Novel Glycosyl Hydrolase Enzymes And Uses Thereof

Abstract

The present disclosure is generally directed to glycosyl hydrolase enzymes, compositions comprising such enzymes, and methods of using the enzymes and compositions, for example for the saccharification of cellulosic and hemicellulosic materials into sugars.

Description

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Applications 61/245,273, filed Sep. 23, 2009, and 61/289,886, filed Dec. 23, 2009, the disclosures of which are incorporated herein by reference.

2. TECHNICAL FIELD

[0002] The present disclosure generally pertains to glycosyl hydrolase enzymes, compositions comprising such enzymes, and methods of using the enzymes and compositions, for example for the saccharification or conversion of cellulosic and hemicellulosic materials into sugars.

3. BACKGROUND

[0003] Bioconversion of renewable lignocellulosic biomass to a fermentable sugar that is subsequently fermented to produce alcohol (e.g., ethanol) as an alternative to liquid fuels has attracted the intensive attention of researchers since the 1970s, when the oil crisis occurred because OPEC decreased the output of petroleum (Bungay, H. R., "Energy: the biomass options". NY: Wiley; 1981; Olsson L, Hahn-Hagerdal B. Enzyme Microb Technol 1996, 18:312-31; Zaldivar, J et al., Appl Microbiol Biotechnol 2001, 56: 17-34; Galbe, M et al., Appl Microbiol Biotechnol 2002, 59:618-28). Ethanol has been widely used as a 10% blend to gasoline in the USA or as a neat fuel for vehicles in Brazil in the last two decades. The importance of fuel bioethanol will increase in parallel with skyrocketing prices for oil and gradual depletion of its sources. Additionally, fermentable sugars are increasingly used to produce plastics, polymers and other biobased products, and this industry is expected to expand substantially in the coming years. Thus, the demand for abundant low cost fermentable sugars, which can be used as a feed stock in lieu of petroleum based feedstocks, continues to grow.

[0004] Chiefly among the useful renewable feedstocks are cellulose and hemicellulose (xylans), which can be converted into fermentable sugars. The enzymatic conversion of these polysaccharides to soluble sugars, for example glucose, xylose, arabinose, galactose, mannose, and/or other hexoses and pentoses, occurs due to combined actions of various enzymes. For example, endo-1,4-.beta.-glucanases (EG) and exo-cellobiohydrolases (CBH) catalyze the hydrolysis of insoluble cellulose to cellooligosaccharides (e.g., with cellobiose being a main product), while .beta.-glucosidases (BGL) catalyzes the conversion of the oligosaccharides to glucose. Xylanases together with other accessory proteins (non-limiting examples of which include L-.alpha.-arabinofuranosidases, feruloyl and acetylxylan esterases, glucuronidases, and .beta.-xylosidases) catalyze the hydrolysis of hemicelluloses.

[0005] The cell walls of plants are composed of a heterogeneous mixture of complex polysaccharides that interact through covalent and noncovalent means. Complex polysaccharides of higher plant cell walls include, for example, cellulose (.beta.-1,4 glucan), which generally constitutes 35-50% of carbon found in cell wall components. Cellulose polymers self associate through hydrogen bonding, van der Waals interactions and hydrophobic interactions to form semi-crystalline cellulose microfibrils. These microfibrils also include noncrystalline regions, generally known as amorphous cellulose. The cellulose microfibrils are embedded in a matrix formed of hemicelluloses (including, e.g., xylans, arabinans, and mannans), pectins (e.g., galacturonans and galactans), and various other .beta.-1,3 and .beta.-1,4 glucans. These matrix polymers are often substituted with, for example, arabinose, galactose and/or xylose residues to yield highly complex arabinoxylans, arabinogalactans, galactomannans, and xyloglucans. The hemicellulose matrix is, in turn, surrounded by polyphenolic lignin.

[0006] The complexity of the matrix makes it difficult to degrade by microorganisms as lignin and hemicellulose components must be broken down before enzymes can act on the core cellulose microfibrils. Ordinarily, a consortium of different enzymatic activities is required to break down cell wall polymers to release the constituent monosaccharides. For saccharification of plant cell walls, the lignin must be permeabilized and hemicellulose disrupted to allow cellulose-degrading enzymes to act on their substrate.

[0007] Regardless of the type of cellulosic feedstock, the cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of biomass bioconversion processes. The production costs of microbially produced enzymes are tightly connected with the productivity of the enzyme-producing strain and the final activity yield in the fermentation broth. The hydrolytic efficiency of a multienzyme complex depends both on properties of individual enzymes, the synergies among them, and their ratio in the multienzyme blend.

[0008] There exists a need in the art to identify enzyme and/or enzymatic blends/compositions that are capable of converting plant and/or other cellulosic or hemicellulosic materials into fermentable sugars with improved efficacy and yield.

4. SUMMARY

[0009] The disclosure provides certain glycosyl hydrolase polypeptides having hemicellulolytic activity, including, e.g., xylanases (e.g., endoxylanases), xylosidases (e.g., .beta.-xylosidases), arabinofuranosidases (e.g., L-.alpha.-arabinofuranosidases), nucleic acids encoding these polypeptides, and methods for making and using the polypeptides and/or nucleic acids. The disclosure is based, in part, on the discovery of novel enzymes and variants having xylanase, .beta.-xylosidase, and/or L-.alpha.-arabinofuranosidase activities. The disclosure is also based on the identification of enzyme blends (or compositions) that efficiently catalyze the hydrolysis of cellulosic and hemicellulosic materials. For purpose of this disclosure, an enzyme can be defined either as a polypeptide having the particular enzymatic activity or as that enzyme. For example, a xylanase can be referred to as a polypeptide having xylanase activity or as a xylanase enzyme, and a .beta.-xylosidase can be referred to as either a polypeptide having .beta.-xylosidase activity or a .beta.-xylosidase enzyme.

[0010] The enzymes and/or enzyme blends/compositions of the disclosure can be used to produce sugars from biomass. The sugars so produced can be used by microorganisms for ethanol production or can be used to produce other bioproducts in various industrial applications. Therefore, the disclosure also provides industrial applications (e.g., saccharification processes in ethanol production) using the enzymes and/or enzyme blends/compositions described herein. The enzymes and/or enzyme blends/compositions of the present disclosure can be used to decrease enzyme costs in biofuel production.

[0011] In one aspect, the invention of the disclosure pertains to enzymes (including variants thereof), or enzyme blends/compositions that are useful for hydrolyzing the major components of a lignocellulosic biomass (including, e.g., cellulose, hemicellulose, and lignin) or any material comprising cellulose and/or hemicellulose. Such lignocellulosic biomass and/or material comprising cellulose and/or hemicellulose include, e.g., seeds, grains, tubers, plant waste or byproducts of food processing or industrial processing (e.g., stalks), corn (including, e.g., cobs, stover, and the like), grasses (e.g., Indian grass, such as Sorghastrum nutans; or, switchgrass, e.g., Panicum species, such as Panicum virgatum), wood (including, e.g., wood chips, processing waste), paper, pulp, recycled paper (e.g., newspaper).

[0012] The enzyme blends/compositions of the invention can be used to hydrolyze cellulose comprising a linear chain of .beta.-1,4-linked glucose moieties, or hemicellulose, of a complex structure that varies from plant to plant.

