281 
                 QGEWSFDPEF WPDPEAMVKE LKELKVELMV SIWPTVENAS 
               
               
                   
               
               
                 321 
                 ENFPEMLEQG LLIRHDRGMR VAMQCDGDIT HFDATNPAAR 
               
               
                   
               
               
                 361 
                 KFIWSKAKQN YYDIGIKTFW LDEAEPEYSI YDFDIYRYHA 
               
               
                   
               
               
                 401 
                 GSNLQIGNTY PKEYARGFYE GMTAEGQTNI VNLLRCAWAG 
               
               
                   
               
               
                 441 
                 SQKYGALVWS GDIASSWSSF RNQLAAGLNM GLAGIPWWTT 
               
               
                   
               
               
                 481 
                 DIGGFHGGNP DDPLFRELFT RWFQWGTFCP VMRLHGDREP 
               
               
                   
               
               
                 521 
                 KPEGQPTASG ADNEIWSYGD EVYEICKRYI GIREKLREYT 
               
               
                   
               
               
                 561 
                 RGLMREAHEK GTPVMRTLFY EFPSDERAWE VETQYMFGSK 
               
               
                   
               
               
                 601 
                 YLVVPVLEPG QRTVKVYLPA GASWKLWDEK DVLHEGGRNV 
               
               
                   
               
               
                 641 
                 EIECPIENMP VFCRQ 
               
            
           
         
       
     
     Another α-xylosidase from  Penicillium chrysogenum  has a Joint Genome Institute (JGI; see jgi.doe.gov) accession number JGI 85065 and has a signal peptide and amino acid sequence SEQ ID NO:16. 
     
       
         
           
               
               
            
               
                 1 
                 MRLALIALGA IWASSSVASP VQQTTYHKPT SKGFRMQHGF 
               
               
                   
               
               
                 41 
                 ETVLVQPFGY DGFRVRAWPF RAPTGHEIGF VYDPPLEGPE 
               
               
                   
               
               
                 81 
                 NGEAHGMTFD TAFNGNRSEE LRNGNMIVRT SGWGGSPGGY 
               
               
                   
               
               
                 121 
                 RLAFYRVEAN GSETLLTNEY APLKSLNPRY YSWTGPGSEF 
               
               
                   
               
               
                 161 
                 AAEFSFSTTP EEQIYGTGTQ QDHLVNKKGL TIDLINFNTH 
               
               
                   
               
               
                 201 
                 IPTPVFMSNK GYGFIWNMAS TGRMEFGPLR NRFTADAASV 
               
               
                   
               
               
                 241 
                 VDYVIVSSDP SDYDTLQQRL SALVGRAPTP PDWSLGYLQS 
               
               
                   
               
               
                 281 
                 KLRYENQSEV IQLAQQFHDR KIPVSMIVID YQSWAHQGDW 
               
               
                   
               
               
                 321 
                 GLDPALWPDV AEMARQVKDL TNAEMMASLW PSVADDSVNY 
               
               
                   
               
               
                 361 
                 LEMMAQGFLS ATRSGPGTTD SWNGSYIRNY DSTNPGARRF 
               
               
                   
               
               
                 401 
                 LWNTLKRNYF DKGIKNFWID QADGGSLGEA YENNGQSDYI 
               
               
                   
               
               
                 441 
                 QSLPFPMPDV LYAAGTQRNV GKLYPWAHQQ AIEEGFRNAT 
               
               
                   
               
               
                 481 
                 STDMGSPCNY LSLSRSGYIG SQRFCSMIWS GDITSVWETL 
               
               
                   
               
               
                 521 
                 SAQVASGLSA AATGWGWWTL DAGGFQADPT VPWSGNIDSP 
               
               
                   
               
               
                 561 
                 EYRELYVRWF QWSTFLPFMR THGSRTCDFQ DAYTCANEPW 
               
               
                   
               
               
                 601 
                 SYGSENTPIL VSYINLRYQL SAYLRAVFAQ LHKSGRMIMR 
               
               
                   
               
               
                 641 
                 PLYMDFEKSD PHVARWTSAN TNITTQQYMF GPRLLVSPVT 
               
               
                   
               
               
                 681 
                 IPNVTEWSVY LPQTAGDDSK PWTYWWSNQT YSGGQTVTVP 
               
               
                   
               
               
                 721 
                 APKEHIPLFH LGTRADIVDG RVFA 
               
            
           
         
       
     
     An α-xylosidase from  Aspergillus carbonarius  has JGI accession number jgi|Aspca3|209950, has a signal peptide sequence, and has amino acid sequence SEQ ID NO:17. 
     
       
         
           
               
               
            
               
                 1 
                 MYFPSLLALG ALVQAAAATY IAPNSTGLRL QHGFETILIQ 
               
               
                   
               
               
                 41 
                 PFGYDGFRVR AWPFRPPSGN EISFIYDPPL EGFEDSAHGM 
               
               
                   
               
               
                 81 
                 SYDTATTGSE PRTLRNGNMI LRTTGWGGET GGYRLSFSRV 
               
               
                   
               
               
                 121 
                 NEDGSETLLT NEYAPLKSLN PRYYHWPGPG PEFSAEFSFS 
               
               
                   
               
               
                 161 
                 ATPDEQIYGT GTQQDHMINK KGQVIDMVNF NTHIPTPVFM 
               
               
                   
               
               
                 201 
                 SNKGYAFIWN MPAEGRMEFG PLRTRFTAAT TTLVDYVIVA 
               
               
                   
               
               
                 241 
                 SAPGDYDTLQ RRISALTGRA PVPPDFALGY IQSKLRYENE 
               
               
                   
               
               
                 281 
                 TEVELLAQNF HDRGIPVAMI VIDYQSWAHQ GDWALDPRLW 
               
               
                   
               
               
                 321 
                 PNVGQMSARV KNLTGAEMMA SLWPSVADNS VNYAALQANG 
               
               
                   
               
               
                 361 
                 LLSATRDGPG TTDSWNGSYI RNYDSTNPSA RQFLWSMLKK 
               
               
                   
               
               
                 401 
                 NYYDKGIKNF WIDQADGGAL GEAYENNGQS TYIESIPFAL 
               
               
                   
               
               
                 441 
                 PNVLYAAGTQ LSVGKLYPWA HQQAIDEGFR NATDTEEGSA 
               
               
                   
               
               
                 481 
                 CDHVSLSRSG YIGSQRFCSM IWSGDTTSVW DTLAVQVASG 
               
               
                   
               
               
                 521 
                 LSAAATGWGW WTVDAGGFQA DPTVWWSGNI DTPEFRELYV 
               
               
                   
               
               
                 561 
                 RWLSWTTFLP FMRTHGSRAC YFQDAYTCAN EPWSYGEANT 
               
               
                   
               
               
                 601 
                 PIIVSYIHLR YQLGAYLRSI FKQFHLTGRS IMRPLYMDFE 
               
               
                   
               
               
                 641 
                 KTDPKISTLT ASNSNYTTQQ YMFGPRLLVS PVTLPNVTEW 
               
               
                   
               
               
                 681 
                 PVYLPQTGGN STKPWTYWWT NETYAGGQVV TVSAPVQHIP 
               
               
                   
               
               
                 721 
                 VFHLGSREEL LTGNVF 
               
            
           
         
       
     
     An α-xylosidase from  Aspergillus brasiliensis  has JGI accession number jgi|Aspbr1|131273, has a signal peptide sequence, and has amino acid sequence SEQ ID NO:18. 
     
       
         
           
               
               
            
               
                 1 
                 MYFSSFFALG ALVQAAAATY FAPNSTGLRI QHGFETILVQ 
               
               
                   
               
               
                 41 
                 PFGYDGFRVR AWPFRPPSGN EISFIYDPPI EGYEDTAHGM 
               
               
                   
               
               
                 81 
                 SYDTATTGAE PRTLRNGNII LRTTGWGGDT AGYRLSFYRV 
               
               
                   
               
               
                 121 
                 NEDGSETLLT NEYAPLKSLN PRYYSWPGPG AEFSAEFSFS 
               
               
                   
               
               
                 161 
                 ATPDEQIYGT GTQQDHMINK KGSVIDMVNF NTHIPTPVFM 
               
               
                   
               
               
                 201 
                 SNKGYAFIWN MPAEGRMEFG TLRTRFTAAS TTLVDYVIVA 
               
               
                   
               
               
                 241 
                 AQPGDYDTLQ QRISALTGRA PTPPDFSLGY IQSKLRYENQ 
               
               
                   
               
               
                 281 
                 TEVELLAQNF HDRNIPVSMI VIDYQSWAHQ GDWALDPRLW 
               
               
                   
               
               
                 321 
                 PNVAQMSARV KNLTGAEMMA SLWPSVEDNS VNYATLQANG 
               
               
                   
               
               
                 361 
                 LLSATRDGPG TTDSWNGSYI RNIDSTNPAA RKFLWSTLKK 
               
               
                   
               
               
                 401 
                 NYYDKGIKNF WIDQADGGAL GEAYENNGQS TYIQSIPYTL 
               
               
                   
               
               
                 441 
                 PNVNYAAGTQ LGVGKLYPWA HQQAIEEGFR NATDTKEGSA 
               
               
                   
               
               
                 481 
                 CDHVSLSRSG YIGSQRFCSM IWSGDTTSVW DTLAVQVASG 
               
               
                   
               
               
                 521 
                 LSAAATGWGW WTVDAGGFEV DSTVWWSGNI DTPEFRELYV 
               
               
                   
               
               
                 561 
                 RWLAWTTFLP FMRTHGSRTC YYQDAYTCAN EPWSYGASNT 
               
               
                   
               
               
                 601 
                 PIIVSYIHLR YQLGAYLKSI FNQFHLTGRS IMRPLYMDFE 
               
               
                   
               
               
                 641 
                 KTDPKISQLV SSNSNYTTQQ YMFGPRLLVS PVTLPNVTEW 
               
               
                   
               
               
                 681 
                 PVYLPQTGEN NTKPWTYWWT NETYAGGQVV KVPAPVQHIP 
               
               
                   
               
               
                 721 
                 VFHLGSREEL LSGDVF 
               
            
           
         
       
     
     An α-xylosidase from  Aspergillus acidus  has JGI accession number jgi|Aspfo1|143652, has a signal peptide sequence, and has amino acid sequence SEQ ID NO:19. 
                        1   MYFSSFLALG ALIQAAAATY LAPNSTGLRI QHGFETILIQ               41   PFGYDGFRVR AWPFRPPSGN EISFIYDPPI EGYEDTAHGM               81   SYDTATTGTE PRTLRNGNII LRTTGWGGTT AGYRLSFYRV               121   NDDGSETLLT NEYAPLKSLN PRYFSWPGPG AEFSAEFSFS               161   ATPDEQIYGT GTQQDHMINK KGSVIDLVNF NTHIPTPVFM               201   SNKGYAFIWN MPAEGRMEFG SLRTRFTAAS TTLVDYVIVA               241   AQPGDYDTLQ QRISALTGRA PTPPDFSLGY IQSKLRYENQ               281   TEVELLAQNF HDRDIPVSMI VIDYQSWAHQ GDWALDPRLW               321   PNVAQMSATV KNLTGAEMMA SLWPSVADDS VNYAALQANG               361   LLSATRDGPG TTDSWNGSYI RNYDSTNPSA RKFLWSMLKK               401   NYYDKGIKNF WIDQADGGAL GEAYENNGQS TYIQSIPYTL               441   PNVNYAAGTQ LGVGKLYPWA HQQAIEEGFR NATDTKEGSA               481   CDHVSLSRSG YIGSQRFCSM IWSGDTTSVW DTLAVQVASG               521   LSAAATGWGW WTVDAGGFEVDSTVWWSGNIDTPEFRELYV               561   RWLAWTTFLP FMRTHGSRAC YYQDAYTCAN EPWSYGASNT               601   PIIVSYIHLR YQLGAYLKSI FNQFHLTGRS IMRPLYMDFE               641   KTDPKISQLV SSNSNYTTQQ YMFGPRLLVS PVTLPNVTEW               681   PVYLPQTGDN STKPWTYWWT NETYAGGQVV KVPAPVQHIP               721   VFHLGSREEL LSGDVF            
A cDNA encoding the  Aspergillus acidus  α-xylosidase has SEQ ID NO:20.
 
     
       
         
           
               
               
            
               
                 1 
                 ATGTATTTTT CTTCCTTTTT GGCCCTAGGG GCCCTGATTC 
               
               
                   
               
               
                 41 
                 AGGCAGCAGC AGCAACCTAT CTCGCCCCCA ACTCTACCGG 
               
               
                   
               
               
                 121 
                 TCTCCGTATC CAGCATGGCT TCGAGACCAT CCTCATCCAG 
               
               
                   
               
               
                 161 
                 CCGTTTGGGT ACGACGGATT CCGCGTGCGC GCATGGCCCT 
               
               
                   
               
               
                 201 
                 TCCGTCCGCC TTCGGGCAAC GAGATTAGCT TCATCTATGA 
               
               
                   
               
               
                 241 
                 TCCCCCGATT GAAGGTTATG AGGACACCGC ACATGGCATG 
               
               
                   
               
               
                 281 
                 AGCTATGACA CCGCAACAAC CGGCACGGAG CCTCGCACCT 
               
               
                   
               
               
                 321 
                 TGCGCAACGG CAATATCATC CTGCGCACCA CTGGCTGGGG 
               
               
                   
               
               
                 361 
                 TGGCACCACC GCCGGATACC GCCTGTCCTT CTACCGCGTC 
               
               
                   
               
               
                 401 
                 AATGATGATG GGAGTGAGAC CCTGCTCACA AACGAATATG 
               
               
                   
               
               
                 441 
                 CTCCGCTGAA GTCTCTCAAC CCCCGATACT TTTCCTGGCC 
               
               
                   
               
               
                 481 
                 GGGACCTGGG GCCGAATTCT CTGCCGAGTT CTCCTTCAGT 
               
               
                   
               
               
                 521 
                 GCGACTCCGG ATGAGCAGAT TTATGGCACG GGCACGCAAC 
               
               
                   
               
               
                 561 
                 AAGACCATAT GATCAACAAG AAGGGTTCCG TTATCGACTT 
               
               
                   
               
               
                 601 
                 GGTCAACTTC AACACCCACA TCCCTACCCC AGTCTTCATG 
               
               
                   
               
               
                 641 
                 AGCAACAAAG GCTATGCCTT TATCTGGAAC ATGCCGGCCG 
               
               
                   
               
               
                 681 
                 AGGGGCGTAT GGAGTTTGGC AGCCTGCGCA CCAGGTTCAC 
               
               
                   
               
               
                 721 
                 CGCGGCGTCC ACGACGCTTG TCGACTATGT AATCGTCGCC 
               
               
                   
               
               
                 761 
                 GCTCAGCCAG GTGATTACGA CACCCTCCAG CAGCGGATTT 
               
               
                   
               
               
                 801 
                 CGGCCCTGAC AGGACGGGCA CCGACCCCGC CCGACTTTTC 
               
               
                   
               
               
                 841 
                 TCTCGGGTAC ATCCAGTCCA AGCTACGATA TGAGAACCAA 
               
               
                   
               
               
                 881 
                 ACGGAGGTGG AGCTGCTGGC TCAGAACTTC CATGATAGAG 
               
               
                   
               
               
                 921 
                 ACATCCCGGT GTCCATGATC GTTATTGACT ACCAGTCGTG 
               
               
                   
               
               
                 961 
                 GGCTCATCAG GGTGACTGGG CGCTCGATCC GCGCCTGTGG 
               
               
                   
               
               
                 1001 
                 CCCAATGTCG CGCAGATGTC GGCGACAGTC AAGAATCTGA 
               
               
                   
               
               
                 1041 
                 CCGGAGCCGA AATGATGGCG TCTCTATGGC CCAGTGTTGC 
               
               
                   
               
               
                 1081 
                 CGATGACAGT GTCAACTACG CAGCCCTGCA GGCGAACGGT 
               
               
                   
               
               
                 1121 
                 CTGCTCTCAG CCACCCGCGA CGGCCCTGGT ACCACTGACT 
               
               
                   
               
               
                 1161 
                 CCTGGAACGG ATCATACATC CGGAACTATG ACTCCACCAA 
               
               
                   
               
               
                 1201 
                 CCCCTCGGCG CGGAAATTCC TCTGGAGCAT GCTGAAGAAA 
               
               
                   
               
               
                 1241 
                 AACTACTACG ACAAGGGTAT TAAGAACTTT TGGATTGATC 
               
               
                   
               
               
                 1281 
                 AGGCCGATGG CGGAGCATTG GGCGAGGCTT ATGAGAACAA 
               
               
                   
               
               
                 1321 
                 CGGCCAGAGC ACATACATTC AGTCCATTCC GTATACCCTG 
               
               
                   
               
               
                 1361 
                 CCGAACGTGA ACTACGCCGC TGGCACGCAG CTCGGCGTGG 
               
               
                   
               
               
                 1401 
                 GTAAGTTGTA CCCCTGGGCG CAGCAACAGG CAATCGAAGA 
               
               
                   
               
               
                 1441 
                 AGGCTTCCGC AATGCGACAG ACACCAAGGA AGGAAGCGCT 
               
               
                   
               
               
                 1481 
                 TGCGATCACG TCTCCCTGAG TCGGTCCGGA TACATCGGAT 
               
               
                   
               
               
                 1521 
                 CTCAGCGGTT CTGCAGCATG ATCTGGTCTG GAGACACCAC 
               
               
                   
               
               
                 1561 
                 CTCTGTTTGG GACACACTGG CAGTGCAGGT CGCCAGTGGT 
               
               
                   
               
               
                 1601 
                 CTGTCCGCCG CAGCAACAGG CTGGGGTTGG TGGACCGTCG 
               
               
                   
               
               
                 1641 
                 ATGCTGGCGG CTTCGAAGTC GACTCGACAG TTTGGTGGAG 
               
               
                   
               
               
                 1681 
                 TGGAAACATT GACACGCCCG AATTCCGGGA GTTGTATGTG 
               
               
                   
               
               
                 1721 
                 CGCTGGCTGG CCTGGACGAC CTTCCTGCCA TTCATGCGCA 
               
               
                   
               
               
                 1761 
                 CTCATGGTAG TCGGGCCTGC TACTACCAGG ACGCCTACAC 
               
               
                   
               
               
                 1801 
                 TTGTGCCAAT GAGCCATGGT CCTATGGTGC AAGCAACACC 
               
               
                   
               
               
                 1841 
                 CCCATTATTG TCTCGTATAT CCACCTGCGT TACCAATTGG 
               
               
                   
               
               
                 1881 
                 GTGCTTATCT GAAGTCGATT TTCAACCAGT TCCACCTCAC 
               
               
                   
               
               
                 1921 
                 GGGTCGCAGT ATCATGCGCC CGTTGTACAT GGATTTCGAG 
               
               
                   
               
               
                 1961 
                 AAGACCGACC CGAAGATCTC TCAGCTGGTG TCGTCGAACA 
               
               
                   
               
               
                 2001 
                 GAGACACCAC AACTCAACAG TACATGTTTG GTCCACGTCT 
               
               
                   
               
               
                 2041 
                 CCTAGTCTCT CCAGTGACCT TGCCAAACGT CACTGAGTGG 
               
               
                   
               
               
                 2081 
                 CCTGTGTATC TTCCGCAGAC GGGAGATAAT AGCACTAAGC 
               
               
                   
               
               
                 2121 
                 CTTGGACGTA CTGGTGGACG AATGAGACGT ATGCGGGAGG 
               
               
                   
               
               
                 2161 
                 ACAGGTCGTC AAGGTTCCTG CGCCCGTGCA GCATATCCCG 
               
               
                   
               
               
                 2201 
                 GTATTCCATC TGGGATCGCG CGAGGAGCTT CTGTCGGGTG 
               
               
                   
               
               
                 2241 
                 ATGTATTCTA G 
               
            
           
         
       
     
     An α-xylosidase from  Aspergillus tubingensis  has JGI accession number jgi|Asptu1|396136, has a signal peptide sequence, and has amino acid sequence SEQ ID NO:21. 
     
       
         
           
               
               
            
               
                 1 
                 MYFSSLLALG ALVQAAAATY FAPNSTGLRI QHGFETILIQ 
               
               
                   
               
               
                 41 
                 PFGYDGFRVR AWPFRPPSGN EISFIYDPPI EGYEDTAHGM 
               
               
                   
               
               
                 121 
                 SYDTATTGTE PRTLRNGNII LRTTGWGGTT AGYRLSFYRV 
               
               
                   
               
               
                 161 
                 NDDGSETLLT NEYAPLKSLN PRYYYWPGPG AEFSAEFSFS 
               
               
                   
               
               
                 201 
                 ATPDEQIYGT GTQQDHMINK KGSVIDLVNF NTHIPTPVFM 
               
               
                   
               
               
                 241 
                 SNKGYAFIWN MPAEGRMEFG SLRTRFTAAS TTLVDYVIVA 
               
               
                   
               
               
                 281 
                 AQPGDYDTLQ QRISALTGRA PTPPDFSLGY IQSKLRYENQ 
               
               
                   
               
               
                 321 
                 TEVELLAQNF HDRDIPVSMI VIDYQSWAHQ GDWALDPRLW 
               
               
                   
               
               
                 361 
                 PNVAQMSATV KNLTGAEMMA SLWPSVADDS VNYAALQANG 
               
               
                   
               
               
                 401 
                 LLSATRDGPG TTDSWNGSYI RNYDSTNPSA RKFLWSMLKK 
               
               
                   
               
               
                 441 
                 NYYDKGIKNF WIDQADGGAL GEAYENNGQS TYIQSIPYTL 
               
               
                   
               
               
                 481 
                 PNVNYAAGTQ LGVGKLYPWA HQQAIEEGFR NATDTKKGSA 
               
               
                   
               
               
                 521 
                 CDHVSLSRSG YIGSQRFCSM IWSGDTTSVW DTLAVQVASG 
               
               
                   
               
               
                 561 
                 LSAAATGWGW WTVDAGGFEV DSTVWWSGNI DTPEFRELYV 
               
               
                   
               
               
                 601 
                 RWLAWTTFLP FMRTHGSRTC YYQDAYTCAN EPWSYGASNT 
               
               
                   
               
               
                 641 
                 PIIVSYIHLR YQLGAYLKSI FNQFHLTGRS IMRPLYMDFE 
               
               
                   
               
               
                 681 
                 KTDPKISQLV SSNSNYTTQQ YMFGPRLLVS PVTLPNVTEW 
               
               
                   
               
               
                 721 
                 PVYLPQTGDN STKPWTYWWT NETYAGGQVV KVPAPVQHIP 
               
               
                   
               
               
                 761 
                 VFHLGSREEL LSGDVF 
               
            
           
         
       
     
     The following AN7505 (GenBank XP 680774 or, DQ490509.1, or ABF50885.1 with SEQ ID NO:22) polypeptide sequence from  Aspergillus nidulans  has about 25% amino acid identity to Ax1A, lacks a predicted signal peptide, and is thereby most likely an intracellular, cytosolic α-xylosidase enzyme. See, also, Bauer et al,  Proc. Natl. Acad. Sci. U.S.A.  103:11417-11422 (2006). 
                        1   MKFTEGMWLL REGIRIDWMS NVERLNVDKD TVNLLLNKFQ               41   RHRGDTLNSS TVSARVTSPL EGIIGVKLVH WAGGLDNGPH               81   YELNTSAGHT EITHEKGKNL KYTSGRLELD INIAPNELAF               121   TFTTGADGQD KRKKLTGHSF RSIGYVGDST TPKSQLSDGI               161   FYERQGYTLA ELDLSVGEKL YGLGERFGPF VKNGQSVNIW               201   NEDGGTSSEL AYKNIPFYIS SNGYGVFVNH PGKVSLELQS               241   ERTTRVNVSV EGEELEYFVI EGKNPKEILK RWTDLTGKPA               281   LVPAWSYGLW LTTSFTTNYS ERTVTGFLDG FKDRNLPLSV               321   FHFDCFWMKS YQWCDFEFDA DMFPDAAGYL ARLKERGLKL               361   SIWINPYVGQ ASPLFEIGKR EGYFIKRIDG SVWQWDLWQA               401   GMAVVDFTNP AACSWYTGHL KRLMDLGIDT FKTDFAERIP               441   FKNITYHDGS DPARMHNYYA LLYNKVVYET MTSISGKSNS               481   LLFARSTSVG GQKYPVHWGG DCESTYEAMA ESLRGGLSLG               521   LAGYIFWASD IGGFEGTPPP ALYKRWVQFG LLSSHSRLHG               561   SSSFRVPWIY GEDCSDVLRD CVKRKISLTP YLLAEALNGH               601   RSGTPLMRPM FMEFPEDLNT YPLDTQYMFG SNLLVAPVFS               641   DEGIVTFYVP RTPEEEGRKQ WISWFDHGKK YEGGRWYTET               681   HGFDTLPILI RPGSVTPINY KLEKPEGNPL DGLEILVNGS               721   IDKEVEIEIV DPETTHKVLK VMTVSERETE NGVEVIARLD               761   GVDGNENSVK VNWVGHGVTK            
Therefore, even if native AN7505 is an α-xylosidase, the data highly suggest that the enzyme is localized within the intracellular cytoplasmic space.
 
     The α-xylosidase described herein with SEQ ID NO:1 is also referred to as Ax1A. The Ax1A is present in both sequenced strains of  A. niger , ATCC1015 and CBS 513.88, with 100% amino acid identity and 99% nucleotide identity in the coding region. Pel et al., Nat. Biotechnol. 25: 221-231 (2007); and Andersen et al., Genome Res. 21:885-897 (2011).  A. nidulans  has 10 predicted GH31 genes, five of which have signal peptides. Of these, AN7120 (XP_664724) has the best amino acid identity to Ax1A (30%), but no signal peptide.  A. niger  ATCC 1015 and CBS 513.88 both have seven predicted GH31 genes, the best of which (after Ax1A itself) being ANI_1_620014 (also known as Aspni5|55419), with 32% identity. 
     The Ax1A mRNA and protein expression have been reported to be induced by  A. niger  growth on xylose as compared with maltose. Gonzalez-Vogel et al.,  Appl. Microbial. Biotechnol.  89:145-155 (2011); Jørgensen et al.,  BMC Genomics  10:44 (2009); and de Oliveira et al., PLoS ONE 6:e20865 (2011). Ax1A was not included in a genome-wide microarray expression study comparing  A. nidulans, A. oryzae , and  A. niger , presumably because it is not common to all three species. Andersen et al.,  Proc. Natl. Acad. Sci. U.S.A.  105, 4387-4392 (2008). 
     After the first tier Ax1A orthologs were identified in species of  Aspergillus , approximately the next best 20 hits to Ax1A in GenBank™, have E-values ranging from e-97 to e-23 and percent identities ranging from 22% to 52%, encompassing a much wider variety of fungi. All of these proteins are hypothetical, and it is not known whether they have α-xylosidase, β-glucosidase, or any other catalytic activities. However, the majority of these second tier orthologs lack predicted signal peptides. This is a strong indication that they are not extracellularly secreted and are probably functional orthologs of the cytoplasmic α-xylosidase enzymes of  A. flavus, A. niger , and  P. wortmanii.    
       T. reesei  has only two poor (E-value&gt;e-10 and &lt;25% amino acid identity) BLASTP hits to Ax1A (Trire2|121351 and Trir2|69944 [JGI numbering]), and neither of these has a predicted signal peptide. It appears that  T. reesei  does not have the genetic potential to biosynthesize a secreted α-xylosidase related to Ax1A, which is consistent with the observed lack of this enzymatic activity in commercial enzyme mixtures derived from  T. reesei .  FIG. 1B . 
     Taken together, the evidence indicates that only a small subset of fungi have the genetic potential to biosynthesize secreted enzymes with α-xylosidase activity. 
     Proteins and nucleic acids related to those specifically described herein can be isolated and identified by a variety of methods. For example, any of SEQ ID NO:1-6 can be mutated and/or can be isolated by hybridization to DNA and/or RNA isolated from other species (e.g., other fungal species) using any of the SEQ ID NO:2, 4 or 6 nucleic acids as probes. The sequence of the α-xylosidase enzyme (e.g., SEQ ID NO:1, 3, 5, 7-19, 19, 21 and/or 22) can also be examined and used as a basis for designing alternative α-xylosidase nucleic acids that encode related α-xylosidase polypeptides. 
     In one embodiment, the α-xylosidase nucleic acids of the invention include any nucleic acid that can selectively hybridize to SEQ ID NO:2, 4, 6 and/or 20. 
     The term “selectively hybridize” includes hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence (e.g., SEQ ID NO:2, 4, 6 and/or 20) to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences. Such selective hybridization substantially excludes non-target nucleic acids. Selectively hybridizing sequences typically have about at least 40% sequence identity, at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 60-90% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 98-99% sequence identity, or 100% sequence identity (or complementarity) with each other. In some embodiments, a selectively hybridizing sequence has about at least about 90% sequence identity or complementarity with SEQ ID NO:2, 4, 6 and/or 20. 
     Thus, the nucleic acids of the invention include those with about 500 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 700 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 900 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 1000 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 1200 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 1400 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 1600 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 1800 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 2000 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 2100 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 2150 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20, or about 700-2150 of the same nucleotides as SEQ ID NO:2, 4, 6 and/or 20. The identical nucleotides or amino acids can be distributed throughout the nucleic acid or the protein, and need not be contiguous. 
     Note that if a value of a variable that is necessarily an integer, e.g., the number of nucleotides or amino acids in a nucleic acid or protein, is described as a range, e.g., or 90-99% sequence identity, what is meant is that the value can be any integer between 90 and 99 inclusive, i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. 
     The terms “stringent conditions” or “stringent hybridization conditions” include conditions under which a probe will hybridize to its target sequence to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are somewhat sequence-dependent and can vary in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified with up to 100% complementarity to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of sequence similarity are detected (heterologous probing). The probe can be approximately 20-500 nucleotides in length, but can vary greatly in length from about 18 nucleotides to equal to the entire length of the target sequence. In some embodiments, the probe is about 10-50 nucleotides in length, or about 18-25 nucleotides in length, or about 18-50 nucleotides in length, or about 18-100 nucleotides in length. 
     Typically, stringent conditions will be those where the salt concentration is less than about 1.5 M Na ion (or other salts), typically about 0.01 to 1.0 M Na ion concentration (or other salts), at pH 7.0 to 8.3 and the temperature is at least about 30° C. for shorter probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longer probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt&#39;s solution. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1×SSC to 2×SSC (where 20×SSC is 3.0 M NaCl, 0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.5×SSC to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Specificity is typically a function of post-hybridization washes, where the factors controlling hybridization include the ionic strength and temperature of the final wash solution. 
     For DNA-DNA hybrids, the T m  can be approximated from the equation of Meinkoth and Wahl (Anal. Biochem. 138:267-84 (1984)):
 
 T   m =81.5° C.+16.6(log  M )+0.41(%  GC )−0.61(% formamide)−500/L
 
where M is the molarity of monovalent cations; % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T m  is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. The T m  is reduced by about 1° C. for each 1% of mismatching. Thus, the T m , hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired sequence identity. For example, if sequences with greater than or equal to 90% sequence identity are sought, the T m  can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can include hybridization and/or a wash at 1, 2, 3 or 4° C. lower than the thermal melting point (T m ). Moderately stringent conditions can include hybridization and/or a wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting point (T m ). Low stringency conditions can include hybridization and/or a wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting point (T m ). Using the equation, hybridization and wash compositions, and a desired T m , those of ordinary skill can identify and isolate nucleic acids with sequences related to SEQ ID NO:2, 4, 6 and/or 20.
 