[0013] The enzyme blends/compositions of the invention can comprise a number of different enzymes, including, e.g., cellulases and/or hemicellulases. For example, the enzymes blends/compositions of the invention can be used to hydrolyze biomass or a suitable feedstock. The enzyme blends/compositions of the invention desirably comprise mixtures of enzymes, selected from, e.g., xylanases, xylosidases, cellobiohydrolases, arabinofuranosidases, and/or other enzymes that can digest hemicellulose to monomer sugars. An enzyme blend/composition of the invention can comprise a mixture of two or more, three or more, or four or more enzymes selected from one or more xylanases, one or more xylosidases, one or more cellobiohydrolases, one or more arabinofuranosidases, and one or more other enzymes that are capable of converting hemicelluose to monomer sugars. The other enzymes that can digest hemicellulose to monomer sugars include, without limitation, a cellulase, a hemicellulase, or a composition comprising a cellulase or a hemicellulase. An enzyme blend/composition of the invention can comprise a mixture of two or more, three or more, or four or more enzymes selected from a xylanase, a xylosidase, a cellobiohydrolase, an arabinofuranosidase, and at least one other enzyme capable of converting hemicellulose to monomer sugars. A non-limiting example of an enzyme blend/composition of the invention comprises a mixture of a xylanase, a xylosidase, a cellobiohydrolase, an arabinofuranosidase, and a .beta.-glucosidase. The enzyme blend/composition of the invention is suitably one that is non-naturally occurring.

[0014] As used herein, the term "naturally occurring composition" refers to a composition produced by a naturally occurring source, which comprises one or more enzymatic components or activities, wherein each of these components or activities is found at the ratio and level produced by the naturally-occurring source as it is found in nature, untouched and unmodified by the human hand. Accordingly, a naturally occurring composition is, for example, one that is produced by an organism unmodified with respect to the cellulolytic or hemicelluloytic enzymes such that the ratio or levels of the component enzymes are unaltered from that produced by the native organism in its native environment. A "non-naturally occurring composition," on the other hand, refers to a composition produced by: (1) combining component cellulolytic or hemicelluloytic enzymes either in a naturally occurring ratio or a non-naturally occurring, i.e., altered, ratio; or (2) modifying an organism to express, overexpress or underexpress one or more endogeneous or exogenous enzymes; or (3) modifying an organism such that at least one endogenous enzyme is deleted. A "non-naturally occurring composition" can also refer to a composition produced by a naturally-occurring and unmodified organism, but cultured in a man-made medium or environment that is different from the organism's native environment, such that the amounts or weight ratios of particular enzymes in the composition differ from those existing in a composition made by a native organism grown in its native habitat.

[0015] The enzyme blend/composition of the invention described herein is, for example, a fermentation broth. The fermentation broth can be one of a filamentous fungus, including, e.g., a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp. is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is a Penicillium funiculosum. The fermentation broth can be, for example, a cell-free fermentation broth or a whole cell broth.

[0016] The enzyme blend/composition of the invention described herein is, in another example, a cellulase compostion. The cellulase composition is a filamentous fungal cellulase composition, including, for example, a Trichoderma, such as a Trichoderma reesei, cellulase composition. The cellulase composition can be produced by a filamentous fungus, for example, a Trichoderma, such as a Trichoderma reesei.

[0017] For example, an enzyme blend/composition of the invention can comprise (a) one or more xylanase enzyme(s), wherein at least one of said one or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or more .beta.-xylosidase enzyme(s), wherein at least one of said one or more .beta.-xylosidase enzyme(s) is a Group 1 .beta.-xylosidase or a Group 2 .beta.-xylosidase, wherein the Group 1 .beta.-xylosidase is an Fv3A or an Fv43A, and the Group 2 .beta.-xylosidase is a Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, or a Trichoderma reesei Bxl1; (c) one or more L-.alpha.-arabinofuranosidase enzyme(s), wherein at least one of said one or more L-.alpha.-arabinofuranosidase is an Af43A, an Fv43B, a Pf51A, a Pa51A, or an Fv51A; (d) one or more cellulase enzymes; and optionally (e) one or more other components. The enzyme blend/composition is suitably one that is non-naturally occurring. The one or more cellulase enzyme(s) of (d) is desirably able to achieve at least 0.00005 fraction product per mg protein per gram of phosphoric acid swollen cellulose (PASC) as determined by a calcofluor assay. In a non-limiting example, the combined weight of xylanase enzyme(s) in the composition can represent or constitute 5 wt. % to 45 wt. % (e.g., 5 wt. % to 25 wt. %, 5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %) of the combined or total protein weight in the composition, whereas the combined weight of .beta.-xylosidase enzyme(s) can represent or constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total proteins in the composition, whereas the combined weight of L-.alpha.-arabinofuranosidase enzyme(s) can represent or constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 2 wt. % to 20 wt. %, 5 wt. % to 15 wt. %, 5 wt. % to 10 wt. %) of the combined or total protein weight in the composition, whereas the combined weight of cellulase enzyme(s) can represent or constitute 30 wt. % to 80 wt. % (e.g., 40 wt. % to 70 wt. %, 50 wt. % to 60 wt. %) of the combined or total protein weight in the composition. The enzyme blend/composition as described herein is, for example, a fermentation broth composition. The fermentation broth is, for example, one of a filamentous fungus, including, without limitation, a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp. is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is a Penicillium funiculosum. The fermentation can be, for example, a cell-free fermentation broth or a whole cell broth. The enzyme blend/composition as described herein can also be a cellulase composition, for example, a filamentous fungal cellulase composition. The cellulase composition, for example, can be produced by a filamentous fungus, such as by a Trichoderma.

[0018] For example, an enzyme blend/composition of the invention can comprise (a) one or more xylanase enzyme(s) wherein at least one of said one or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or both of Group 1 .beta.-xylosidase enzymes: Fv3A and Fv43A; (c) one or more of Group 2 .beta.-xylosidase enzyme(s) selected from Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reesei Bxl1; (d) one or more cellulase enzyme(s); and optionally (e) one or more other components. The one or more celluase enzyme(s) of (e) is desirably able to achieve at least 0.00005 fraction product per mg protein per gram of phosphoric acid swollen cellulose (PASC) as determined by a calcofluor assay. The enzyme blend/composition is suitably one that is non-naturally occurring. In a non-limiting example, the combined weight of xylanase enzyme(s) in the composition can represent or constitute 5 wt. % to 45 wt. % (e.g., 5 wt. % to 25 wt. %, 5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %) of the combined or total protein weight in the composition, whereas the combined weight of the Group 1 .beta.-xylosidase enzyme(s) can constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight in the composition, whereas the combined weight of the Group 2 .beta.-xylosidase enzyme(s) can constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight in the composition, and wherein the combined weight of the cellulase enzyme(s) can represent or constitute 30 wt. % to 80 wt. % (e.g., 40 wt. % to 70 wt. %, 50 wt. % to 60 wt. %) of the combined or total protein weight in the composition. The ratio of the weight of Group 1 .beta.-xylosidase enzymes to the weight of Group 2 .beta.-xylosidase enzymes can be, for example, 1:10 to 10:1 (e.g., 1:8 to 8:1, 1:6 to 6:1, 1:4 to 4:1, 1:2 to 2:1, or 1:1). The enzyme blend/composition can further comprise additional components, which may be accessory proteins or other protein/non-protein components. The additional components can constitute, for example, 1 wt. % to 50 wt. %, 1 wt. % to 10 wt. %, 2 wt. % to 5 wt. %, 5 wt. % to 10 wt. %, or 5 wt. % to 20 wt. % of the total weight of proteins in the composition. The enzyme blend/composition as described herein is, for example, a fermentation broth composition. The fermentation broth is, for example, one of a filamentous fungus, including, without limitation, a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp. is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is a Penicillium funiculosum. The fermentation can be, for example, a cell-free fermentation broth or a whole cell broth. The enzyme blend/composition as described herein can also be a cellulase composition, for example, a filamentous fungal cellulase composition. The cellulase composition, for example, can be produced by a filamentous fungus, such as by a Trichoderma.

[0019] In further examples, an enzyme blend/composition of the invention can comprise (a) one or more xylanase enzyme(s) wherein at least one of said one or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or more .beta.-xylosidase enzyme(s) wherein at least one of the one or more .beta.-xylosidase enzyme(s) is a Group 1 .beta.-xylosidase enzyme Fv3A or Fv43A or a Group 2 .beta.-xylosidase enzyme Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reesei Bxl1; (c) one or more L-.alpha.-arabinofuranosidase enzyme(s), wherein at least one of said one or more L-.alpha.-arabinofuranosidase enzyme(s) is an Af43A, an Fv43B, a Pf51A, or an Fv51A; (d) one or more .beta.-glucosidase enzyme(s); and optionally (e) one or more other components. The enzyme blend/composition is suitably one that is non-naturally occurring. In a non-limiting example, the combined weight of xylanase enzyme(s) in the composition can represent or constitute 5 wt. % to 45 wt. % (e.g., 5 wt. % to 25 wt. %, 5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %) of the combined or total protein weight in the composition, whereas the combined weight of the .beta.-xylosidase enzyme(s) can constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight in the composition, whereas the combined weight of the L-.alpha.-arabinofuranosidase enzyme(s) can constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight in the composition, and wherein the combined weight of the .beta.-glucosidase enzyme(s) can constitute 2 wt. % to 50 wt. % (e.g., up to 50 wt. %, 2 wt. % to 10 wt. %, or 3 wt. % to 8 wt. %) of the combined or total protein weight in the composition. The enzyme blend/composition can further comprise additional components, which may be accessory proteins or other protein/non-protein components. The additional components can constitute, for example, 1 wt. % to 50 wt. %, 1 wt. % to 10 wt. %, 2 wt. % to 5 wt. %, 5 wt. % to 10 wt. %, or 5 wt. % to 20 wt. % of the total weight of proteins in the composition. The enzyme blend/composition as described herein is, for example, a fermentation broth composition. The fermentation broth is, for example, one of a filamentous fungus, including, without limitation, a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp. is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is a Penicillium funiculosum. The fermentation can be, for example, a cell-free fermentation broth or a whole cell broth. The enzyme blend/composition as described herein can also be a cellulase composition, for example, a filamentous fungal cellulase composition. The cellulase composition, for example, can be produced by a filamentous fungus, such as by a Trichoderma.

[0020] An enzyme blend/composition of the invention can also comprise, for example, (a) one or more xylanase enzyme(s) wherein at least one of said one or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or both of Group 1 .beta.-xylosidase enzymes: Fv3A and Fv43A; (c) one or more of Group 2 .beta.-xylosidase enzyme(s) selected from Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reesei Bxl1; and (d) one or more .beta.-glucosidase enzyme(s); and optionally (e) one or more other components. The enzyme blend/composition is suitably one that is non-naturally occurring. In a non-limiting example, the combined weight of xylanase enzyme(s) constitutes 5 wt. % to 45 wt. % (e.g., 5 wt. % to 25 wt. %, 5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %) of the total weight of proteins in the composition, whereas the combined weight of Group 1 .beta.-xylosidase enzyme(s) constitutes 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total weight of proteins in the composition, whereas the combined weight of Group 2 .beta.-xylosidase enzyme(s) constitutes 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total weight of proteins in the composition, whereas the combined weight of .beta.-glucosidase enzyme(s) constitutes 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total weight of proteins in the composition. The ratio of the weight of Group 1 .beta.-xylosidase enzymes to the weight of Group 2 .beta.-xylosidase enzymes can be, for example, 1:10 to 10:1, for example, 1:8 to 8:1, 1:6 to 6:1, 1:4 to 4:1, 1:2 to 2:1, or 1:1. The enzyme blend/composition can further comprise additional components, which may be accessory proteins or other protein/non-protein components. The additional components can constitute, for example, 1 wt. % to 50 wt. %, 1 wt. % to 10 wt. %, 2 wt. % to 5 wt. %, 5 wt. % to 10 wt. %, or 5 wt. % to 20 wt. % of the total weight of proteins in the composition. The enzyme blend/composition as described herein is, for example, a fermentation broth composition. The fermentation broth is, for example, one of a filamentous fungus, including, without limitation, a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp. is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is a Penicillium funiculosum. The fermentation can be, for example, a cell-free fermentation broth or a whole cell broth. The enzyme blend/composition as described herein can also be a cellulase composition, for example, a filamentous fungal cellulase composition. The cellulase composition, for example, can be produced by a filamentous fungus, such as by a Trichoderma.

[0021] Moreover, an enzyme blend/composition of the invention can comprise, for example, (a) one or more xylanase enzyme(s) wherein at least one of said one or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or more .beta.-xylosidase enzyme(s) wherein at least one of said one or more .beta.-xylosidase enzyme(s) is a Group 1 .beta.-xylosidase or a Group 2 .beta.-xylosidase, wherein Group 1 .beta.-xylosidase can be an Fv3A or an Fv43A, and a Group 2 .beta.-xylosidase can be a Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reesei Bxl1; and (c) one or more L-.alpha.-arabinofuranosidase enzyme(s), wherein at least one of said one or more L-.alpha.-arabinofuranosidase enzyme(s) is an Af43A, an Fv43B, a Pf51A, or an Fv51A; and optionally (d) one or more other components. The enzyme blend/composition is suitably one that is non-naturally occurring. In a non-limiting example, the combined weight of the xylanase enzyme(s) constitutes 5 wt. % to 45 wt. % (e.g., 5 wt. % to 25 wt. %, 5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %) of the total protein weight in the composition, whereas the combined weight of the .beta.-xylosidase enzyme(s) constitutes 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight of the composition, whereas the combined weight of L-.alpha.-arabinofuranosidase enzyme(s) constitutes 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight of the composition. The enzyme blend/composition can further comprise additional components, which may be accessory proteins or other protein/non-protein components. The additional components can constitute, for example, 1 wt. % to 50 wt. %, 1 wt. % to 10 wt. %, 2 wt. % to 5 wt. %, 5 wt. % to 10 wt. %, or 5 wt. % to 20 wt. % of the total weight of proteins in the composition. The enzyme blend/composition as described herein is, for example, a fermentation broth composition. The fermentation broth is, for example, one of a filamentous fungus, including, without limitation, a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp. is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is a Penicillium funiculosum. The fermentation can be, for example, a cell-free fermentation broth or a whole cell broth. The enzyme blend/composition as described herein can also be a cellulase composition, for example, a filamentous fungal cellulase composition. The cellulase composition, for example, can be produced by a filamentous fungus, such as by a Trichoderma.