     Those of skill in the art also understand how to vary the hybridization and/or wash solutions to isolate desirable nucleic acids. For example, if the desired degree of mismatching results in a T m  of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. 
     An extensive guide to the hybridization of nucleic acids is found in Tijssen, L ABORATORY  T ECHNIQUES IN  B IOCHEMISTRY AND  M OLECULAR  B IOLOGY —H YBRIDIZATION WITH  N UCLEIC  A CID  P ROBES , part 1, chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, N.Y. (1993); and in C URRENT  P ROTOCOLS IN  M OLECULAR  B IOLOGY , chapter 2, Ausubel, et al., eds, Greene Publishing and Wiley-Interscience, New York (1995). 
     Unless otherwise stated, in the present application high stringency is defined as hybridization in 4×SSC, 5×Denhardt&#39;s (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65° C., and a wash in 0.1×SSC, 0.1% SDS at 65° C. 
     The following terms are used to describe the sequence relationships between two or more nucleic acids or nucleic acids or polypeptides: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity” and (e) “substantial identity.” 
     As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison (e.g., any of SEQ ID NO:1-6). The reference sequence can be a nucleic acid sequence (e.g., SEQ ID NO:2, 4, 6 and/or 20) or an amino acid sequence (e.g., SEQ ID NO:1, 3, 5, 7-19, 21 and/or 22). A reference sequence may be a subset or the entirety of a specified sequence. For example, a reference sequence may be a segment of a full-length cDNA or of a genomic DNA sequence, or the complete cDNA or complete genomic DNA sequence, or a domain of a polypeptide sequence. 
     As used herein, “comparison window” refers to a contiguous and specified segment of a nucleic acid or an amino acid sequence, wherein the nucleic acid/amino acid sequence can be compared to a reference sequence and wherein the portion of the nucleic acid/amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can vary for nucleic acid and polypeptide sequences. Generally, for nucleic acids, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or more nucleotides. For amino acid sequences, the comparison window is at least about 15 amino acids, and can optionally be 20, 30, 40, 50, 100 or more amino acids. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the nucleic acid or amino acid sequence, a gap penalty is typically introduced and is subtracted from the number of matches. 
     Methods of alignment of nucleotide and amino acid sequences for comparison are well known in the art. The local homology algorithm (BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, may permit optimal alignment of compared sequences; by the homology alignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-53; by the search for similarity method (Tfasta and Fasta) of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG™ programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992) Computer Applications in the Biosciences 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol. 24:307-31. An example of a good program to use for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle, (1987) J. Mol. Evol., 25:351-60, which is similar to the method described by Higgins and Sharp, (1989) CABIOS 5:151-53 (and is hereby incorporated by reference). The BLAST family of programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., eds., Greene Publishing and Wiley-Interscience, New York (1995). 
     GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-53, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP makes a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or greater. 
     GAP presents one member of the family of best alignments. There may be many members of this family. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915). 
     Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-402). 
     As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (C.sub.1-ayerie and States, (1993) Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination. 
     The terms “substantial identity” indicates that a polypeptide or nucleic acid comprises a sequence with between 55-100% sequence identity to a reference sequence, with at least 55% sequence identity, preferably 60%, preferably 70%, preferably 80%, more preferably at least 90% or at least 95% sequence identity to the reference sequence over a specified comparison window. Optimal alignment may be ascertained or conducted using the homology alignment algorithm of Needleman and Wunsch, supra. 
     An indication that two polypeptide sequences are substantially identical is that both polypeptides have α-xylosidase activity, meaning that both polypeptides can hydrolyze α-1,6-linked xylose residues. The polypeptide that is substantially identical to an α-xylosidase with any of SEQ ID NO:1, 3, 5, 7-19, 21 and/or 22 sequence (especially one substantially identical to the SEQ ID NO:1 sequence), may not have exactly the same level of activity as an α-xylosidase with any of SEQ ID NO:1, 3, 5, 7-19, 21 and/or 22. Instead, the substantially identical polypeptide may exhibit greater or lesser levels of α-xylosidase activity than the α-xylosidase with SEQ ID NO:1, 3, 5, 7-19, 21 and/or 22 (especially SEQ ID NO:1), as measured by assays available in the art or described herein (see, e.g., Example II). For example, the substantially identical polypeptide may have at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 100%, or at least about 105%, or at least about 110%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or at least about 200% of the activity of the an α-xylosidase with the SEQ ID NO:1, 3, 5, 7-19, 21 and/or 22 sequences (especially the SEQ ID NO:1 sequence) when measured by similar assay procedures. 
     Alternatively, substantial identity is present when second polypeptide is immunologically reactive with antibodies raised against the first polypeptide (e.g., any of polypeptides with SEQ ID NO:1, 3, 5, 7-19, 19, 21 and/or 22). Thus, a polypeptide is substantially identical to a first polypeptide, for example, where the two polypeptides differ only by a conservative substitution. In addition, a polypeptide can be substantially identical to a first polypeptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical. Polypeptides that are “substantially similar” share sequences as noted above except that some residue positions, which are not identical, may differ by conservative amino acid changes. 
     The α-xylosidase polypeptides of the present invention may include the first 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 N-terminal amino acid residues of any of the SEQ ID NO:1, 3, 5, 7-19, 21 and/or 22 sequences. Alternatively, the α-xylosidase polypeptides of the present invention may include the first 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 C-terminal amino acid residues of the SEQ ID NO:1, 3, 5, 7-19, 21 and/or 22 sequences. 
     2. Properties of Secreted Ax1A 
     The pH optimum of secreted α-xylosidase (Ax1A) on pNPαX was between 3 and 4.  FIG. 12A . The temperature optimum was 55° C. The activity was about 50% of maximum at 65° ( FIG. 12B ). The native protein was approximately as active on isoprimeverose (IP) as on the synthetic substrate pNPαX. See, Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Ax1A enzyme kinetics 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 K m  95% 
                   
                 k cat  95% 
               
               
                 Enzyme 
                   
                 K m   
                 confidence  
                 k cat   
                 confidence 
               
               
                 source 
                 Substrate 
                 μm 
                 interval 
                 min −1   
                 interval 
               
               
                   
               
               
                 Native 
                 pNPαX 
                 3.68 
                 1.83-5.53 
                 1393 
                 1130-1656 
               
               
                 Native 
                 IP 
                 9.78 
                  7.73-11.84 
                  917 
                 834-999 
               
               
                   Pichia - 
                 pNPαX 
                 6.91 
                 4.25-9.56 
                 1234 
                 1047-1420 
               
               
                 expressed 
                   
                   
                   
                   
                   
               
               
                   Pichia - 
                 IP 
                 4.03 
                 3.36-4.71 
                 1337 
                 1265-1409 
               
               
                 expressed 
               
               
                   
               
               
                 Nonlinear curve-fitting software (GraphPad Prism) was used to calculate the parameters and confidence intervals. 
               
            
           
         
       
     
     3. Heterologous Expression of Secreted Ax1A 
     When expressed in  P. pastoris , recombinant secreted Ax1A had an apparent molecular weight of about 110,000, larger than the native protein. Recombinant secreted Ax1A also ran as a more diffuse band than the native protein.  FIG. 11B . Both of these observations suggest that recombinant secreted Ax1A is hyperglycosylated when expressed in  P. pastoris . Secreted Ax1A has at least ten predicted N-glycosylation sites. Nonetheless, the heterologously expressed protein showed kinetic properties similar to the native protein on both pNPαX and isoprimeverose. See, Table 1. The recombinant secreted Ax1A expressed in  P. pastoris  had no detectable β-glucosidase, β-xylosidase, or α-glucosidase activity when assayed with p-nitrophenyl-β-D-glucoside, p-nitrophenyl-β-D-xyloside, or pNPβG, respectively (data not shown). This supports the conclusion that the β-glucosidase activity seen in the “purified” α-xylosidase is due to contamination with another protein.  FIG. 10 . 
     4. Activity of Secreted Ax1A on Xyloglucan Heptasaccharide 
     HPLC-purified native Ax1A (supra) degraded the heptasaccharide XXXG into free glucose and xylose sugar residues (data not shown). However, the above data suggested that this preparation contained residual β-glucosidase (βG) activity.  FIG. 10 . Recombinant secreted Ax1A released about 10 nmol of xylose, a quantity that did not increase with time.  FIG. 13 . This proportion of xylose corresponds to approximately one-third of the total xylose present in the xyloglucan heptasaccharide sample (i.e., for example, about 34 nmol in a 12 μl of a reaction volume of 500 μl). This result is consistent with secreted Ax1A removing a single xylose residue from the heptasaccharide to produce WOW and is further evidence that secreted Ax1A does not have intrinsic β-glucosidase activity. Digestion of the xyloglucan heptasaccharide with no enzyme or β-glucosidase alone released no to little xylose.  FIG. 13 . Digestion with a combination of secreted Ax1A and β-glucosidase released 83.4% of the theoretical maximum of xylose in 10 h.  FIG. 13 . Thus, β-glucosidase and α-xylosidase together are capable of substantially depolymerizing the heptasaccharide, which is the repeating unit of native xyloglucan ( FIG. 1 ). 
     5. Activity of Secreted Ax1A on Tamarind Xyloglucan 
     Because xyloglucan contains β-linked galactose and β-linked glucose in addition to α-linked xylose, four enzymes were included in the experiment: xyloglucanase, β-glucosidase, and β-galactosidase, all from  T. reesei , in addition to secreted Ax1A as described herein. See, Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Optimal proportions of four hemicellulases for release 
               
               
                 of glucose and xylose from tamarind xyloglucan. 
               
               
                 Total protein loading was 15 mg/g glucan 
               
               
                 βG is β-glucosidase 
               
               
                 Optimal enzyme proportions (%) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Product 
                 Ax1A 
                 Xyloglucanase 
                 βG 
                 β-Galactosidase 
                 Sugar Yield % 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 glucose 
                 51 
                 19 
                 5 
                 25 
                 99 
               
               
                 xylose 
                 59 
                 11 
                 5 
                 25 
                 100 
               
               
                   
               
            
           
         
       
     