[0022] An enzyme blend/composition of the invention can also be one that comprises (a) one or more xylanase enzyme(s) wherein at least one of said one or more xylanase enzyme(s) is a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5; (b) one or both of Group 1 .beta.-xylosidase enzymes: Fv3A and Fv43A; (c) one or more of Group 2 .beta.-xylosidase enzyme(s) selected from Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, and/or a Trichoderma reesei Bxl1; and optionally (d) one or more other components. The enzyme blend/composition is suitably one that is non-naturally occurring. In a non-limiting example, the combined weight of xylanase enzyme(s) can constitute 5 wt. % to 45 wt. % (e.g., 5 wt. % to 25 wt. %, 5 wt. % to 15 wt. %, 10 wt. % to 15 wt. %) of the total protein weight in the composition, whereas the combined weight of Group 1 .beta.-xylosidase enzyme(s) can constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight in the composition, wheras the combined weight of Group 2 .beta.-xylosidase enzyme(s) can constitute 2 wt. % to 50 wt. % (e.g., 2 wt. % to 30 wt. %, 5 wt. % to 25 wt. %, 5 wt. % to 10 wt. %) of the total protein weight in the composition. The ratio of the weight of Group 1 .beta.-xylosidase enzymes to the weight of Group 2 .beta.-xylosidase enzymes can be, for example, 1:10 to 10:1, for example, 1:8 to 8:1, 1:6 to 6:1, 1:4 to 4:1, 1:2 to 2:1, or 1:1. The enzyme blend/composition can further comprise additional components, which may be accessory proteins or other protein/non-protein components. The additional components can constitute, for example, 1 wt. % to 50 wt. %, 1 wt. % to 10 wt. %, 2 wt. % to 5 wt. %, 5 wt. % to 10 wt. %, or 5 wt. % to 20 wt. % of the total weight of proteins in the composition. The enzyme blend/composition as described herein is, for example, a fermentation broth composition. The fermentation broth is, for example, one of a filamentous fungus, including, without limitation, a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium. An exemplary fungus of Trichoderma spp. is a Trichoderma reesei. An exemplary fungus of Penicillium spp. is a Penicillium funiculosum. The fermentation can be, for example, a cell-free fermentation broth or a whole cell broth. The enzyme blend/composition as described herein can also be a cellulase composition, for example, a filamentous fungal cellulase composition. The cellulase composition, for example, can be produced by a filamentous fungus, such as by a Trichoderma.

[0023] The enzymes, enzyme blends/compositions of the disclosure can be used in the food industry, e.g., for baking, for fruit and vegetable processing, in breaking down of agricultural waste, in the manufacture of animal feed, in pulp and paper production, in textile manufacture, or in household and industrial cleaning agents. The enzymes, and the enzymes in the enzyme blends/compositions of the disclosure are, for example, each independently produced by a microorganism, e.g., by a fungi or a bacteria.

[0024] The enzymes, enzyme blends/compositions of the disclosure can also be used as commercial enzymes or compositions to digest lignocellulose from any suitable sources, including all biological sources, such as plant biomasses, e.g., corn, grains, grasses (e.g., Indian grass, such as Sorghastrum nutans; or, switchgrass, e.g., Panicum species, such as Panicum virgatum), or, woods or wood processing byproducts, e.g., in the wood processing, pulp and/or paper industry, in textile manufacture, in household and industrial cleaning agents, and/or in biomass waste processing.

[0025] In another aspect, the disclosure provides isolated, synthetic or recombinant nucleic acids having at least about 70%, for example, at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete (100%) sequence identity to a nucleic acid sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 46, 47, 48, 49, or 50, over a region of at least about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or 2000, residues. Relatedly, the disclosure provides isolated, synthetic, or recombinant nucleic acids that are capable of hybridizing, under high stringency conditions, to a complement of 97%, 98%, 99%, or complete (100%) sequence identity to a nucleic acid sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 46, 47, 48, 49, or 50, or to a fragment thereof. The fragment, for example, can be at least 150 contiguous residues in length, for example, at least 200, 250, or 300 contiguous residues in length. The present disclosure provides nucleic acids encoding a polypeptide having hemicellulolytic activity. Exemplary hemicellolytic activity includes, without limitation, xylanase, .beta.-xylosidase, and/or L-.alpha.-arabinofuranosidase activity. Exemplary polypeptides having hemicellulolytic activity include, without limitation, a xylanase, a .beta.-xylosidase, and/or an L-.alpha.-arabinofuranosidase.

[0026] The disclosure further provides isolated, synthetic, or recombinant nucleic acids encoding an enzyme of the disclosure, including a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or 44, or a subsequence thereof (e.g., a catalytic domain ("CD") or carbohydrate binding module ("CBM")), or a suitable variant thereof. In some embodiments, a nucleic acid of the disclosure encodes the mature portion of a protein of amino acid sequence SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or 44, which is optionally operably linked to a heterologous signal sequence, e.g., the Trichoderma reesei CBHI signal sequence. The nucleic acid desirably encodes a polypeptide having hemicellulolytic activity, e.g., xylanase, .beta.-xylosidase, and/or L-.alpha.-arabinofuranosidase activity. The nucleic acid of the disclosure encodes a hemicellulase, for example, a xylanase, a .beta.-xylosidase, and/or an L-.alpha.-arabinofuranosidase, or a suitable variant thereof. Further nucleic acids of the disclosure are described in Section 6.2 below.

[0027] The disclosure additionally provides expression cassettes comprising a nucleic acid of the disclosure or a subsequence thereof. For example, the nucleic acid comprises at least about 70%, e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 46, 47, 48, 49, or 50, over a region of at least about 10 residues, e.g., at least about 10, 20, 30, 40, 50, 75, 90, 100, 150, 200, 250, 300, 350, 400, or 500 residues. In another example, the nucleic acid encodes a polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or 44, wherein the nucleic acid is optionally operably linked to a promoter. The promoter can be, e.g., a fungal, viral, bacterial, mammalian, or plant promoter. The promoter can be a constitutive promoter or an inducible promoter. An exemplary suitable promoter is expressable in filamentous fungi, e.g., Trichoderma reesei. A suitable promoter can be derived from a filamentous fungus, e.g., Trichoderma reesei, e.g., a cellobiohydrolase I ("cbh1") gene promoter from Trichoderma reesei.

[0028] The disclosure further provides a recombinant cell engineered to express a nucleic acid of the disclosure or an expression cassette of the disclosure. For example, the nucleic acid comprises at least about 70%, e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 46, 47, 48, 49, or 50, over a region of at least about 10, e.g., at least about 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 nucleotide residues. The nucleic acid can encode a polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or 44, wherein the nucleic acid is optionally operably linked to a promoter. The expression cassette can comprise the nucleic acid having at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 46, 47, 48, 49, or 50, over a region of at least about 10 nucleotide residues. For example, the expression cassette can comprise the nucleic acid encoding a polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or 44, wherein the nucleic acid is optionally operably linked to a promoter. The recombinant cell is desirably a recombinant bacterial cell, a recombinant mammalian cell, a recombinant fungal cell, a recombinant yeast cell, a recombinant insect cell, a recombinant algal cell, or a recombinant plant cell. For example, the recombinant cell is a recombinant filamentous fungal cell, such as a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium cell.