     An optimized mixture of the four enzymes was developed using GENPLAT at fixed total protein loading. In the first experiment, the lower limit of each enzyme was set to 0%. However, because many combinations failed to yield about 5% of xylose or glucose, a statistically valid model could not be determined. In subsequent experiment, the lower limit of each enzyme was set to 5%, which gave a statistically valid model for both glucose and xylose and a complete digestion of tamarind xyloglucan was achieved (data not shown). The optimized proportions of the four enzymes for glucose and xylose release are shown in Table 2. Of these four enzymes, secreted Ax1A was present in the highest proportion (e.g., 51% for glucose and 59% for xylose). 
     6. Summary 
     A secreted α-xylosidase from  A. niger  was isolated, purified and characterized. Evidence that it is secreted include, but is not limited to: 1) presence of a predicted signal peptide in secreted Ax1A itself; and 2) secretion of Ax1A from  P. pastoris  under the control of its native signal peptide. 
     Previously reported α-xylosidase enzymes from filamentous fungi are usually intracellular. Consistent with this, the large majority of proteins (all of which are hypothetical) annotated as being in glycosyl hydrolase family 31, lack predicted signal peptides. For example, a putative  A. nidulans  cytosolic α-xylosidase (AN7505, GenBank DQ490509.1 or ABF50885) has minimal sequence identity (about 25%) to the secreted Ax1A α-xylosidase described herein. In the report showing that α-xylosidase AN7505 is secreted from  Pichia pastoris , a yeast signal peptide was fused to the amino terminus of the protein. Therefore, secretion under such conditions does not indicate whether the native protein is secreted or not. Bauer et al.,  Proc. Natl. Acad. Sci. U.S.A.  103:11417-11422 (2006). Like most other known and presumed fungal α-xylosidase enzymes, the native cellular location of AN7505 is most likely the cytoplasm. 
     Despite the abundance of α-linked xylose in plant cell wall polysaccharides, there has been relatively little previous work on α-xylosidase enzymes. van den Brink, J. et al.,  Appl. Microbial. Biotechnol.  91:1477-1492 (2011). The investigations described herein indicate that this may be because secreted microbial α-xylosidase enzymes are rare. The available data from both bacteria and fungi suggest that even though most lignocellulolytic microorganisms secrete enzymes that can degrade xyloglucan to isoprimeverose, they transport and degrade isoprimeverose intracellularly. That is, although α-xylosidases are made by many micro-organisms, as a general rule they do not secrete the enzyme. The rarity of secreted α-xylosidase enzymes in fungi is illustrated by the example of the commercial enzyme product known as Driselase®, which comes from the basidiomycete  Irpex lacteus . Although Driselase® contains dozens of cell wall-active enzymes, it lacks α-xylosidase activity. This has made it a useful diagnostic tool for studying xyloglucan because treatment of plant cell walls with Driselase® completely degrades xyloglucan into isoprimeverose molecules, which can be quantitated by several methods including chromatography. Lorences et al.,  Carbohydr. Res.  263:285-293 (1994). 
     The hypothesis that secreted α-xylosidase enzymes are rare among microorganisms is consistent with the preponderance of predicted GH31 proteins without signal peptides in the genomes of sequenced filamentous fungi and with the existence of isoprimeverose utilization operons in bacteria such as  L. pentosus . Chaillou et al.,  J. Bacterial.  180:2312-2320 (1998). The best BLASTP hits of Ax1A to the GenBank™ database are to α-xylosidase enzymes that have signal peptides, but this is only a small subset of all of the putative fungal GH31 proteins. Orthologs of secreted Ax1A with signal peptides are from species of  Aspergillus  and several basidiomycetes.  Aspergillus  species have many additional predicted GH31 proteins without signal peptides. 
     Secreted Ax1A has activity against pNPαX, isoprimeverose, xyloglucan heptasaccharide, and tamarind xyloglucan. As a naturally secreted protein, it should be able to tolerate a variety of environmental conditions. Secreted Ax1A is therefore predicted to be a versatile α-xylosidase enzyme that should find utility in biotechnological applications such as deconstruction of lignocellulosic materials into free, fermentable sugar residues (e.g., xylose, glucose) to support biofuel production. Because herbaceous dicotyledonous plants contain higher amounts of xyloglucan than grasses, Ax1A may be particularly useful for processing biomass from dicot species. Ax1A has a pH optimum of about 4.0, whereas most cellulase mixtures perform better at pH 4.5-5.0. 
     III. Secreted α-Xylosidase Enhanced Plant Biomass Degradation 
     Xylose (Xyl) is usually present in an isoprimeverose (IP) disaccharide molecule linked by an α-1,6 bond with a glucose (Glc) molecule. α-Xylosidases, either cytosolic or secreted, can cleave the xylose-glucose isoprimeverose molecule and/or xyloglucan oligosaccharides (i.e., for example, the heptasaccharide, XXXG). Fry et al.  Plant Physiol  89:1-3 (1993); and  FIG. 1 . Some embodiments of the present invention are commercially applicable because: i) xyloglucan is a major component of plant cell walls; ii) complete breakdown of xyloglucan is enhanced by α-xylosidase, preferably a secreted α-xylosidase; iii) in the absence of a secreted α-xylosidase, xyloglucan may remain in a non-fermentable form of isoprimeverose, thereby reducing the efficiency of the plant biomass degradation process into fermentable sugars; and iv) most, if not all, commercial enzyme preparations lack secreted α-xylosidase enzymes. Although it is not necessary to understand the mechanism of an invention, it is believed that both glucose and xylose are desirable for fermentation, and a secreted α-xylosidase is useful for improving the effectiveness of any enzyme mixture for biomass degradation. 
     Conventionally used enzyme mixtures for biomass deconstruction (supra) do not contain α-xylosidase enzymes because the α-xylosidase gene is not naturally present in most fungal genomes that are usually the source of commercial enzyme mixtures (i.e., for example,  Trichoderma reesei ). The data presented herein demonstrates that the efficiency of most commercially available enzyme mixtures is increased when a secreted α-xylosidase is added. This increased efficiency results in the production of higher sugar yields. For example, use of a secreted α-xylosidase enhances glucose (Glc) and xylose (Xyl) yields when mixed with a commercially available cellulase enzyme mixtures (CTec2, Novozyme; Accellerase 1000, Genencor). Use of a secreted α-xylosidase also enhances glucose and xylose yields from corn stover that has been pretreated with alkaline hydrogen peroxide (infra). 
     The secreted Ax1A described herein is a true extracellular fungal α-xylosidase, which can be expressed by  Picha pastoris  and which exhibits degradation activity on isoprimeverose molecules. The data presented herein demonstrates that secreted Ax1A is an α-xylosidase active on a range of substrates including natural substrates such as isoprimeverose and tamarind xyloglucan. Specifically, Ax1A enhances the release of glucose and xylose from natural lignocellulosic materials, especially when combined with commercial mixtures of cellulase enzymes. The biomass used in these experiments was AHP (alkaline-hydrogen peroxide) pretreated corn stover. Hydrolysis conditions were 0.2% glucan loading, 48 hr, 50° C., unless otherwise indicated. 
     Secreted Ax1A increased glucose release from this alkaline-hydrogen peroxide treated biomass by 9%, from 76% to 85% when combined with Accellerase 1000 (a Genencor product). See,  FIG. 3 . Similarly, secreted Ax1A enhanced glucose release from 83% to 90% when combined with a CTec2:HTec2 enzyme mixture (Novozymes product). See,  FIGS. 4 and 5B . When using CTec2 alone, a secreted Ax1A dose response curve enhanced glucose release from AHP by 7%, from 82 to 89%. See,  FIG. 5 . When CTec2 and HTec2 were combined in various proportions, a secreted Ax1A dose response curve showed enhanced glucose release of about 0.5-7% in all combinations, except the 25:75 mixture of CTec2 and HTec2. See,  FIG. 6 . A time course at two different Ctec2:Htec2 loading concentrations showed enhanced glucose release of about 2-10% in the presence of secreted Ax1A as compared to the absence of Ax1A. See,  FIG. 8 . 
     Secreted Ax1A enhanced xylose release from 56% to 60% in a dose response fashion when in combination with CTec2 and HTec2 (75:25) at a 2.5 mg/g glucan loading dose. See,  FIG. 7 . Similar enhancement of xylose release was seen in a dose response fashion with either CTec2, alone, or a CTec2:Htec2 combination (75:25) at a 1.0 mg/g glucan loading dose. See,  FIG. 9 . These data indicate that secreted Ax1A increases the ultimate yields of glucose and xylose when used in sufficient concentration and over time. Although it is not necessary to understand the mechanism of an invention, it is believed that these data suggest that the secreted Ax1A acts on the final step(s) of xyloglucan degradation. 
     IV. Compositions 
     Compositions of the α-xylosidase(s) described herein are also provided. Such compositions are also referred to as converting enzyme mixtures, or simply enzyme mixtures. Such compositions can include any of the α-xylosidase(s) described herein. For example, the compositions can include a carrier, α-xylosidase(s), and at least 5% weight percentage cellulase(s). 
     The carrier can include a convenient solvent such as an aqueous medium. The carrier can also include agents such as protease inhibitors, chelation agents, sugars, oligosaccharides, polyols, osmolytes, protein stabilizers, buffers, salts, and the like. In some instances, the carrier is a microbial fermentation or growth medium that has been employed to grow the microbial host cells that express the α-xylosidase(s). After fermentation and/or growth of the microbial host cells, the host cells are removed, and a microbial fermentation medium can be filtered, diluted, proteins in the medium can be concentrated, and/or agents such as those listed above can be added. 
     The α-xylosidase(s) in the compositions and enzyme mixtures can include any of those described herein For example, the α-xylosidase(s) in the compositions and enzyme mixtures can include polypeptides with sequences having at least 40% sequence identity with any of SEQ ID NO:1, 3, 5, 7-19, or 21. The α-xylosidase(s) in the compositions and enzyme mixtures can also include polypeptides with sequences having other percentages of sequence identity with any of SEQ ID NO:1, 3, 5, 7-19, or 21. Such percentages of sequence identity can be any of the percentages described herein (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and/or at least 95% sequence identity with any of SEQ ID NO:1, 3, 5, 7-19, or 21). In some embodiments, the compositions, enzyme mixtures and converting enzyme mixtures do not include a polypeptide having SEQ ID NO:22. 
     The α-xylosidase(s) can be present in the compositions and enzyme mixtures in varying amounts. For example, the compositions and enzyme mixtures can include about 0.1%, about 0.25%, about 0.5%, about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 15%, or about 20% by weight α-xylosidase(s). In some instances, the compositions and enzyme mixtures can include about 0.1% to about 20% by weight α-xylosidase(s), or about 0.2% to about 10% by weight α-xylosidase(s), or about 0.5% to about 5% by weight α-xylosidase(s), or about 0.5% to about 3% by weight α-xylosidase(s). 
     The cellulases included in the compositions and enzyme mixtures can include any cellulase or lignocellulosic depolymerizing enzyme available to those of skill in the art. For example, the compositions and enzyme mixtures can include a cellobiohydrolase, a polysaccharide oxidase (e.g., cel61, see NCBI accession no. AY094489.1 GI:21694046), an endoxylanase, a β-glucosidase, a β-1,4-glucanase, a β-galactosidase, an α-fucosidase, a β-galactosidase, an endoxylanase, a β-xylosidase, α-arabinosidase, α-glucuronidase, an esterase and combinations thereof. 
     The cellulase or cellulase mixture is present in the compositions and enzyme mixtures at weight percentages of at least 5%, or at least 10%, or at least 15% cellulase or at least 20%, or at least 25% cellulase, or at least 30% cellulase, or at least 40% cellulase, or at least 50%. 
     The following non-limiting Examples illustrate aspects of the invention. 
     EXPERIMENTAL 
     Example I 
     Fungal Strains, Enzymes, and Substrates 
       Aspergillus niger  strain FGSC A1144 (ATCC 1015) was obtained from the Fungal Genetics Stock Center (Kansas City, Mo.),  Trichoderma reesei  (also known as  Hypocrea jecorina ) strain QM9414 was obtained from the United States Department of Agriculture National Center for Agricultural Utilization Research (Peoria, Ill.),  Fusarium graminearum  ( Gibberella zeae ) strain PH-1 was obtained from Dr. L. P. Hart (Department of Plant Pathology, Michigan State University), and  Phanerochaete chrysosporium  strain RP-78 was obtained from Dr. D. Cullen (United States Department of Agriculture Forest Products Laboratory, Madison, Wis.).  P. pastoris  strain X-33 and plasmid pPicZB were obtained from Invitrogen. 
     Commercial enzyme preparations (Multifect Pectinase, Multifect Xylanase, Accellerase XY, Accellerase 1000, Accellerase 1500, and Stargen) were obtained from Dupont/Danisco, Inc. (Genencor Division (Rochester, N.Y.)). CTec2 and HTec2 were obtained from Novozymes, Inc. (Franklinton, N.C.). Isoprimeverose (catalog no. 0-IPRM), xyloglucan from tamarind (catalog no. P-XYGLN), and borohydride-reduced xyloglucan-derived heptasaccharide (catalog no. O-X3G4R) were purchased from Megazyme Intl. (Wicklow, Ireland). The monosaccharide composition of the xyloglucan heptasaccharide and the tamarind xyloglucan were reanalyzed by the alditol acetate method. Foster et al.,  J. Vis. Exp . doi:10.3791/1837 (2010). For the xyloglucan heptasaccharide assay, total recovery of sugars was 101±2% of the mass and the molar percent composition was 0.2% arabinose, 43.2% xylose, 0.8% galactose, and 55.8% glucose. This is very close to a 4:3 ratio of xylose:glucose, which is consistent with the manufacturer&#39;s stated structure of XXXG. Fry et al.,  Physiol. Plant.  89:1-3 (1993). Reanalysis of the tamarind xyloglucan indicated that it contains 2.3% arabinose, 35.1% xylose, 15.5% galactose, and 47.1% glucose, on a molar basis. This is in good agreement with the manufacturer&#39;s stated composition of 4% arabinose, 38% xylose, 16% galactose, and 42% glucose. 
     Example II 
     Enzyme Assays 
     p-Nitrophenyl-α-D-glucoside (pNP α G), p-nitrophenyl-α-D-xyloside (pNP α X), p-nitrophenyl-α-D-xyloside, and p-nitrophenyl-α-D-glucoside were purchased from Sigma. Enzyme reactions were performed in 96-well microtiter plates in a total volume of 0.2 ml and the absorbance of reaction mixtures were read on a SpectraMax Plus microplate reader (Molecular Devices, Sunnyvale, Calif.). The influence of pH on α-xylosidase activity was measured at 37° C. in Mcllvaine buffers adjusted to pH values from 2.5 to 7.5. Mcllvaine T. C.,  J. Biol. Chem.  49:183-186 (1921). Free glucose and xylose were measured colorimetrically using enzyme-linked assays in 96-well plates. Banerjee et al.,  Biotechnol. Bioengineer.  106:707-720 (2010). Enzyme kinetics were analyzed by nonlinear curve fitting using GraphPad Prism™ software (La Jolla, Calif.). 
     Example III 
     Purification of α-Xylosidase 
     A column of DEAE-cellulose (Sigma D0909), 3-ml bed volume in a 5-ml syringe, was equilibrated with 25 mM sodium acetate, pH 4.0, and 1 ml of Multifect Pectinase applied and eluted with 25 mM sodium acetate, pH 4.0. Active fractions were combined and loaded onto a cation exchange HPLC column (TSK-Gel SP-5PW, Tosoh Bioscience, Montgomeryville, Pa.), equilibrated in the same buffer, and eluted with a gradient of 0-0.6 M NaCl in 30 min at a flow rate of 1 ml/min. Fractions containing α-xylosidase activity were combined, and dry NH 4 SO 4  was added to 1.7 M. This material was applied to a hydrophobic interaction column (TSK-gel Phenyl-5PW, Tosoh BioScience) equilibrated in 25 mM sodium acetate, pH 4.0+1.7 M NH 4 SO 4 . Proteins were eluted with a 30 min linear gradient to 100% water followed by 20 min of water at a flow rate of 1 ml/min. In some experiments, an additional fractionation step on hydroxyapatite CHT5-1 (10×64 mm, Bio-Rad) was included between the cation exchange and hydrophobic interaction steps. Elution conditions were 10 to 500 mM Na 2 HPO 4 , pH 7.0, in 30 min at 1 ml/min. 
     HPLC fractions were analyzed by SDS-PAGE (4-20% acrylamide, Tris-HCl, Bio-Rad). Proteins were visualized with ProtoBlueSafe (National Diagnostics, Atlanta, Ga.). Proteins were quantitated using Bio-Rad protein assay reagent and bovine IgG as standard. Bradford, M. M.,  Anal. Biochem.  72:248-254 (1976). 
     For mass spectrometric proteomics, proteins were excised from SDS-PAGE gels, digested with trypsin, and analyzed at the Michigan State University Proteomics Facility. For the proteomics analysis of Multifect Pectinase, 100 g of protein were separated by SDS-PAGE, the gel was divided into four equal portions, and each was processed individually as described Nagendran et al.,  Fung. Genet. Biol.  46:427-435 (2009). The mass spectral data were analyzed using Scaffold software and the  A. niger  proteome as the query database (version 3.0, Department of Energy Joint Genome Institute, Walnut Creek, Calif.). Signal peptides were predicted using the SignalP server (version 4.0). 
     Example IV 
     α-Xylosidase Gene Expression in  P. pastoris    
     A cDNA corresponding to Aspni5|43342 from  A. niger  (Department of Energy Joint Genome Institute numbering) was synthesized by GeneArt (Invitrogen) with the addition of restriction sites for PmlI (5′ end) and XbaI (3′ end) and cloned into pPICZB (Invitrogen). The amino acid sequence of the encoded α-xylosidase is provided below (SEQ ID NO:1). 
     
       
         
           
               
               
            
               
                 1 
                 MYFSSFLALG ALVQAAAATY FAPNSTGLRI QHGFETILIQ 
               
               
                   
               
               
                 41 
                 PFGYDGFRVR AWPFRPPSGN EISFIYDPPI EGYEDTAHGM 
               
               
                   
               
               
                 81 
                 SYDTATTGTE PRTLRNGNII LRTTGWGGTT AGYRLSFYRV 
               
               
                   
               
               
                 121 
                 NDDGSETLLT NEYAPLKSLN PRYYYWPGPG AEFSAEFSFS 
               
               
                   
               
               
                 161 
                 ATPDEQIYGT GTQQDHMINK KGSVIDMVNF NSYIPTPVFM 
               
               
                   
               
               
                 201 
                 SNKGYAFIWN MPAEGRMEFG TLRTRFTAAS TTLVDYVIVA 
               
               
                   
               
               
                 241 
                 AQPGDYDTLQ QRISALTGRA PAPPDFSLGY IQSKLRYENQ 
               
               
                   
               
               
                 281 
                 TEVELLAQNF HDRNIPVSMI VIDYQSWAHQ GDWALDPRLW 
               
               
                   
               
               
                 321 
                 PNVAQMSARV KNLTGAEMMA SLWPSVADDS VNYAALQANG 
               
               
                   
               
               
                 361 
                 LLSATRDGPG TTDSWNGSYI RNYDSTNPSA RKFLWSMLKK 
               
               
                   
               
               
                 401 
                 NYYDKGIKNF WIDQADGGAL GEAYENNGQS TYIESIPFTL 
               
               
                   
               
               
                 441 
                 PNVNYAAGTQ LSVGKLYPWA HQQAIEEGFR NATDTKEGSA 
               
               
                   
               
               
                 481 
                 CDHVSLSRSG YIGSQRFCSM IWSGDTTSVW DTLAVQVASG 
               
               
                   
               
               
                 521 
                 LSAAATGWGW WTVDAGGFEV DSTVWWSGNI DTPEYRELYV 
               
               
                   
               
               
                 561 
                 RWLAWTTFLP FMRTHGSRTC YFQDAYTCAN EPWSYGASNT 
               
               
                   
               
               
                 601 
                 PIIVSYIHLR YQLGAYLKSI FNQFHLTGRS IMRPLYMDFE 
               
               
                   
               
               
                 641 
                 KTDPKISQLV SSNSNYTTQQ YMFGPRLLVS PVTLPNVTEW 
               
               
                   
               
               
                 681 
                 PVYLPQTGQN NTKPWTYWWT NETYAGGQVV KVPAPLQHIP 
               
               
                   
               
               
                 721 
                 VFHLGSREEL LSGNVF 
               
            
           
         
       
     
       P. pastoris  was grown and induced as previously described, except with the addition of 1% Casamino acids (Difco Laboratories), which enhanced yield and stability of Ax1A. Banerjee et al.,  Bioresour. Technol.  101:9097-9105 (2010). Secretion was driven by the native signal peptide of Ax1A. 
     Twenty independent  P. pastoris  transformants were confirmed by colony PCR, purified by single colony isolation, and grown in 10-ml cultures. The three isolates exhibiting the highest activity on pNPαX were grown in 500-ml cultures and then concentrated and desalted. Banerjee et al.,  Bioresour. Technol.  101:9097-9105 (2010). In some cases, Ax1A was further purified by cation exchange HPLC as described above. 
     Xyloglucanase (also known as Cel74A; Trire2|49081 [JGI numbering]) and β-galactosidase (Trire2|80240) from  T. reesei  were expressed as previously reported for the expression of β-glucosidase (βG) from  T. reesei  (Trire2|76672) in  P. pastoris . Banerjee et al.,  Biotechnol. Bioengineer.  106:707-720 (2010). 
     Example V 
     Digestion of Xyloglucan Heptasaccharide 
     Each reaction contained 0.5 mg xyloglucan-derived heptasaccharide (Megazyme) in a reaction volume of 0.5 ml of sodium acetate (50 mM, pH 5.0). The Ax1A and β-glucosidase were produced in  P. pastoris . The final total enzyme concentration was 30 μg/ml, and the reactions were run at 50° C.  FIG. 13  illustrates the amount (nmol) of xylose released as a function of time. 
     Example VI 
     Digestion of Tamarind Xyloglucan and Optimization with GENPLAT® 
     For digestion of tamarind xyloglucan with commercial enzymes, the reaction volume was 0.5 ml, the total protein loading in each assay was 15 μg/g glucan, the reaction time was 24 h, and the reaction temperature was 50° C. 
     The mixture optimization experiments with enzymes active on xyloglucan used Design Expert™ software (State-Ease, Inc., Minneapolis, Minn.) and robotic handling of biomass and enzymes in an integrated platform called GENPLAT. A four component quadratic model was used, which involved 15 reactions performed in duplicate. The four components were α-xylosidase, β-glucosidase, xyloglucanase, and β-galactosidase. The stock solution of tamarind xyloglucan was 2.5 mg/ml in 50 mM citrate buffer, pH 4.8, and the final concentration was 1 mg/ml in a volume of 500 μl. The total protein loading in each reaction was fixed at 15 μg. The reaction plates were incubated at 50° C. for 48 h with end-over-end mixing at 10 rpm, after which 200 μl was transferred to a fresh 96-well plate. Glucose and xylose were measured by enzyme-linked colorimetric assays. Banerjee et al.,  Bioresour. Technol.  101:9097-9105 (2010); and Banerjee et al.,  Biotechnol. Bioengineer.  106:707-720 (2010). 
     Example VII 
     Identification and Purification of α-Xylosidase 
     Several fungi grown on a variety of substrates were tested for α-xylosidase activity. These included  Cochliobolus carbonum, F. graminearum, T. reesei, A. niger , and  P. chrysosporium . The fungi were grown on ground tamarind seed, corn ( Zea mays ) stover, pea ( Pisum sativum ) cell walls, carrot ( Daucus carona ) cell walls, lactose, or xylose for 5-14 days in still culture. No activity against pNPαX was seen in any of the resulting culture filtrates. An assortment of commercial enzyme products was also examined, including Accellerase 1000, Accellerase XY, Multifect Xylanase, Multifect Pectinase, Novozyme 188, CTec2, and HTec2. Activity against pNPαX was not seen in any of them except Multifect Pectinase, which had a specific activity of 0.197 μmol/min/mg. Consistent with the presence of α-xylosidase activity this preparation, and only in this preparation, could degrade tamarind xyloglucan to free xylose and glucose ( FIG. 1B ). Among all of the commercial enzyme mixtures tested, Multifect Pectinase was also the only one that showed activity against IP. 
     The protein responsible for α-xylosidase activity was purified by HPLC, the final step of which is shown in  FIG. 10 . Through three high resolution purification stages, a low level of βG activity was consistently associated with the peak of α-xylosidase activity ( FIG. 10 ). The peak of α-xylosidase activity did not contain any α-glucosidase or β-xylosidase activity as measured using pNPαG and p-nitrophenyl-β-D-xyloside, respectively. Later experiments indicated that the β-glucosidase activity was probably due to co-purification of a separate enzyme. Their co-elution through multiple purification steps suggests that the two enzymes might form a complex in vivo. Although the secreted proteins of aerobic filamentous fungi are generally considered to be “noncomplexed,” evidence for the formation of complexes between the secreted enzymes of a filamentous fungus has been reported recently (Gonzalez-Vogel et al.,  Appl. Microbiol. Biotechnol.  89, 145-155 (2011). 
     The molecular weight of α-xylosidase by SDS-PAGE was about 85 kDa ( FIG. 11A ). The dominant band was excised and subjected to tryptic digestion and mass spectrometric proteomics based on the whole predicted proteome of  A. niger  ATCC 1015 as the query database. (Multifect Pectinase is produced by fermentation of  A. niger .) Eight unique peptides amounting to 16% coverage of Aspni5|43342 were detected at greater than 95% probability. The only other protein detected, at a lower level, was Aspni5|50997 (two unique peptides, 6% coverage), which is a β-glucosidase in GH family 3. This might account for the residual βG activity co-eluting with α-xylosidase ( FIG. 10 ), a conclusion that was supported by heterologous expression (described below). 
     Unfractionated Multifect Pectinase was also analyzed by mass spectrometric proteomics. At high confidence (95% probability according to Scaffold, and at least two peptides), 132 proteins were identified. More than 90% of the proteins have predicted signal peptides. Both Aspni5|43342 and Aspni5|50997 were detected. Aspni5|56782, not Aspni5|50997, is the most abundant βG in Multifect Pectinase. See Table 4. 
     Aspni5|43342 is a predicted protein in GH family 31, which includes known α-xylosidases. Alternate designations for this gene and its product are XP_001393647, An09g03300, and CAK40270. On the basis of its weak amino acid similarity to AN7055 of  A. nidulans  and its induction by growth on xylose, Yuan et al. (Mol. Genet. Genomics 279, 545-561 (2008)) hypothesized that this protein is an α-xylosidase and named it Ax1A. The results provided herein are the first experimental evidence that Aspni5|43342 is, in fact, an α-xylosidase. The name Ax1A is used herein. 
     By BLASTP against the GenBank™ nonredundant database, Ax1A has many orthologs throughout the higher fungi (both ascomycetes and basidiomycetes). Many of these orthologs are annotated as belonging to GH family 31 and as having α-glucosidase or α-xylosidase activity, but with the exception of AN7505 of  A. nidulans , there is no supporting biochemical evidence for any of these annotations (Bauer et al.,  Proc. Natl. Acad. Sci. U.S.A.  103, 11417-11422 (2006)). The top BLASTP hits (all with E-values of 0.0 and percent identities ranging from 52 to 81%) are from several species of  Aspergillus , the closely related species  Neosartorya fischeri , and two basidiomycetes ( Schizophyllum commune  XP_003031084 and  Serpula lachrymans  EG001163) (see Table 3). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Best BLASTP hits of Ax1A against GenBank nr and 
               
               
                 against GenBank “fungi”. All are annotated as putative GH31 proteins. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Accession 
                 Species 
                 score 
                 E value 
                 % identity  
                 SP? 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 XP_001217011.1 
                 Aspergillus terreus 
                 1259 
                 0.0 
                 81 
                 yes 
               
               
                 XP_001265600.1 
                 Neosartorya fischeri 
                 1234 
                 0.0 
                 79 
                 yes 
               
               
                 XP_002378848.1 
                 Aspergillus flavus 
                 1232 
                 0.0 
                 79 
                 yes* 
               
               
                 XP_001823456.1 
                 Aspergillus oryzae 
                 1230 
                 0.0 
                 78 
                 yes 
               
               
                 gb|EGO01163.1 
                 Serpula lacrymans 
                 751 
                 0.0 
                 52 
                 no 
               
               
                 XP_003031084.1 
                 Schizophyllum 
                 708 
                 0.0 
                 51 
                 yes 
               
               
                   
                 commune 
                   
                   
                   
                   
               
               
                 ZP_07294496.1 
                 Streptomyces 
                 376 
                 8e-118 
                 35 
                 no 
               
               
                   
                 hygroscopicus 
                   
                   
                   
                   
               
               
                 gb|EGP91994.1 
                 Mycosphaerella 
                 374 
                 3e-117 
                 33 
                 no 
               
               
                   
                 graminicola 
                   
                   
                   
                   
               
               
                 ZP_08605161.1 
                 Lachnospiraceae 
                 369 
                 3e-115 
                 34 
                 no 
               
               
                   
                 bacterium 
                   
                   
                   
                   
               
               
                 YP_003842451.1 
                 Clostridium 
                 369 
                 5e-115 
                 32 
                 no 
               
               
                   
                 cellulovorans 
                   
                   
                   
                   
               
               
                 gb|ADI06537.1 
                 Streptomyces 
                 368 
                 le-114 
                 34 
                 no 
               
               
                   
                 bingchenggensis 
                   
                   
                   
                   
               
               
                 ZP_06576342.1 
                 Streptomyces 
                 366 
                 2e-114 
                 33 
                 no 
               
               
                   
                 ghanaensis 
                   
                   
                   
                   
               
            
           
           
               
            
               
                 Against fungi only: 
               
            
           
           
               
               
               
               
               
               
            
               
                 XP_001217011.1 
                 Aspergillus terreus 
                 1259 
                 0.0 
                 81 
                 yes 
               
               
                 XP_001265600.1 
                 Neosartorya fischeri 
                 1234 
                 0.0 
                 79 
                 yes 
               
               
                 XP_002378848.1 
                 Aspergillus flavus 
                 1232 
                 0.0 
                 79 
                 yes* 
               
               
                 XP_001823456.1 
                 Aspergillus oryzae 
                 1230 
                 0.0 
                 78 
                 yes 
               
               
                 gb|EGO01163.1 
                 Serpula lacrymans 
                 751 
                 0.0 
                 52 
                 Yes* 
               
               
                 XP_003031084.1 
                 Schizophyllum 
                 708 
                 0.0 
                 51 
                 yes 
               
               
                   
                 commune 
                   
                   
                   
                   
               
               
                 gb|EGP91994.1 
                 Mycosphaerella 
                 374 
                 3e-121 
                 33 
                 no 
               
               
                   
                 graminicola 
                   
                   
                   
                   
               
               
                 XP_003048593.1 
                 Nectria 
                 359 
                 1e-115 
                 33 
                 no 
               
               
                   
                 haematococca 
                   
                   
                   
                   
               
               
                 XP_388973.1 
                 Gibberella zeae 
                 358 
                 4e-115 
                 32 
                 no 
               
               
                 XP_003047209.1 
                 Nectria 
                 355 
                 5e-114 
                 33 
                 no 
               
               
                   
                 haematococca 
                   
                   
                   
                   
               
               
                 gb|AEO58673.1 
                 Myceliophthora 
                 354 
                 1e-113 
                 33 
                 No 
               
               
                   
                 thermophila 
                   
                   
                   
                   
               
               
                 XP_364756.1 
                 Magnaporthe grisea 
                 352 
                 1e-112 
                 32 
                 no 
               
               
                   
               
               
                 *reannotation to use a different ATG translational start site reveals a signal peptide 
               
            
           
         
       
     
     Among species of  Aspergillus , orthologs with strong E-values and percent amino acid identity to Ax1A are present in  A. flavus, Aspergillus oryzae, Aspergillus terreus, Aspergillus aculeatus , and  Aspergillus carbonarius , but not  A. fumigatus, A. clavatus , or  A. nidulans  ( Aspergillus  Comparative Database (Broad Institute) and DOE Joint Genome Institute) (Table 3). All of the orthologs in  Aspergillus  have strongly predicted signal peptides, like Ax1A itself (Reannotation of protein XP_002378848 from  A. flavus  by reassigning the ATG start codon indicates that it probably also has a signal peptide). Ax1A is present in both sequenced strains of  A. niger , ATCC1015 and CBS 513.88, with 100% amino acid identity and 99% nucleotide identity in the coding region.  A. nidulans  has 10 predicted GH31 genes, five of which have signal peptides. Of these, AN7120 (XP 664724) has the best amino acid identity to Ax1A (30%) but no signal peptide.  A. niger  ATCC 1015 and CBS 513.88 both have seven predicted GH31 genes, the best of which (after Ax1A itself) being ANI_1_620014 (also known as Aspni5|55419), with 32% identity. 
     The Ax1A mRNA and protein are induced by growth of  A. niger  on xylose compared with maltose. Ax1A was not included in a genome-wide microarray expression study comparing  A. nidulans, A. oryzae , and  A. niger , presumably because it is not common to all three species (Andersen et al., Proc. Natl. Acad. Sci. U.S.A. 105, 4387-4392 (2008)). 
     After the orthologs in species of  Aspergillus , the next best approximate 20 hits to Ax1A in GenBank™, with E-values ranging from e-97 to e-23 and percent identities ranging from 22% to 52%, are to a much wider variety of fungi. All of these proteins are hypothetical, and it is not known whether they have α-xylosidase, α-glucosidase, or other catalytic activities. However, the majority lack predicted signal peptides. This is a strong indication that they are not secreted and are probably functional orthologs of the cytoplasmic α-xylosidase enzymes of  A. flavus, A. niger , and  P. wortmanii  (Matsuo et al., Biosci. Biotechnol. Biochem. 60, 341-343 (1996); Matsushita et al., Agric. Biol. Chem. 51: 2015-2016 (1987); Yoshikawa et al. Biosci. Biotechnol. Biochem. 58, 1392-1398 (1994)). Note that greater than 90% of the proteins in Multifect Pectinase have predicted signal peptides (Table 4). To the best of our knowledge, the encoding genes of the cytoplasmic α-xylosidase fungal enzymes have not been identified. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Proteins identified in Multifect Pectinase by proteomics 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 CAZy Family 
                   
                   
                   
               
               
                   
                   
                   
                   
                 (GH 
                   
                 Signal 
               
               
                   
                 Protein ID 
                 Molecular 
                   
                 unless 
                   
                 Peptide? 
               
               
                   
                 (JGI  A. niger   
                 Weight 
                 Protein 
                 otherwise 
                   
                 (cleavage 
                 Total Spectral 
               
               
                 Protein # 
                 ATCC 1015 v. 3) 
                 (kDa) 
                 Name 
                 indicated) 
                 CBM? 
                 site) 
                 Counts 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 51764 
                 110 
                 β-galactosidase, lacA, 
                  2 or 35 
                 No 
                 18/ 
                 318 
               
               
                   
                   
                   
                   
                   
                   
                 19 
               
               
                 2 
                 200605 
                 53 
                 α-L-arabinofuranosidase B, abfB 
                 54 
                 No 
                 18/ 
                 256 
               
               
                   
                   
                   
                   
                   
                   
                 19 
               
               
                 3 
                 56782 
                 93 
                 β-glucosidase, bglA 
                 3 
                 No 
                 19/ 
                 250 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 4 
                 205670 
                 87 
                 β-xylosidase, xlnD 
                 3 
                 No 
                 26/ 
                 182 
               
               
                   
                   
                   
                   
                   
                   
                 27 
               
               
                 5 
                 43342 
                 83 
                 GH31 glucoside hydrolase 
                 31 
                 No 
                 18/ 
                 124 
               
               
                   
                   
                   
                   
                   
                   
                 19 
               
               
                 6 
                 57436 
                 35 
                 xylanase 
                 10 
                 No 
                 19/ 
                 123 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 7 
                 213597 
                 68 
                 glucoamylase (amyloglucosidase), 
                 15 
                 No 
                 18/ 
                 106 
               
               
                   
                   
                   
                 glaA 
                   
                   
                 19 
               
               
                 8 
                 55136 
                 36 
                 α-L-arabinofuranosidase, axhA 
                 62 
                 No 
                 26/ 
                 101 
               
               
                   
                   
                   
                   
                   
                   
                 27 
               
               
                 9 
                 46065 
                 42 
                 exo-polygalacturonase 
                 28 
                 No 
                 18/ 
                 100 
               
               
                   
                   
                   
                   
                   
                   
                 19 
               
               
                 10 
                 44585 
                 36 
                 Pectin methylesterase 
                 CE8 
                 No 
                 17/ 
                 91 
               
               
                   
                   
                   
                   
                   
                   
                 18 
               
               
                 11 
                 177434 
                 106 
                 β-galactosidase 
                 35 
                 No 
                 19/ 
                 81 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 12 
                 55604 
                 50 
                 aldose 1-epimerase 
                   
                 No 
                 No 
                 80 
               
               
                 13 
                 41815 
                 40 
                 pectin 
                 PL1 
                 No 
                 20/ 
                 72 
               
               
                   
                   
                   
                 lyase 
                   
                   
                 21 
               
               
                 14 
                 138876 
                 104 
                 β-mannosidase; Mannanase 
                 2 
                 No 
                 21/ 
                 70 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 15 
                 56619 
                 94 
                 α-glucuronidase, aguA 
                 67 
                 No 
                 20/ 
                 63 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 16 
                 203143 
                 34 
                 endo-1,5-α-L-arabinosidase A 
                 43 
                 No 
                 19/ 
                 62 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 17 
                 205517 
                 56 
                 α-mannosidase 
                 47 
                 No 
                 21/ 
                 54 
               
               
                   
                   
                   
                   
                   
                   
                 22 
               
               
                 18 
                 206387 
                 68 
                 α-N-arabinofuranosidase A, ABF A 
                 51 
                 No 
                 25/ 
                 49 
               
               
                   
                   
                   
                   
                   
                   
                 26 
               
               
                 19 
                 50997 
                 86 
                 β-glucosidase 
                 3 
                 No 
                 22/ 
                 47 
               
               
                   
                   
                   
                   
                   
                   
                 23 
               
               
                 20 
                 209376 
                 37 
                 endoglucanase B 
                 5 
                 No 
                 18/ 
                 46 
               
               
                   
                   
                   
                   
                   
                   
                 19 
               
               
                 21 
                 42917 
                 46 
                 exo-rhamnogalacturonase C 
                 28 
                 No 
                 20/ 
                 44 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 22 
                 214233 
                 109 
                 Alpha-glucosidase, Maltase 
                 31 
                 No 
                 19/ 
                 44 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 23 
                 46429 
                 112 
                 β-galactosidase 
                 35 
                 No 
                 20/ 
                 43 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 24 
                 206333 
                 90 
                 endoglucanase C, EglC 
                 74 
                 C-term 
                 19/ 
                 41 
               
               
                   
                   
                   
                 (xyloglucanase) 
                   
                   
                 20 
               
               
                 25 
                 174365 
                 43 
                 Pectin methylesterase 
                   
                 No 
                 19/ 
                 38 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 26 
                 187227 
                 39 
                 β-1,4-endogalactanase A 
                 53 
                 No 
                 17/ 
                 36 
               
               
                   
                   
                   
                   
                   
                   
                 18 
               
               
                 27 
                 214608 
                 52 
                 endo-1,4-β-glucanase 
                 5 
                 C-term 
                 18/ 
                 35 
               
               
                   
                   
                   
                   
                   
                   
                 19 
               
               
                 28 
                 212716 
                 57 
                 1,3-β-glucanosyltransferase, 
                   
                 No 
                 19/ 
                 31 
               
               
                   
                   
                   
                 membrane 
                   
                   
                 20 
               
               
                   
                   
                   
                 anchor 
               
               
                 29 
                 42916 
                 74 
                 α-L-rhamnosidase 
                 78 
                 No 
                 19/ 
                 30 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 30 
                 194447 
                 46 
                 Glycoside hydrolase 
                 5 
                 No 
                 19/ 
                 30 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 31 
                 54830 
                 72 
                 hypothetical protein 
                   
                 No 
                 20/ 
                 29 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 32 
                 53702 
                 91 
                 AMP dependent synthetase/ligase 
                   
                 No 
                 No 
                 29 
               
               
                 33 
                 46255 
                 40 
                 Polygalacturonase-4 
                 28 
                 No 
                 19/ 
                 25 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 34 
                 41596 
                 48 
                 hypothetical protein 
                   