[0029] The disclosure provides transgenic plants comprising a nucleic acid of the disclosure or an expression cassette of the disclosure.

[0030] The disclosure provides isolated, synthetic or recombinant polypeptides comprising an amino acid sequence having at least about 80%, e.g., at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or complete (100%) sequence identity to a polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or 44, over a region of at least about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 residues, or over the full length immature polypeptide, the full length mature polypeptide, the full length CD, or the full length CBM. Exemplary polypeptides include fragments of at least about 10, for example, at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 residues in length. In specific embodiments, the fragments comprise a CD and/or a CBM. Where a fragment comprises both a CD and a CBM of an enzyme of the disclosure, the fragment optionally includes a linker separating the CD and the CBM. The linker can be a native linker or a heterologous linker. In certain embodiments, the polypeptides of the disclosure have one or more hemicellulase activities. Polypeptides or peptide sequences of the disclosure include sequences encoded by the nucleic acids of the disclosure. Exemplary polypeptides are described in Section 6.1.

[0031] The disclosure additionally provides a chimeric or fusion protein comprising at least one domain of a polypeptide of the disclosure (e.g., the CD, the CBM, or both). The at least one domain can be operably linked to a second amino acid sequence, e.g., a signal peptide sequence.

[0032] Conversely, the disclosure provides a chimeric or fusion protein comprising a signal sequence of a polypeptide of the disclosure operably linked to a second sequence, e.g., encoding the amino acid sequence of a heterologous polypeptide that is not naturally associated with the signal sequence. Accordingly, the disclosure provides a recombinant polypeptide comprising residues 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, or 1 to 40 of a polypeptide of, e.g., SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, or 44. Further exemplary chimeric or fusion polypeptides are described in Section 6.1.1.

[0033] The disclosure also provides methods of producing a recombinant polypeptide comprising: (a) culturing a host cell engineered to express a polypeptide of the disclosure; and (b) recovering the polypeptide. The recovery of the polypeptide includes, e.g., recovery of the fermentation broth comprising the polypeptide. In certain embodiments, recovery of the polypeptide can include further purification step(s).

[0034] The disclosure provides methods for hydrolyzing, breaking up, or disrupting a cellooligosaccharide, an arabinoxylan oligomer, or a glucan- or cellulose-comprising composition comprising contacting the composition with an enzyme, enzyme blend/composition of the disclosure under suitable conditions, wherein the enzyme, or enzyme blend/composition hydrolyzes, breaks up, or disrupts the cellooligosaccharide, arabinoxylan oligomer, or glucan- or cellulose-comprising composition.

[0035] The disclosure provides enzyme "blends" or compositions (also termed "enzyme blend/composition" herein), comprising a polypeptide of the disclosure, or a polypeptide encoded by a nucleic acid of the disclosure. In some embodiments, the polypeptide of the disclosure has one or more activities selected from xylanase, .beta.-xylosidase, and L-.alpha.-arabinofuranosidase activities. In certain embodiments, the enzyme blends/compositions are used or are useful for depolymerization of cellulosic and hemicellulosic polymers to metabolizable carbon moieties. The enzyme blends of the disclosure can be in the form of a composition e.g., as a product of manufacture. The composition can be, e.g., a formulation, and can take the physical form of, e.g., a liquid or a solid. In exemplary embodiments, an enzyme blend/composition of the disclosure includes a cellulase comprising at least three different enzyme types selected from (1) an endoglucanase, (2) a cellobiohydrolase, and (3) a .beta.-glucosidase; or at least three different enzymatic activities selected from (1) an endoglucanase activity catalyzing the cleavage of internal .beta.-1,4 linkages of cellulosic or hemicellulosic materials, resulting in shorter glucooligosaccharides, (2) a cellobiohydrolase activity catalyzing the cleavage and release, in an "exo" manner, of cellobiose units (e.g., .beta.-1,4 glucose-glucose disaccharide), and (3) a .beta.-glucosidase activity catalyzing the release of glucose monomers from short cellooligosaccharides (e.g., cellobiose). Exemplary enzyme blends/compositions of the disclosure are described in Section 6.3.4. below.

[0036] In another aspect, the disclosure provides methods for processing a biomass material comprising lignocellulose comprising contacting a composition comprising a cellulose and/or a fermentable sugar with a polypeptide of the disclosure, or a polypeptide encoded by a nucleic acid of the disclosure, or an enzyme blend/composition (e.g., a product of manufacture) of the disclosure. Suitable biomass material comprising lignocellulose can be derived from an agricultural crop, a byproduct of a food or feed production, a lignocellulosic waste product, a plant residue, or a waste paper or waste paper product. The polypeptides of the disclosure can have one or more enzymatic activities selected from cellulase, endoglucanase, cellobiohydrolase, .beta.-glucosidase, xylanase, mannanase, .beta.-xylosidase, arabinofuranosidase, and other hemicellulase activities. Suitable plant residue can comprise grain, seeds, stems, leaves, hulls, husks, corncobs, corn stover, straw, grasses, wood, wood chips, wood pulp and sawdust. The grasses can be, e.g., Indian grass, or switchgrass. The grasses can also be, for example, Miscanthus. The paper waste can be, e.g., discarded or used photocopy paper, computer printer paper, notebook paper, notepad paper, typewriter paper, newspapers, magazines, cardboard, and various paper-based packaging materials. The paper waste can also be, for example, pulp.

[0037] The disclosure provides compositions (including enzymes, enzyme blends/compositions, e.g., products of manufacture of the disclosure) comprising a mixture of hemicellulose- and cellulose-hydrolyzing enzymes, and at least one biomass material. Optionally the biomass material comprises a lignocellulosic material derived from an agricultural crop. Alternatively the biomass material is a byproduct of a food or feed production. Suitable biomass material can also be a lignocellulosic waste product, a plant residue, a waste paper or waste paper product, or comprises a plant residue. The plant residue can, e.g., be one comprising grains, seeds, stems, leaves, hulls, husks, corncobs, corn stover, grasses, straw, wood, wood chips, wood pulp, or sawdust. Exemplary grasses include, without limitation, Indian grass or switchgrass. Exemplary grasses can also include Miscanthus. Exemplary paper waste include, without limitation, discarded or used photocopy paper, computer printer paper, notebook paper, notepad paper, typewriter paper, newspapers, magazines, cardboard and paper-based packaging materials. Exemplary paper waste can also include, e.g., pulp.

[0038] All publically available information as of the filing date, including, e.g., publications, patents, patent applications, GenBank sequences, and ATCC deposits cited herein are hereby expressly incorporated by reference.

5. BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0039] Tables 1A-1B: Table 1A provides a summary of the sequence identifiers used in the present disclosure for glycosyl hydrolase enzymes; Table 1B provides accession numbers for additional glycosyl hydrolase enzymes referred to in the Examples.

[0040] Table 2: Activity of expressed proteins on synthetic substrates pNPA and pNPX (as defined in Section 7.1.6. below) in terms relative to the Trichoderma reesei Quad delete host background activity. The Quad delete host background (or "XQuad") is defined as the activity of the expressed protein(s) in the Quad delete strain divided by the activity of the Quad delete background strain without the expressed protein(s). For example, a value of >1 indicates that the expressed protein have an activity greater than that of the background.