                 No 
                 No 
                 24 
               
               
                 35 
                 41606 
                 72 
                 α-D-galactosidase 
                 27 
                 No 
                 17/ 
                 24 
               
               
                   
                   
                   
                   
                   
                   
                 18 
               
               
                 36 
                 54398 
                 59 
                 β-N-acetylhexosaminidase 
                 20 
                 No 
                 No 
                 24 
               
               
                 37 
                 52011 
                 25 
                 xyloglucanase 2 
                 2 
                 No 
                 15/ 
                 24 
               
               
                   
                   
                   
                   
                   
                   
                 16 
               
               
                 38 
                 214460 
                 61 
                 carboxypeptidase C(cathepsin A) 
                   
                 No 
                 18/ 
                 24 
               
               
                   
                   
                   
                   
                   
                   
                 19 
               
               
                 39 
                 52418 
                 61 
                 sugar transporter 
                   
                 No 
                 20/ 
                 24 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 40 
                 45030 
                 23 
                 hypothetcial protein 
                   
                 No 
                 21/ 
                 4 
               
               
                   
                   
                   
                   
                   
                   
                 22 
               
               
                 41 
                 47911 
                 64 
                 1,4-α-D-glucan glucanohydrolase 
                 13 
                 starch 
                 24/ 
                 23 
               
               
                   
                   
                   
                   
                   
                   
                 25 
               
               
                 42 
                 213462 
                 36 
                 Cel 45/expansin 
                   
                 No 
                 22/ 
                 22 
               
               
                   
                   
                   
                   
                   
                   
                 23 
               
               
                 43 
                 50161 
                 51 
                 endo-polygalacturonase D 
                 28 
                 No 
                 17/ 
                 21 
               
               
                   
                   
                   
                   
                   
                   
                 18 
               
               
                 44 
                 214857 
                 35 
                 pectinmethylesterase 
                   
                 No 
                 22/ 
                 21 
               
               
                   
                   
                   
                   
                   
                   
                 23 
               
               
                 45 
                 197446 
                 44 
                 endochitinase 
                 18 
                 No 
                 No 
                 21 
               
               
                 46 
                 43957 
                 41 
                 endopolygalacturonase C 
                 28 
                 No 
                 19/ 
                 20 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 47 
                 50979 
                 68 
                 α-L-arabinofuranosidase 
                   
                 No 
                 20/ 
                 20 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 48 
                 55270 
                 99 
                 exo-β-1,3-glucanase 
                 55 
                 No 
                 20/ 
                 19 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 49 
                 189722 
                 47 
                 rhamnogalacturonase 
                 28 
                 No 
                 18/ 
                 19 
               
               
                   
                   
                   
                   
                   
                   
                 19 
               
               
                 50 
                 214598 
                 39 
                 endo-polygalacturonase A 
                 28 
                 No 
                 19/ 
                 19 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 51 
                 211032 
                 66 
                 tripeptidyl peptidase 
                   
                 No 
                 26/ 
                 18 
               
               
                   
                   
                   
                   
                   
                   
                 27 
               
               
                 52 
                 52219 
                 38 
                 endo-polygalacturonase B 
                 28 
                 No 
                 20/ 
                 18 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 53 
                 53361 
                 45 
                 secretory lipase 
                   
                 No 
                 19/ 
                 17 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 54 
                 172944 
                 48 
                 exo-polygalacturonase 
                 28 
                 No 
                 19/ 
                 17 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 55 
                 44517 
                 47 
                 Glycoside hydrolase 
                 17 
                 No 
                 22/ 
                 17 
               
               
                   
                   
                   
                   
                   
                   
                 23 
               
               
                 56 
                 184037 
                 83 
                 α-fucosidase 
                 65 or 95 
                 No 
                 20/ 
                 17 
               
               
                   
                   
                   
                   
                   
                   
                 21 
               
               
                 57 
                 206342 
                 81 
                 Catalase R 
                   
                 No 
                 16/ 
                 17 
               
               
                   
                   
                   
                   
                   
                   
                 17 
               
               
                 58 
                 196122 
                 31 
                 Glycoside hydrolase 
                 16 
                 No 
                 19/ 
                 17 
               
               
                   
                   
                   
                   
                   
                   
                 20 
               
               
                 59 
                 45801 
                 48 
                 Glycoside hydrolase 
                 30 
                 No 
                 18/ 
                 17 
               
               
                   
                   
                   
                   
                   
                   
                 19 
               
               
                 60 
                 53159 
                 48 
                 1,4-β-D-glucan cellobiohydrolase A 
                 7 
                 No 
                 17/ 
                 16 
               
               
                   
                   
                   
                   
                   
                   
                 18 
               
               
                 61 
                 119858 
                 93 
                 α-glucosidase 
                 31 
                 No 
                 14/ 
                 16 
               
               
                   
                   
                   
                   
                   
                   
                 15 
               
               
                 62 
                 49710 
                 59 
                 hypothetical carboxylesterase (type 
                   
                 No 
                 21/ 
                 16 
               
               
                   
                   
                   
                 B) 
                   
                   
                 22 
               
               
                 63 
                 201655 
                 41 
                 extracellular aspartic protease, 
                   
                 No 
                 20/ 
                 16 
               
               
                   
                   
                   
                 pepA 
                   
                   
                 21 
               
               
                 64 
                 51773 
                 52 
                 1,4-β-D-glucan cellobiohydrolase B 
                 7 
                 C-term 
                 21/22 
                 15 
               
               
                 65 
                 51478 
                 57 
                 Feruloyl esterase B 
                   
                 No 
                 17/18 
                 14 
               
               
                 66 
                 205580 
                 43 
                 endo-β-1,4-glucanase 
                 5 
                 C-term 
                 19/20 
                 14 
               
               
                 67 
                 122978 
                 47 
                 glycosyl hydrolase 
                 43 
                 No 
                 17/18 
                 14 
               
               
                 68 
                 44858 
                 58 
                 hypothetical protein 
                   
                 No 
                 23/24 
                 14 
               
               
                 69 
                 191158 
                 48 
                 exo-polygalacturonase B 
                 28 
                 No 
                 15/16 
                 13 
               
               
                 70 
                 54860 
                 44 
                 purine nucleoside permease 
                   
                 No 
                 24/25 
                 13 
               
               
                 71 
                 209408 
                 55 
                 glycoside hydrolase 
                 71 
                 No 
                 21/22 
                 13 
               
               
                 72 
                 179265 
                 82 
                 glycoside hydrolase 
                 3 
                 No 
                 19/20 
                 13 
               
               
                 73 
                 173481 
                 81 
                 hypothetical protein 
                   
                 No 
                 17/18 
                 13 
               
               
                 74 
                 182100 
                 35 
                 Glycoside hydrolase 
                 43 
                 No 
                 21/22 
                 13 
               
               
                 75 
                 185301 
                 65 
                 extracellular carboxylesterase 
                   
                 No 
                 17/18 
                 12 
               
               
                 76 
                 50148 
                 24 
                 hypothetical protein 
                   
                 No 
                 18/19 
                 12 
               
               
                 77 
                 202490 
                 46 
                 exo-β-1,3 glucanase 
                 5 
                 No 
                 22/23 
                 11 
               
               
                 78 
                 57027 
                 51 
                 inositol polyphosphate phosphatase, 
                   
                 No 
                 21/22 
                 11 
               
               
                   
                   
                   
                 phyA 
               
               
                 79 
                 141677 
                 37 
                 polygalacturonase 
                 28 
                 No 
                 18/19 
                 10 
               
               
                 80 
                 198063 
                 64 
                 beta fructofuranosidase, invertase 
                 32 
                 No 
                 15/16 
                 10 
               
               
                 81 
                 42184 
                 47 
                 glycoside hydrolase, 
                 28 
                 No 
                 22/23 
                 10 
               
               
                   
                   
                   
                 polygalacturonase 
               
               
                 82 
                 214786 
                 57 
                 hypothetical protein 
                   
                 No 
                 21/22 
                 10 
               
               
                 83 
                 51662 
                 31 
                 feruloyl esterase A; cinnamoyl 
                   
                 No 
                 21/22 
                 10 
               
               
                   
                   
                   
                 esterase, 
               
               
                   
                   
                   
                 faeA 
               
               
                 84 
                 173684 
                 62 
                 extracellular carboxylesterase, type B 
                   
                 No 
                 17/18 
                 10 
               
               
                 85 
                 51794 
                 29 
                 Ribonuclease T2 
                   
                 No 
                 23/24 
                 10 
               
               
                 86 
                 52688 
                 37 
                 glycoside hydrolase 
                 61 
                 No 
                 25/26 
                 10 
               
               
                 87 
                 38973 
                 61 
                 FAD/FMN-containing dehydrogenase 
                   
                 No 
                 20/21 
                 10 
               
               
                 88 
                 199085 
                 37 
                 glycoside hydrolase 
                 16 
                 No 
                 21/22 
                 10 
               
               
                 89 
                 50378 
                 41 
                 β-mannanase 
                 5 
                 No 
                 16/17 
                 9 
               
               
                 90 
                 135787 
                 27 
                 Lipolytic enzyme, G-D-S-L 
                   
                 No 
                 No 
                 9 
               
               
                 91 
                 56161 
                 60 
                 Peptidase S10, serine 
                   
                 No 
                 23/24 
                 9 
               
               
                   
                   
                   
                 carboxypeptidase 
               
               
                 92 
                 35378 
                 50 
                 Phosphoesterase 
                   
                 No 
                 18/19 
                 9 
               
               
                 93 
                 54734 
                 63 
                 Peptidase S10, serine 
                   
                 No 
                 19/20 
                 9 
               
               
                   
                   
                   
                 carboxypeptidase 
               
               
                 94 
                 209830 
                 54 
                 FAD/FMN-containing dehydrogenase 
                   
                 No 
                 20/21 
                 9 
               
               
                 95 
                 170172 
                 70 
                 α-rhamnosidase 
                   
                 No 
                 17/18 
                 8 
               
               
                 96 
                 41679 
                 26 
                 necrosis-inducing proteins 
                   
                 No 
                 19/20 
                 8 
               
               
                 97 
                 182156 
                 38 
                 Endopolygalacturonase-2 
                 28 
                 No 
                 21/22 
                 8 
               
               
                 98 
                 46876 
                 56 
                 hypothetical protein 
                   
                 No 
                 No 
                 8 
               
               
                 99 
                 50599 
                 41 
                 hypothetical protein 
                   
                 No 
                 19/20 
                 8 
               
               
                 100 
                 206560 
                 156 
                 possible dynactin 
                   
                 No 
                 No 
                 7 
               
               
                 101 
                 209490 
                 65 
                 glycosyl hydrolase 
                 76 
                 No 
                 22/23 
                 7 
               
               
                 102 
                 172825 
                 42 
                 hypothetical protein 
                   
                 No 
                 No 
                 7 
               
               
                 103 
                 207264 
                 49 
                 α-galactosidase 
                 27 
                 No 
                 16/17 
                 7 
               
               
                 104 
                 55665 
                 65 
                 Peptidase S8 and S53, subtilisin, kexin 
                   
                 No 
                 20/21 
                 7 
               
               
                 105 
                 47780 
                 73 
                 rhamnogalacturonan lyase 
                 PL4 
                 No 
                 19/20 
                 7 
               
               
                 106 
                 52703 
                 59 
                 Peptidase S28 
                   
                 No 
                 22/23 
                 6 
               
               
                 107 
                 177169 
                 45 
                 lactonohydrolase 
                   
                 No 
                 18/19 
                 6 
               
               
                 108 
                 197735 
                 34 
                 arabinanase 
                 43 
                 No 
                 15/16 
                 6 
               
               
                 109 
                 48594 
                 200 
                 Cytokinesis protein sepA 
                   
                 No 
                 No 
                 6 
               
               
                 110 
                 42242 
                 63 
                 saponin hydrolase 
                   
                 No 
                 26/27 
                 6 
               
               
                 111 
                 124618 
                 38 
                 predicted protein 
                   
                 No 
                 16/17 
                 6 
               
               
                 112 
                 37735 
                 17 
                 predicted protein 
                   
                 No 
                 17/18 
                 6 
               
               
                 113 
                 133986 
                 43 
                 Cellobiohydrolase 
                 6 
                 No 
                 28/29 
                 5 
               
               
                 114 
                 207829 
                 87 
                 α-1,2-mannosidase 
                 92 
                 No 
                 25/26 
                 5 
               
               
                 115 
                 57215 
                 67 
                 Metallophosphoesterase 
                   
                 No 
                 20/21 
                 5 
               
               
                 116 
                 52849 
                 12 
                 predicted protein 
                   
                 No 
                 19/20 
                 5 
               
               
                 117 
                 189254 
                 28 
                 rhamnogalacturonan acetylesterase 
                   
                 No 
                 17/18 
                 4 
               
               
                 118 
                 210947 
                 57 
                 rhamnogalacturonan lyase A 
                 PL4 
                 No 
                 20/21 
                 4 
               
               
                 119 
                 46979 
                 58 
                 extracellular serine carboxypeptidase 
                   
                 No 
                 18/19 
                 4 
               
               
                   
                   
                   
                 S10 
               
               
                 120 
                 52460 
                 76 
                 glutaminase A 
                   
                 No 
                 19/20 
                 4 
               
               
                 121 
                 131668 
                 52 
                 hypothetical protein 
                   
                 No 
                 No 
                 4 
               
               
                 122 
                 208679 
                 109 
                 hypothetical protein 
                   
                 No 
                 19/20 
                 4 
               
               
                 123 
                 40102 
                 74 
                 extracellular carboxylesterase (type B) 
                   
                 No 
                 21/22 
                 4 
               
               
                 124 
                 45021 
                 34 
                 pectate lyase A 
                   
                 No 
                 20/21 
                 4 
               
               
                 125 
                 52700 
                 62 
                 Peptidase S8 and S53, subtilisin, kexin, 
                   
                 No 
                 19/20 
                 4 
               
               
                   
                   
                   
                 sedolisin 
               
               
                 126 
                 171242 
                 41 
                 predicted protein 
                   
                 No 
                 No 
                 4 
               
               
                 127 
                 37736 
                 60 
                 α-galactosidase 
                 27 
                 No 
                 31/32 
                 3 
               
               
                 128 
                 53620 
                 54 
                 Phosphoesterase 
                   
                 No 
                 17/18 
                 3 
               
               
                 129 
                 56664 
                 59 
                 Glycoside hydrolase, exo-inulinase 
                 32 
                 No 
                 19/20 
                 3 
               
               
                 130 
                 56689 
                 63 
                 Peptidase S28 
                   
                 No 
                 17/18 
                 3 
               
               
                 131 
                 128537 
                 11 
                 Allergen Asp F7 
                   
                 No 
                 No 
                 2 
               
               
                 132 
                 50333 
                 55 
                 Histidine acid phosphatase 
                   
                 No 
                 20/21 
                 2 
               
               
                   
               
            
           
         
       
     
       T. reesei  has only two poor (E-value greater than e-10 and less than 25% amino acid identity) BLASTP hits to Ax1A (Trire2|121351 and Trir2|69944), and neither of these has a predicted signal peptide. Therefore,  T. reesei  does not have the genetic potential to biosynthesize a secreted α-xylosidase-related to Ax1A, which explains the lack of this enzymatic activity in commercial enzyme mixtures derived from  T. reesei  ( FIG. 1B ). 
     AN7505 (XP_680774) of  A. nidulans  has less than 25% amino acid identity to Ax1A and lacks a predicted signal peptide. When expressed in  P. pastoris  fused to a yeast signal peptide, AN7505 was secreted and showed activity against pNPαX but was not further characterized (Bauer et al., Proc. Natl. Acad. Sci. U.S.A. 103, 11417-11422 (2006)). Therefore AN7505 is not an extracellular α-xylosidase. 
     Properties of Ax1A— 
     The pH optimum of AX on pNPX was between 3 and 4 ( FIG. 12A ). The temperature optimum was 55° C. ( FIG. 12B ). The activity was about 50% of maximum at 65°. The native protein was approximately as active on IP as on the synthetic substrate pNPαX (Table 1). 
     Heterologous Expression of Ax1A— 
     When expressed in  P. pastoris , Ax1A had an apparent molecular weight of about 110,000, larger than the native protein. It also ran as a more diffuse band than the native protein ( FIG. 11B ). Both of these observations suggest that recombinant Ax1A is hyperglycosylated when expressed in  P. pastoris . (Ax1A has 10 predicted N-glycosylation sites.) Nonetheless, the heterologously expressed protein showed kinetic properties similar to the native protein on both pNPαX and IP (Table 1). The protein expressed in  P. pastoris  had no detectable βG, β-xylosidase, or α-glucosidase activity when assayed with p-nitrophenyl-β-D-glucoside, p-nitrophenyl-β-D-xyloside, or pNPαG, respectively (data not shown). 
     This supports the conclusion that the βG activity seen in the α-xylosidase assay shown in  FIG. 10  was due to impurities. 
     Activity of Ax1A on Xyloglucan Heptasaccharide— 
     Ax1A purified by HPLC ( FIG. 11 ), without addition or other enzymes, was able to degrade the heptasaccharide XXXG to free glucose and xylose (data not shown). However, this preparation contained residual β-glucosidase activity ( FIG. 10 ). Recombinant Ax1A released about 10 nmol of xylose, and the quantity did not increase with time ( FIG. 13 ). This proportion of xylose corresponds to approximately one-third of the total xylose present in the xyloglucan heptasaccharide sample (34 nmol in 12 μl of a reaction volume of 500 μl). This result is consistent with Ax1A removing a single xylose residue from the heptasaccharide to produce WOW and is further evidence that Ax1A does not have intrinsic β-glucosidase activity. Digestion of the xyloglucan heptasaccharide with no enzyme or β-glucosidase alone released no to little xylose ( FIG. 13 ). Digestion with a combination of Ax1A and β-glucosidase released 83.4% of the theoretical maximum of xylose in 10 h ( FIG. 13 ). Thus, β-glucosidase and Ax1A together are capable of substantially depolymerizing this heptasaccharide repeating unit of native xyloglucan. 
     Activity of Ax1A on Tamarind Xyloglucan— 
     Because xyloglucan contains β-linked galactose and β-linked glucose in addition to α-linked xylose, four enzymes were included in the experiment: xyloglucanase, β-glucosidase, and β-galactosidase, all from  T. reesei , in addition to Ax1A (Table 2). An optimized mixture of the four enzymes was developed using GENPLAT at fixed total protein loading (Banerjee et al.  Bioresour. Technol.  101, 9097-9105 (2010); Banerjee et al. Biotechnol. Bioengineer. 106, 707-720 (2010)). In the first experiment (Table 5) the lower limit of each enzyme was set to 0%. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 First experimental design and experimental results for optimization of a cocktail 
               
               
                 of four enzymes for deconstruction of tamarind XG. The lower proportion of each enzyme 
               
               
                 was set to 0%. This gave a statistically invalid model (see Table 7). Glucose (Glc) yields 
               
               
                 are expressed as a percentage of total glucose in the biomass, ±1 SD of the mean (n = 8). 
               
            
           
           
               
               
               
               
               
            
               
                 β-glucosidase 
                 β-galactosidase 
                 xyloglucanase 
                 Ax1A 
                 Glc yield, % 
               
               
                   
               
               
                 1.00 
                 0.00 
                 0.00 
                 0.00 
                 3.1 ± 0.6 
               
               
                 0.00 
                 1.00 
                 0.00 
                 0.00 
                 3.2 ± 0.7 
               
               
                 0.00 
                 0.00 
                 1.00 
                 0.00 
                 4.2 ± 0.5 
               
               
                 0.00 
                 0.00 
                 0.00 
                 1.00 
                 3.1 ± 0.6 
               
               
                 0.50 
                 0.50 
                 0.00 
                 0.00 
                 3.8 ± 0.5 
               
               
                 0.50 
                 0.00 
                 0.50 
                 0.00 
                 4.5 ± 0.2 
               
               
                 0.50 
                 0.00 
                 0.00 
                 0.50 
                 11.8 ± 0.1  
               
               
                 0.00 
                 0.50 
                 0.50 
                 0.00 
                 4.3 ± 0.7 
               
               
                 0.00 
                 0.50 
                 0.00 
                 0.50 
                 3.1 ± 0.8 
               
               
                 0.00 
                 0.00 
                 0.50 
                 0.50 
                 4.1 ± 0.8 
               
               
                 0.63 
                 0.13 
                 0.13 
                 0.13 
                 93.3 ± 0.0  
               
               
                 0.13 
                 0.63 
                 0.13 
                 0.13 
                 97.1 ± 0.0  
               
               
                 0.13 
                 0.13 
                 0.63 
                 0.13 
                 99.4 ± 0.0  
               
               
                 0.13 
                 0.13 
                 0.13 
                 0.63 
                 100.0 ± 0.0  
               
               
                 0.25 
                 0.25  
                 0.25 
                 0.25 
                 100.0 ± 0.0  
               
               
                   
               
            
           
         
       
     
     However, many combinations failed to yield greater than 5% of xylose or glucose. In the second experiment (Table 6), the lower limit of each enzyme was set to 5%, which gave a statistically valid model for both glucose and xylose. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Second experimental design and experimental results for optimization of a 
               
               
                 cocktail of four enzymes for deconstruction of tamarind XG. The lower limit of each 
               
               
                 enzyme was set to 5% (mandating the upper limit of each enzyme at 85%). This 
               
               
                 experiment gave a valid model (see Table 7). Glucose (Glc) and xylose (Xyl) yields are 
               
               
                 expressed as a percentage of total glucose or xylose in the biomass, ±1 SD of the mean 
               
               
                 (n = 8). 
               
            
           
           
               
               
               
               
               
               
            
               
                 β-glucosidase 
                 β-galactosidase 
                 xyloglucanase 
                 Ax1A 
                 Glc yield, % 
                 Xyl yield, % 
               
               
                   
               
               
                 0.85 
                 0.05 
                 0.05 
                 0.05 
                 78.9 ± 2.0 
                 80.8 ± 0.8 
               
               
                 0.05 
                 0.85 
                 0.05 
                 0.05 
                 65.6 ± 0.5 
                 63.1 ± 1.2 
               
               
                 0.05 
                 0.05 
                 0.85 
                 0.05 
                 80.9 ± 1.3 
                 71.7 ± 0.8 
               
               
                 0.05 
                 0.05 
                 0.05 
                 0.85 
                 97.1 ± 0.4 
                 97.7 ± 0.1 
               
               
                 0.45 
                 0.45 
                 0.05 
                 0.05 
                 73.2 ± 0.6 
                 70.6 ± 0.1 
               
               
                 0.45 
                 0.05 
                 0.45 
                 0.05 
                 80.6 ± 0.5 
                 76.8 ± 0.7 
               
               
                 0.45 
                 0.05 
                 0.05 
                 0.45 
                 95.5 ± 0.7 
                 100.6 ± 2.2  
               
               
                 0.05 
                 0.45 
                 0.45 
                 0.05 
                 75.2 ± 1.3 
                 71.4 ± 2.1 
               
               
                 0.05 
                 0.45 
                 0.05 
                 0.45 
                 98.4 ± 0.0 
                 100.4 ± 0.3  
               
               
                 0.05 
                 0.05 
                 0.45 
                 0.45 
                 99.1 ± 0.6 
                 100.5 ± 0.1  
               
               
                 0.55 
                 0.15 
                 0.15 
                 0.15 
                 98.4 ± 2.6 
                 94.5 ± 0.9 
               
               
                 0.15 
                 0.55 
                 0.15 
                 0.15 
                 97.4 ± 1.2 
                 88.1 ± 1.7 
               
               
                 0.15 
                 0.15 
                 0.55 
                 0.15 
                 97.7 ± 0.0 
                 89.1 ± 0.3 
               
               
                 0.15 
                 0.15 
                 0.15 
                 0.55 
                 96.3 ± 2.0 
                 85.7 ± 0.0 
               
               
                 0.25 
                 0.25 
                 0.25 
                 0.25 
                 96.9 ± 0.5 
                 87.4 ± 0.2 
               
               
                   
               
            
           
         
       
     
     Complete digestion of tamarind xyloglucan was achieved (Table 7). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Statistical values for the models shown in Tables 5 and 6. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Adj 
                 Adeq 
               
               
                   
                 Sugar 
                 p-value 
                 F-value 
                 R{circumflex over ( )}2 
                 Pred R{circumflex over ( )}2 
                 R{circumflex over ( )}2 
                 Precision 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Table 5 
                 Glc 
                 0.4 
                 1.1 
                 0.34 
                 0.04 
                 −0.6 
                 2.9 
               
               
                 Table 6 
                 Glc 
                 &lt;0.0001 
                 7.1 
                 0.77 
                 0.66 
                 0.53 
                 9.0 
               
               
                 Table 6 
                 Xyl 
                 &lt;0.0001 
                 8.2 
                 0.80 
                 0.70 
                 0.64 
                 9.0 
               
               
                   
               
            
           
         
       
     
     The optimized proportions of the four enzymes for glucose and xylose release are shown in Table 2. Of the four enzymes, Ax1A was needed in the highest proportions (51% for glucose and 59% for xylose). The need for a high proportion of Ax1A might reflect a lower specific activity, steric hindrance, or the fact that the reactions were run at a suboptimal pH for Ax1A (see  FIG. 12B ). 
     Example VIII 
     Enhancement of Fermentable Sugar Yields by α-Xylosidase Supplementation 
     This Example illustrates that α-xylosidase supplementation improves glucose yields from real biomass substrates. 
     Methods 
     Plant Materials and Pretreatments 
     Stover of corn ( Zea mays  L.) was ground to 0.5 mm particle size with a Wiley mill before pretreating with alkaline hydrogen peroxide (AHP) as described by Banerjee et al.,  Biotechnol Biofuels  2011, 4:16. AHP conditions were 10% biomass loading, 0.5 g H 2 O 2 /g biomass, and shaking at 90 rpm and 24° C. for 24 hr. Peas ( Pisum sativum  L. “Little Marvel”) were soaked in water for 24 hr with bubbling air and grown in vermiculite in either total darkness for 5-7 days (“etiolated peas”) or for 9-14 days in a greenhouse (“green peas”). After freeze-drying, the etiolated plants were ground in liquid nitrogen. The green peas were freeze-dried and then ground in a Wiley mill to pass a 0.5-mm screen. Both were then pretreated by the same AHP conditions used for corn stover.  Chenopodium album  L. (lamb&#39;s quarters) was collected from local abandoned fields in mid-August. Plants were dried at 50° C. and ground in a Wiley mill to pass a 0.5 mm screen and pretreated by AHP. 
     Pea xyloglucan was prepared as described by Paper et al. ( Appl Microbiol Biotechnol  2012, in press) and Zablackis et al. ( Plant Physiol  1995, 107:1129-1138). After such preparation, the pea xyloglucan composition was analyzed by the alditol acetate method (Foster et al.  J Vis Exp  2010). It was judged to be partially pure by its atypical content of arabinose and because the sum of the neutral sugars did not add up to 100% (Table 8). Tamarind xyloglucan was purchased from Megazyme, Inc. (Wicklow, Ireland), and its composition is reported in Scott-Craig et al. ( J Biol Chem  2011, 286:42848-42854). 
                     TABLE 8                  Monomer sugar composition of plant materials used in this study. All       values (±1 SD, n = 3) are mg/g dry weight. Materials were dried but otherwise       not processed by washing or other fractionation before acid hydrolysis.       “Total” indicates the percentage of the original dry weight accounted for by       the indicated neutral sugars.                                         Plant                               Material   Glc   xylose   Ara a     Man a     Gal   Total               pea     226 ± 19.0     287 ± 25.2   127 ± 8.1   8.5 ± 0.6     75 ± 6.0   72.4%       xyloglucan       tamarind    471 ± 8.3    351 ± 9.2    23 ± 2.1   0.0    155 ± 5.3    100%       xyloglucan b                                       etiolated   281.6 ± 12.0   49.4 ± 4.4   55.8 ± 4.8   48.3 ± 2.2   43.5%       peas       green peas   106.7 ± 12.0   19.6 ± 0.7   30.8 ± 2.0   23.7 ± 2.7   18.1%       corn stover   391.5 ± 0.35   194.7 ± 10.9   33.3 ± 5.3    9.4 ± 2.3   62.9%       lamb&#39;s   170.1 ± 1.7     30.2 ± 0.03    24.6 ± 0.64    14.4 ± 0.14   23.9%       quarters                 a The HPLC protocol could not resolve arabinose and mannose. Pea and tamarind xyloglucans (XGs) were analyzed by gas chromatography of alditol acetates after hydrolysis with trifluoroacetic acid (Foster et al.  J Vis Exp  2010).         b From Scott-Craig et al. ( J Biol Chem  2011, 286: 42848-42854).            
Cell Wall Analysis
 