[0041] Table 3: Xylanase activity of purified candidate endo-xylanases with birchwood xylan, incubated at 50.degree. C., pH 5.

[0042] Table 4: Percent conversion of cob arabinoxylan oligomers to monomer products based on total sugar available as determined by acid hydrolysis. See, Example 4.

[0043] Table 5: Experiment results (as described in Example 7) defining the level of hemicellulase activity for hydrolyzing treated corncob to monomer sugars. The column entitled "run #" indicates the randomized experimental order. The column entitled "trial #" indicates the standard experimental design order. The column entitled "Quad" indicates fraction of the total protein that is from the culture supernatant for a growth of the Quad deleted T. reesei strain. The column entitled "Xyn3" indicates fraction of the total protein that is Trichoderma reesei Xyn3. The column entitled "Fv43D" indicates fraction of the total protein that is Fv43D. The column entitled "Fv51A" indicates fraction of the total protein that is Fv51A. The column entitled "Fv43A" indicates fraction of the total protein that is Fv43A. The column entitled "Fv43B" indicates fraction of the total protein that is Fv43B. The column entitled "loading (ug/mg carbo)" indicates the protein loaded into the saccharification reaction in units of micrograms of protein per milligram of carbohydrate. The columns entitled "Xyl mg/mL, Glu mg/mL, Arab mg/mL, and G+X+A mg/mL" indicate the concentration of xylose, glucose, arabinose, and the combination of those three sugar products that is detected at the end of the saccharification reaction. The columns entitled "Xyl % theor, Glu % theor, and Arab % theor" indicate the percent of theoretical yield of xylose, glucose, and arabinose reached at the end of the saccharification reaction.

[0044] Table 6: Calculated ratios of the seven enzymatic components for predicted maximal yield of glucose, xylose and arabinose from hydrolysis of corncob. The column "loading total mg/gr carb" indicates the total enzyme dose at which the predictions are calculated. The rows entitled "Total mg/ml G+X+A, % Yield Glucose, % Yield Xylose, and % Yield Arabinose" indicate the response for which the optimum has been calculated. The column entitled "r2 data fit to model (includes both loadings)" indicates the r-squared statistical parameter for the model fit to data presented in Table 5. The columns entitled "fraction Accellerase, fraction Quad del sup, fraction purified Trichoderma reesei Xyn3, fraction purified Fv43D, fraction purified Fv51A, fraction purified Fv43A, and fraction purified Fv43B" indicate the fraction of that component that is calculated to be optimal by the model fitted to the data in Table 5.

[0045] Table 7: Refinement of enzyme loadings for maximal hydrolytic conversion of corncob using blends including Fv3A and Fv43D enzymes in 1.06 g, 14% dry solids pretreated cob reactions. The conditions for saccharification were as described in Example 7. Columns marked with enzyme names indicate the mg of each of the listed enzyme per gram of glucan or xylan used.

[0046] Table 8: Refinement of enzyme loadings for maximal conversion of corncob using blends containing Fv51A as the only L-.alpha.-arabinofuranosidase in 1.06 gr, 14% dry solids pretreated cob reactions. The conditions used for saccharification were as described in Example 7. Columns marked with enzyme names indicate the mg of each of the listed enzymes per gram of glucan or xylan used. Columns marked with carbohydrates indicate the mg per mL of each carbohydrate product produced based on measurements made with size exclusion chromatography. The >dp2 column includes all oligomers larger than a disaccharide.

[0047] Table 9: Sugar yields obtained from mixes A, B, C of purified hemicellulases tested in 1.06 g, 14% dry solids pretreated cob reactions. The conditions used for saccharification were as described in Example 7. Mix A: 6 mg Trichoderma reesei Xyn3, 4 mg Fv3A, 1 mg Fv51A per gram xylan. Mix B: 6 mg Trichoderma reesei Xyn3, 1 mg Fv43D, 3 mg Fv43A, 3 mg Fv43B per gram xylan. Mix C: 6 mg Trichoderma reesei Xyn3, 3 mg Fv3A, 1 mg Fv43D, 1 mg Fv51A per gram xylan. Columns marked with monomer sugars indicate the % yield of each of the listed monomer.

[0048] Table 10: Sugar yields from treatment of hemicellulose preparations made from corncob, sorghum, switchgrass and sugar cane bagasse by hemicellulase mixes A, B, C. The reactions were run at 100-.mu.L scale in 50 mM pH 5.0 Sodium Acetate buffer for 6 h at 48.degree. C. as described in Example 8. The % yield for each monomer sugar is shown.

[0049] Table 11: Concentration of the majority enzymes expressed by various T. reesei integrated expression strains (designated H3A, 39A, 69A, A10A, G6A, 102, 44A, 11A, G9A) as determined by percent of the integrated HPLC area.

[0050] Table 12: List of switchgrass pretreatment parameters and saccharification results from the various pretreatments.

[0051] Table 13: Saccharification results of a hardwood pulp with the enzyme composition produced by a T. reesei integrated strain, H3A, in reactions with different amounts of solids, enzymes, and incubation time.

[0052] Table 14: Saccharification of a hardwood pulp with the enzyme composition produced by an integrated strain H3A over a temperature range and a pH range.

[0053] Signal sequences listed below and in the figures were predicted. The predictions were made with the SignaIP algorithm (available at http://www.cbs.dtu.dk). Domain predictions were made based on one or more of the Pfam, SMART, or NCBI databases.

[0054] FIGS. 1A-1B: FIG. 1A: Fv3A nucleotide sequence (SEQ ID NO:1). FIG. 1B: Fv3A amino acid sequence (SEQ ID NO:2). SEQ ID NO:2 is the sequence of the immature Fv3A. Fv3A has a predicted signal sequence corresponding to positions 1 to 23 of SEQ ID NO:2 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 24 to 766 of SEQ ID NO:2. The predicted conserved domain is in boldface type.

[0055] FIGS. 2A-2B: FIG. 2A: Pf43A nucleotide sequence (SEQ ID NO:3). FIG. 2B: Pf43A amino acid sequence (SEQ ID NO:4). SEQ ID NO:4 is the sequence of the immature Pf43A. Pf43A has a predicted signal sequence corresponding to positions 1 to 20 of SEQ ID NO:4 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 21 to 445 of SEQ ID NO:4. The predicted conserved domain is in boldface type, the predicted carbohydrate binding module ("CBM") is in uppercase type, and the predicted linker separating the CD and CBM is in italics.

[0056] FIGS. 3A-3B: FIG. 3A: Fv43E nucleotide sequence (SEQ ID NO:5). FIG. 3B: Fv43E amino acid sequence (SEQ ID NO:6). SEQ ID NO:6 is the sequence of the immature Fv43E. Fv43E has a predicted signal sequence corresponding to positions 1 to 18 of SEQ ID NO:6 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 19 to 530 of SEQ ID NO:6. The predicted conserved domain is in boldface type.

[0057] FIGS. 4A-4B: FIG. 4A: Fv39A nucleotide sequence (SEQ ID NO:7). FIG. 4B: Fv39A amino acid sequence (SEQ ID NO:8). SEQ ID NO:8 is the sequence of the immature Fv39A. Fv39A has a predicted signal sequence corresponding to positions 1 to 19 of SEQ ID NO:8 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 20 to 439 of SEQ ID NO:8. The predicted conserved domain is in boldface type.