     Cell wall sugar composition (of materials other than pea xyloglucan) was determined by two-stage hydrolysis with sulfuric acid without prior removal of extractives (Sluiter et al., U.S. Department of Energy National Renewable Energy Laboratory, 2011). Sugars were separated by HPLC using a Bio-Rad (Hercules, Calif.) Aminex HPX-87P column at 80° C. with 1 ml/min water as mobile phase and detection by refractive index. Each run took about 20 min. Under these conditions, arabinose and mannose could not be resolved and are reported together. Because the biomass was not washed to remove extractives prior to acid hydrolysis, the compositional analysis includes any contributions from starch, sucrose, free monomeric sugars, or acid-labile conjugated glucose and xylose. Recovery from the acid hydrolysis step was calculated to be 95% for glucose, arabinose, and galactose, and 85% for xylose. 
     Enzymes 
     Cellic CTec2 (lot number VCPI0004) and HTec2 (lot number VHN00002) were obtained from Novozymes, Inc. (Davis, Calif.) and typically used at a ratio of 3:1 on a protein mass basis. The protein concentrations of CTec2 and HTec2 were determined to be 130 mg/ml and 101 mg/ml, respectively, by the dye-binding assay of Bradford ( Anal Biochem  1976, 72:248-254) using bovine IgG as standard. The CTec2:HTec2 enzyme mixture was typically diluted 500-fold with 50 mM sodium citrate, pH 4.8, on the day of use and used at a final protein concentration of 2.5 mg/g glucan. Accellerase 1000 (lot number 1600844643; 69 mg protein/ml) was obtained from Genencor, Inc. (now DuPont Industrial Biosciences, Palo Alto, Calif.) and diluted similarly. Ax1A was prepared by expression in  Pichia pastoris  as described herein and stored in aliquots at −80° C. in 50 mM sodium acetate+20% glycerol, pH 5 (see also, Scott-Craig et al.,  J Biol Chem  2011, 286:42848-42854). The other pure enzymes, all derived from  T. reesei , were obtained commercially or prepared by expression in  P. pastoris  as described by Banerjee et al. ( Biotechnol Bioengineer  2010, 106:707-720) and Banerjee et al. ( Bioresour Technol  2010, 101:9097-9105). 
     Enzyme Assays 
     Unless other specified, enzyme hydrolysis reactions were performed in 96-well deep-well plates in a reaction volume of 0.5 ml, as described by Banerjee et al. ( Biotechnol Bioengineer  2010, 106:707-720). Glucan concentration was typically 2 mg/ml. The buffer was 50 mM sodium citrate, pH 4.8, containing 25 μg/ml each of tetracycline and cycloheximide. Assays were run in duplicate, sampled twice, and the glucose and xylose levels measured twice. Therefore, each data point represents the mean of eight values. All error bars represent ±one standard deviation of the mean. 
     Glucose and xylose were measured using enzyme-linked colorimetric assays (Megazyme kits K-GLUC and K-XYLOSE, respectively). These assays detect only free glucose and xylose and not cellobiose or oligomeric sugars. 
     Results 
     Commercial Cellulases do not Degrade Xyloglucan Because they Lack α-Xylosidase 
     In mixtures of pure enzymes (i.e., β-glucosidase, β-galactosidase, and xyloglucanase), Ax1A was required for release of free glucose and xylose from isolated pea xyloglucan fragments and from tamarind xyloglucan (Scott-Craig et al.  J Biol Chem  286:42848-42854 (2011)). Similarly, supplementation with Ax1A was required for the release of free glucose from intact pea xyloglucan in response to the commercial cellulase cocktails CTec2 and HTec2 ( FIG. 14 ). Addition of Ax1A enhanced glucose yield by 18-fold in 30 hours compared to CTec2:HTec2 alone. These results are consistent with the earlier results showing the absence of α-xylosidase activity in CTec2 or HTec2 against the model substrate pNPαX and against the disaccharide isoprimeverose (Scott-Craig et al.,  J Biol Chem  286:42848-42854 (2011)). These results furthermore indicate that a combination of CTec2 and HTec2 has all of the necessary enzymes to degrade pea xyloglucan except α-xylosidase. In this regard CTec2 and HTec2 are similar to the commercial product Driselase from the basidiomycetous fungus  Irpex lacteus , which degrades xyloglucan only to isoprimeverose (Zeng et al.  Plant Physiol  2008, 147:78-91). Ax1A supplementation was also necessary for complete depolymerization of tamarind xyloglucan by CTec2:HTec2 ( FIG. 15 ). Tamarind xyloglucan is less fucosylated but more heavily galactosylated than pea xyloglucan, but both contain α-linked xylose. In the absence of Ax1A, CTec2:HTec2 released almost no glucose, even in 48 hr ( FIG. 15 ). An Ax1A to CTec2:HTec2 ratio of 1 to 3 (on a protein mass basis) was near saturating for release of glucose in 48 hr ( FIG. 15 ). 
     Although addition of Ax1A to CTec2:HTec2 greatly stimulated release of free glucose from tamarind xyloglucan, yields of glucose were still only about half of the maximal possible ( FIG. 15 ). Tamarind xyloglucan is partially substituted with galactose (Gal) on some of the xylose side chains (Paper et al., 2012, Appl Microbiol Biotechnol 2012 Sep. 26, in press. [PMID: 23011349]. A possible explanation for the approximate half-possible yield is that β-galactosidase activity was limiting in these reactions, and therefore any glucose moiety substituted with galactose as well as xylose would not be released. In fact, addition of both β-galactosidase and Ax1A to CTec2:HTec2 strongly stimulated glucose release compared to reactions without β-galactosidase ( FIG. 16 ). This experiment indicates that CTec2:HTec2 is sub-optimal in regard to β-galactosidase as well as α-xylosidase for the digestion of tamarind xyloglucan. Supplementation of commercial cellulases such as CTec2 and HTec2 with α-xylosidase might improve the usefulness of these cellulases for releasing of fermentable sugars from biomasses rich in xyloglucan. 
     Ax1A Supplementation Improves Glucose Yields from Real Biomass Substrates 
     The effect of Ax1A supplementation of CTec2:HTec2 on digestion of a biofuels-relevant biomass substrate, AHP-pretreated corn stover, is shown in  FIG. 17 . Because cellulose is the major form of glucose in corn stover and CTec2:HTec2 has strong cellulase activity, as expected glucose yields even without Ax1A supplementation were high ( FIG. 17 ). At lower CTec/HTec2 loadings (i.e., 0.4 and 1.0 mg/g glucan), there was no apparent enhancement of glucose release by addition of Ax1A ( FIG. 17 ). At the highest CTec2:HTec2 loading tested (2.5 mg/g glucan), however, there was a statistically significant increase in glucose yield after hydrolysis for 24 hr (data not shown) and 48 hr ( FIG. 17 ). At 48 hours, glucose yields increased from ˜82% to ˜88% of the maximum possible glucose content at Ax1A loadings above 8 mg/g glucan.  FIG. 18A  shows the results from  FIG. 17  in expanded scale to accentuate the enhancement effect. Xylose yields were also increased by Ax1A supplementation, as shown in expanded scale in  FIG. 18B . The Ax1A effect on xylose yield (about 5% absolute increase) was statistically significant only at the highest CTec2:HTec2 (2.5 mg/g glucan) and Ax1A loadings (16 mg/g glucan) tested. 
     Ax1A also enhanced yields of glucose and xylose from pretreated corn stover in response to another commercial cellulase, Accellerase 1000 ( FIG. 19 ). In 48 hr, Ax1A increased glucose yields by 9% (from 76% to 85% of maximum possible yield) and xylose yields by 1.8% ( FIG. 19 ). 
     Time Course of Glucose Release 
     The release of glucose was monitored over 95 hours at two CTec2:HTec2 (75:25) loadings, with and without Ax1A. As expected, the higher CTec2:HTec2 loading released more glucose more quickly ( FIG. 8 ). At the lower CTec2:HTec2 loading, Ax1A caused a small enhancement of glucose yield only at the highest Ax1A loading, and this was not statistically significant ( FIG. 8 ). At the higher CTec2:HTec2 loading, a stimulatory effect of Ax1A was seen at 95 hour that was statistically significant. Under these conditions, Ax1A supplementation resulted in an 8.3% absolute increase in glucose yield, from 84% to 92.3% ( FIG. 8 ). 
     The Enhancement by Ax1A is not a General Protein Effect 
     Addition of nonenzymatic proteins, such as bovine serum albumin (BSA), enhances apparent hydrolysis activity, probably by reducing nonspecific and/or nonproductive binding of cellulases and other enzymes to lignin (Yang &amp; Wyman,  Biotechnol Bioeng  2006, 94:611-617). To test whether the enhancement by Ax1A might be due to a nonspecific protective effect on cellulases as opposed to its intrinsic enzymatic activity, we compared the effect on hydrolysis enhancement of Ax1A against BSA and bovine gamma-globulin. As shown in  FIG. 20 , neither BSA nor IgG stimulated glucose yields in response to CTec2:HTec2, nor did either protein affect the enhancement by Ax1A ( FIG. 20 ). Furthermore, Ax1A that had been boiled to destroy its activity did not stimulate glucose or xylose release from corn stover (data not shown). These data indicated that the Ax1A enhancement is due to the α-xylosidase activity of Ax1A and is not a general nonspecific protein effect. 
     Response of Herbaceous Dicotyledons to Ax1A Supplementation 
     Corn, like other plants in the Poaceae family, is generally considered to have lower levels of xyloglucan than dicotyledons and non-graminaceous monocotyledons (Vogel,  Curr Opin Plant Biol  2008, 11:301-307). To test whether herbaceous dicotyledons might therefore respond differently to Ax1A supplementation, we tested dark-grown (etiolated) peas, light-grown (green) peas, and wild lamb&#39;s quarters. Peas were chosen because their primary wall xyloglucan has been well-characterized (Talbott &amp; Ray,  Curr Opin Plant Biol  2008, 11:301-307). Lamb&#39;s quarters was chosen because, as a soft annual, it should have a high primary wall content. This is consistent with its glucose/xylose ratio of about 5.7, which is very close to etiolated and green peas (ratios of 5.4 and 5.6, respectively) and much higher than corn stover (ratio 2.0) (Table 3). 
     Yields of glucose from etiolated or green (light brown) pea were generally lower than 50% of available glucose content ( FIG. 21 ). Under no conditions tested did Ax1A increase glucose yields from either kind of pea ( FIG. 21 ). However, Ax1A supplementation did have a strong positive effect on glucose yields from lamb&#39;s quarters ( Chenopodium album ) ( FIG. 22 ), although higher loadings of CTec2:HTec2 were useful to obtain optimal glucose yields (e.g., glucose yields like those obtained using corn stover as biomass). At 30 mg/g CTec2:HTec2, 8 μg Ax1A enhanced glucose yields from 82.2% to 96.5% (an absolute increase of 14.3%) and xylose yields by 65.9% to 75.5% (an absolute increase of 9.6%) ( FIG. 22 ). Therefore, although Ax1A supplementation did not significantly affect glucose yields from pea biomass, Ax1A supplementation did enhance both glucose and xylose yields from another herbaceous dicotyledon. 
     Discussion 
     All plant cell walls contain significant levels of α-linked xylose, and commercial cellulase preparations derived from  T. reesei  lack α-xylosidase activity (Scott-Craig et al.,  J Biol Chem  2011, 286:42848-42854). 
     This Example describes tests to evaluate whether supplementation of commercial cellulase mixtures with the secreted α-xylosidase of  A. niger  (known as Ax1A) would improve glucose and xylose yields under otherwise identical hydrolysis conditions. The data provided herein show that supplementation of two commercial cellulase cocktails with Ax1A resulted in higher yields of glucose and xylose from corn stover and lamb&#39;s quarters. The results also indicate that in some conditions β-galactosidase activity in current commercial cellulases might also be limiting. By supplementing lignocellulosic digestion mixtures with the right types of enzymes in optimal amounts, higher ethanol yields can be obtained from a given mass of lignocellulosic material. 
     These experiments indicate that the stimulatory effect of Ax1A supplementation was more apparent when higher CTec2:HTec2 levels were employed ( FIGS. 15, 17, 22 ), longer hydrolysis times were employed ( FIGS. 8, 14 ), or lower biomass recalcitrance was present (compare  FIG. 21  with  FIGS. 17, 19 and 22 ). The complex carbon sources in many biomass sources can more effectively be digested when several enzymes are present in an enzymatic digestion mixture, including other xyloglucan-active enzymes such as xyloglucanase and β-galactosidase. Such enzymes can work in concert to increasingly digest the biomass and make the substrate for all enzymes (including Ax1A) more available. Access to xyloglucan may also be occluded by other wall polymers (e.g., including cellulose). In a recently proposed model of the structure of the primary wall, most of the xyloglucan polymers are hypothesized to be appressed between or embedded within cellulose microfibrils rather than spanning cellulose microfibrils as in the original “tethered network” model (Park &amp; Cosgrove,  Plant Physiol  22012, 158:1933-1943). If so, then the cellulases (i.e., cellobiohydrolases and endo-β1,4-glucanases) would have to act before enzymes active on xyloglucan could gain access. Hence, Ax1A may most optimally increase glucose and xylose yields at the terminal stages of wall deconstruction. This could also explain the lack of an effect of Ax1A supplementation on glucose yields from pea cell walls, postulating that insufficient hydrolysis of cellulose by cellulases (manifested by relatively low yields of glucose) blocks access by the xyloglucan-active enzymes, including Ax1A, to the xyloglucan. 
     These studies also illuminate the levels of Ax1A protein relative to the levels of commercial enzymes that are more effective to achieve increased release of sugars. The ratio of Ax1A to commercial enzyme (on a protein mass basis) varied from 0.3 to 6.4 in different experiments. Depending upon the time for enzymatic digestion, enzymes mixtures containing at least about 2 mg Ax1A/g glucan, or at least about 3 mg Ax1A/g glucan, or at least about 4 mg Ax1A/g glucan, or at least about 5 mg Ax1A/g glucan were useful. Some biomasses were more optimally treated using at least about 6 mg Ax1A/g glucan, or at least about 7 mg Ax1A/g glucan, or at least about 8 mg Ax1A/g glucan. 
     Even though grasses are alleged to contain smaller amounts of xyloglucan compared to dicotyledonous plants, Ax1A supplementation was as effective on corn stover as it was on lamb&#39;s quarters. Pea biomass was somewhat recalcitrant to enzymatic digestion. 
     In conclusion, the data described herein shows that addition of α-xylosidase to enzymatic mixtures such as those currently available for commercial use (e.g., various cellulase preparations) can significantly increase glucose and xylose yields from biomass, thereby improving the overall efficiency of biofuels production from lignocellulosic materials. 
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         20. Yokoyama R, Rose J K C, Nishitani K: A surprising diversity and abundance of xyloglucan endotransglucosylase/hydrolases in rice. Classification and expression analysis.  Plant Physiol  2004, 134:10808-1099. 
       
    
     All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications. 
     The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a nucleic acid” or “a polypeptide” includes a plurality of such nucleic acids or polypeptides (for example, a solution of nucleic acids or polypeptides or a series of nucleic acid or polypeptide preparations), and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants. 
     The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention. 
     The following statements of the invention are intended to describe and summarize various aspects of the invention according to the foregoing description in the specification. 
     Statements: 
     1. A composition comprising an enzyme mixture comprising an isolated α-xylosidase and at least 5% cellulase. 
     2. The composition of statement 1, wherein the isolated α-xylosidase is a secreted α-xylosidase. 
     3. The composition of statement 1 and 2, wherein the isolated α-xylosidase is a purified α-xylosidase. 
     4. The composition of any of statements 1-3, wherein the isolated α-xylosidase is about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% pure. 
     5. The composition of any of statements 1-4, wherein the isolated α-xylosidase lacks a quaternary structure. 
     6. The composition of any of statements 1-5, wherein the isolated α-xylosidase has a pH optimum of approximately 4.0 and/or has a temperature optimum of approximately 50° C.-60° C. 
     7. The composition of any of statements 1-6, wherein the isolated α-xylosidase has an amino acid sequence with at least about 55%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% sequence identity with any of SEQ ID NO:1, 3, 5, 7-19, or 22.
 
8. The composition of any of statements 1-7, wherein the isolated α-xylosidase is derived from a fungal extracellular extract.
 
9. The composition of any of statements 1-8, wherein the isolated α-xylosidase is an  Aspergillus niger  extracellular extract.
 
10. The composition of any of statements 1-9, wherein the isolated α-xylosidase is identified by Aspni5|43342 or accession number GenBank DAA35002.1.
 
11. The composition of any of statement 1-10, further comprising at least 5%, or at least 10%, or at least 15% cellulase or at least 20%, or at least 25% cellulase, or at least 30% cellulase, or at least 40% cellulase, or at least 50% cellulase.
 
12. The composition of any of statements 1-11, further comprising a cellulase, wherein said cellulase is at least one enzyme selected from the group consisting of cellobiohydrolase, endoxylanase, β-glucosidase, β-1,4-glucanase, β-galactosidase, α-fucosidase, β-galactosidase, β-xylosidase, α-arabinosidase, α-glucuronidase, polysaccharide mono-oxygenase, esterase and combinations thereof
 
13. A kit comprising a container comprising the composition of any of statements 1-12, and instructions for incubating a plant biomass with the composition for a time and under conditions sufficient to create a degraded hemicellulose material from the plant biomass.
 
14. A method, comprising:
         a) providing;
           i) a plant biomass comprising a hemicellulose material; and   ii) an enzyme mixture comprising an isolated α-xylosidase and at least 5% cellulase; and   
           b) incubating said biomass with said enzyme mixture for a time and under conditions sufficient to create a depolymerized hemicellulose material;
           wherein said depolymerized hemicellulose material comprises a plurality of free fermentable xylose and glucose residues.
 
15. The method of statement 14, further comprising a pretreatment step performed before step (a).
 
16. The method of statement 14 or 15, further comprising a pretreatment step performed before step (a), wherein the pretreatment step decreases noncovalent interactions between polysaccharides and/or between cell wall polymers of the plant biomass.
 
17. The method of any of statements 14-16, wherein the method further comprises pretreating said plant biomass with alkaline hydrogen peroxide, acid, ammonia, ionic liquids, steam or a combination thereof.
 
18. The method of any of statements 14-17, wherein said degraded hemicellulose material is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% degraded into the plurality of free fermentable xylose and glucose residues.
 
19. The method of any of statements 14-18, wherein the isolated α-xylosidase is a secreted α-xylosidase.
 
20. The method of any of statements 14-19, wherein the isolated α-xylosidase is a purified α-xylosidase.
 
21. The method of any of statements 14-20, wherein the isolated α-xylosidase is about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% pure.
 
22. The method of any of statements 14-21, wherein the isolated α-xylosidase lacks a quaternary structure.
 
23. The method of any of statements 14-22, wherein the isolated α-xylosidase has a pH optimum of approximately 4.0 and/or has a temperature optimum of approximately 50° C.-60° C.
 
24. The method of any of statements 14-23, wherein the isolated α-xylosidase has an amino acid sequence with at least about 55%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% sequence identity with any of SEQ ID NO:1, 3, 5, 7-19, or 22.
 
25. The method of any of statements 14-24, wherein the isolated α-xylosidase is derived from a fungal extracellular extract.
 
26. The method of any of statements 14-25, wherein the isolated α-xylosidase is an  Aspergillus niger  extracellular extract.
 
27. The method of any of statements 14-26, wherein the isolated α-xylosidase is identified by Aspni5|43342 or accession number GenBank DAA35002.1.
 
28. The method of any of statements 14-27, further comprising at least 5%, or at least 10%, or at least 15% cellulase or at least 20%, or at least 25% cellulase, or at least 30% cellulase, or at least 40% cellulase, or at least 50% cellulase.
 
29. The method of any of statements 14-28, further comprising a cellulase, wherein said cellulase is at least one enzyme selected from the group consisting of cellobiohydrolase, endoxylanase, β-glucosidase, β-1,4-glucanase, β-galactosidase, α-fucosidase, β-galactosidase, β-xylosidase, α-arabinosidase, α-glucuronidase, polysaccharide mono-oxygenase, esterase and combinations thereof
 
30. The method of any of statements 14-29, wherein said plant biomass comprises a dicot xyloglucan.
 
31. The method of any of statements 14-30, wherein said plant biomass comprises a monocot xyloglucan.
 
32. The method of any of statements 14-31, wherein said plant biomass comprises grass xyloglucan or corn stover.
 
33. The method of any of statements 14-32, wherein said incubating is performed at a temperature ranging of approximately 40°-50° C.
 
34. The method of any of statements 14-33, wherein said incubating is performed at a pH of approximately 4-5.
   
               

     The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Other embodiments are described within the following claims.