[0058] FIGS. 5A-5B: FIG. 5A: Fv43A nucleotide sequence (SEQ ID NO:9). FIG. 5B: Fv43A amino acid sequence (SEQ ID NO:10). SEQ ID NO:10 is the sequence of the immature Fv43A. Fv43A has a predicted signal sequence corresponding to positions 1 to 22 of SEQ ID NO:10 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 23 to 449 of SEQ ID NO:10. The predicted conserved domain is in boldface type, the predicted CBM is in uppercase type, and the predicted linker separating the conserved domain and CBM is in italics.

[0059] FIGS. 6A-6B: FIG. 6A: Fv43B nucleotide sequence (SEQ ID NO:11). FIG. 6B: Fv43B amino acid sequence (SEQ ID NO:12). SEQ ID NO:12 is the sequence of the immature Fv43B. Fv43B has a predicted signal sequence corresponding to positions 1 to 16 of SEQ ID NO:12 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 17 to 574 of SEQ ID NO:12. The predicted conserved domain is in boldface type.

[0060] FIGS. 7A-7B: FIG. 7A: Pa51A nucleotide sequence (SEQ ID NO:13). FIG. 7B: Pa51A amino acid sequence (SEQ ID NO:14). SEQ ID NO:14 is the sequence of the immature Pa51A. Pa51A has a predicted signal sequence corresponding to positions 1 to 20 of SEQ ID NO:14 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 21 to 676 of SEQ ID NO:14. The predicted L-.alpha.-arabinofuranosidase conserved domain is in boldface type. For expression purposes, the genomic DNA was codon optimized for expression in T. reesei (see FIG. 60B).

[0061] FIGS. 8A-8B: FIG. 8A: Gz43A nucleotide sequence (SEQ ID NO:15). FIG. 8B: Gz43A amino acid sequence (SEQ ID NO:16). SEQ ID NO:16 is the sequence of the immature Gz43A. Gz43A has a predicted signal sequence corresponding to positions 1 to 18 of SEQ ID NO:16 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 19 to 340 of SEQ ID NO:16. The predicted conserved domain is in boldface type. For expression purposes, the Gz43A predicted signal sequence was replaced by the T. reesei CBH1 signal sequence (myrklavisaflatara (SEQ ID NO: 51)) in T. reesei (see FIG. 61).

[0062] FIGS. 9A-9B: FIG. 9A: Fo43A nucleotide sequence (SEQ ID NO:17). FIG. 9B: Fo43A amino acid sequence (SEQ ID NO:18). SEQ ID NO:18 is the sequence of the immature Fo43A. Fo43A has a predicted signal sequence corresponding to positions 1 to 20 of SEQ ID NO:18 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 21 to 348 of SEQ ID NO:18. The predicted conserved domain is in boldface type. For expression purposes, the Fo43A predicted signal sequence was replaced by the T. reesei CBH 1 signal sequence (myrklavisaflatara (SEQ ID NO:51)) (see FIG. 62).

[0063] FIGS. 10A-10B: FIG. 10A: Af43A nucleotide sequence (SEQ ID NO:19). FIG. 10B: Af43A amino acid sequence (SEQ ID NO:20). SEQ ID NO:20 is the sequence of the immature Af43A. The predicted conserved domain is in boldface type.

[0064] FIGS. 11A-11B: FIG. 11A: Pf51A nucleotide sequence (SEQ ID NO:21). FIG. 11B: Pf51A amino acid sequence (SEQ ID NO:22). SEQ ID NO:22 is the sequence of the immature Pf51A. Pf51A has a predicted signal sequence corresponding to positions 1 to 20 of SEQ ID NO:22 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 21 to 642 of SEQ ID NO:22. The predicted L-.alpha.-arabinofuranosidase conserved domain is in boldface type. For expression purposes, the predicted Pf51A signal sequence was replaced by a codon optimized the T. reesei CBH1 signal sequence (myrklavisaflatara (SEQ ID NO:51)) (underlined) and the Pf51A nucleotide sequence was codon optimized for expression in T. reesei (see FIG. 63).

[0065] FIGS. 12A-12B: FIG. 12A: AfuXyn2 nucleotide sequence (SEQ ID NO:23). FIG. 12B: AfuXyn2 amino acid sequence (SEQ ID NO:24). SEQ ID NO:24 is the sequence of the immature AfuXyn2. AfuXyn2 has a predicted signal sequence corresponding to positions 1 to 18 of SEQ ID NO:24 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 19 to 228 of SEQ ID NO:24. The predicted GH11 conserved domain is in boldface type.

[0066] FIGS. 13A-13B: FIG. 13A: AfuXyn5 nucleotide sequence (SEQ ID NO:25). FIG. 13B: AfuXyn5 amino acid sequence (SEQ ID NO:26). SEQ ID NO:26 is the sequence of the immature AfuXyn5. AfuXyn5 has a predicted signal sequence corresponding to positions 1 to 19 of SEQ ID NO:26 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 20 to 313 of SEQ ID NO:26. The predicted GH11 conserved domain is in boldface type.

[0067] FIGS. 14A-14B: FIG. 14A: Fv43D nucleotide sequence (SEQ ID NO:27). FIG. 14B: Fv43D amino acid sequence (SEQ ID NO:28). SEQ ID NO:28 is the sequence of the immature Fv43D. Fv43D has a predicted signal sequence corresponding to positions 1 to 20 of SEQ ID NO:28 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 21 to 350 of SEQ ID NO:28. The predicted conserved domain is in boldface type.

[0068] FIGS. 15A-15B: FIG. 15A: Pf43B nucleotide sequence (SEQ ID NO:29). FIG. 15B: Pf43B amino acid sequence (SEQ ID NO:30). SEQ ID NO:30 is the sequence of the immature Pf43B. Pf43B has a predicted signal sequence corresponding to positions 1 to 20 of SEQ ID NO:30 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 21 to 321 of SEQ ID NO:30. The predicted conserved domain is in boldface type.

[0069] FIGS. 16A-16B: FIG. 16A: Fv51A nucleotide sequence (SEQ ID NO:31). FIG. 16B: Fv51A amino acid sequence (SEQ ID NO:32). SEQ ID NO:32 is the sequence of the immature Fv51A. Fv51A has a predicted signal sequence corresponding to positions 1 to 19 of SEQ ID NO:32 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 20 to 660 of SEQ ID NO:32. The predicted L-.alpha.-arabinofuranosidase conserved domain is in boldface type.

[0070] FIGS. 17A-17B: FIG. 17A: Cg51B nucleotide sequence (SEQ ID NO:33). FIG. 17B: Cg51B amino acid sequence (SEQ ID NO:34). SEQ ID NO:34 is the sequence of the immature Cg51B. Cg51B has a predicted signal sequence corresponding to positions 1 to 20 of SEQ ID NO:34 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 21 to 670 of SEQ ID NO:34. The predicted conserved domain is in boldface type.

[0071] FIGS. 18A-18B: FIG. 18A: Fv43C nucleotide sequence (SEQ ID NO:35). FIG. 18B: Fv43C amino acid sequence (SEQ ID NO:36). SEQ ID NO:36 is the sequence of the immature Fv43C. Fv43C has a predicted signal sequence corresponding to positions 1 to 22 of SEQ ID NO:36 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 23 to 333 of SEQ ID NO:36. The predicted conserved domain is in boldface type.

[0072] FIGS. 19A-19B: FIG. 19A: Fv30A nucleotide sequence (SEQ ID NO:37). FIG. 19B: Fv30A amino acid sequence (SEQ ID NO:38). SEQ ID NO:38 is the sequence of the immature Fv30A. Fv30A has a predicted signal sequence corresponding to positions 1 to 19 of SEQ ID NO:38 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 20 to 537 of SEQ ID NO:38.

[0073] FIGS. 20A-20B: FIG. 20A: Fv43F nucleotide sequence (SEQ ID NO:39). FIG. 20B: Fv43F amino acid sequence (SEQ ID NO:40). SEQ ID NO:40 is the sequence of the immature Fv43F. Fv43F has a predicted signal sequence corresponding to positions 1 to 20 of SEQ ID NO:40 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 21 to 315 of SEQ ID NO:40.

[0074] FIGS. 21A-21B: FIG. 21A: Trichoderma reesei Xyn3 nucleotide sequence (SEQ ID NO:41). FIG. 21B: Trichoderma reesei Xyn3 amino acid sequence (SEQ ID NO:42). SEQ ID NO:42 is the sequence of the immature Trichoderma reesei Xyn3. Trichoderma reesei Xyn3 has a predicted signal sequence corresponding to positions 1 to 16 of SEQ ID NO:42 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to positions 17 to 347 of SEQ ID NO:42. The predicted conserved domain is in bold face type.

[0075] FIG. 22: Amino acid sequence of Trichoderma reesei Xyn2 (SEQ ID NO:43). The signal sequence is underlined. The predicted conserved domain is in bold face type. The coding sequence can be found in Torronen et al. Biotechnology, 1992, 10:1461-65.

[0076] FIG. 23: Amino acid sequence of Trichoderma reesei Bxl1 (SEQ ID NO:44). The signal sequence is underlined. The predicted conserved domain is in bold face type. The coding sequence can be found in Margolles-Clark et al. Appl. Environ. Microbiol. 1996, 62(10):3840-46.

[0077] FIG. 24: Amino acid sequence of Trichoderma reesei Bgl1 (SEQ ID NO:45). The signal sequence is underlined. The predicted conserved domain is in bold face type. The coding sequence can be found in Barnett et al. Bio-Technology, 1991, 9(6):562-567.

[0078] FIG. 25: Cellulase activity assay using PASC hydrolysis with calcofluor detection.

[0079] FIG. 26: Xylanase elution profile.

[0080] FIG. 27: SDS-PAGE detection of the two step separation of AfuXyn 5. Lane 1: Crude sample; Lane 2: Eluate from Phenyl column; Lane 3: Eluate from GF column.

[0081] FIG. 28: pENTR/D-TOPO plasmid.

[0082] FIG. 29: pTrex3gM.

[0083] FIGS. 30A-30B: Performance of different enzymes on corncob substrate. Error bars represent the experimental errors associated with triplicate cob assays. The numbers in parenthesis along the x-axis represent the enzyme doses in mg of protein per g of cellulose.

[0084] FIG. 31: Performance of different enzyme blends/compositions on corncob substrate. Error bars represent the experimental errors associated with triplicate cob assays. The numbers in parentheses along the x-axis represent the enzyme doses in mg of protein per g cellulose.

[0085] FIG. 32: Performance of different enzyme blends/compositions on corncob substrate. Error bars represent the experimental errors associated with triplicate cob assays. The numbers in parentheses along the x-axis represent the enzyme doses in mg of protein per g cellulose.

[0086] FIG. 33: Performance of different enzyme blends/compositions on corncob substrate. Error bars represent the experimental errors associated with triplicate cob assays. The numbers in parentheses along the x-axis represent the enzyme doses in mg of protein per g cellulose.

[0087] FIG. 34: Performance of different enzyme blends/compositions on corncob substrate. Error bars represent the experimental errors associated with triplicate cob assays. The numbers in parentheses along the x-axis represent the enzyme doses in mg of protein per g cellulose.

[0088] FIGS. 35A-35C: Performance of different enzyme blends/compositions on corncob substrate. Error bars represent the experimental errors associated with triplicate cob assays. The numbers along the x-axis represent the enzyme doses in mg of protein per g cellulose.

[0089] FIG. 36: Anomeric proton NMR region of short arabinoxylan oligomers shows cleavage by Fv43A plus Fv43B.

[0090] FIG. 37: Anomeric proton NMR region of short arabinoxylan oligomers shows cleavage of .beta.-1,2-linked xylose from arabinose by Fv3A.

[0091] FIG. 38: Alignment between the amino acid sequences of Trichoderma reesei .beta.-xylosidase and Fv3A.

[0092] FIG. 39: pENTR-TOPO-Bgl1 (943/942) plasmid.

[0093] FIG. 40: pTrex3g 943/942 Bgl1 expression vector.

[0094] FIG. 41: pENTR-Trichoderma reesei Xyn3 plasmid.

[0095] FIG. 42: pTrex3g/Trichoderma reesei Xyn3 expression vector.

[0096] FIG. 43: pENTR-Fv3A plasmid.

[0097] FIG. 44: pTrex6g/Fv3A expression vector.

[0098] FIG. 45: TOPO Blunt/Pegl1-Fv43D plasmid.

[0099] FIG. 46: TOPO Blunt/Pegl1-Fv51A plasmid.

[0100] FIGS. 47A-47D: Glucan (FIG. 47A) and xylan (FIG. 47B) conversions to monomer sugars by secreted enzyme fermentation broths from T. reesei integrated expression strains. The 3-day sample was analyzed for the extent of conversion of glucan and xylan to both monomer and soluble oligomer products (FIG. 47C). FIG. 47D shows a chromatographic comparison of enzyme product from three T. reesei integrated expression strains. The experimental conditions are described in Example 1. Protein ratios differ across transformants and can be quantified as a percentage of the total integrated peak area. The "EGLs" marks the summed area of endoglucanase peaks. EndoH was added to the protein sample in small amounts as a reagent for HPLC analysis.

[0101] FIGS. 48A-48B: Saccharification increased in xylose monomer yield in response to hemicellulase addition to the enzyme composition produced by an integrated strain at 7 mg total protein per gram glucan+xylan in ammonia pretreated cob. FIG. 48A: Constituent Fusarium verticillioides hemicellulases in the enzyme composition produced by the integrated strain. FIG. 48B: Hemicellulases from other fungi.

[0102] FIGS. 49A-49B: Saccharification increased in glucose monomer yield in response to hemicellulase addition to the enzyme composition produced by an integrated strain at 7 mg total protein per gram glucan+xylan in ammonia pretreated cob. FIG. 49A: Constituent Fusarium verticillioides hemicellulases in the enzyme composition produced by the integrated strain. FIG. 49B: Hemicellulases from other fungi.

[0103] FIGS. 50A-50B: Saccharification increased in arabinose monomer yield in response to hemicellulase addition to the enzyme composition produced by an integrated strain at 7 mg total protein per gram glucan+xylan in ammonia pretreated cob. FIG. 50A: Constituent Fusarium verticillioides hemicellulases in the enzyme composition produced by an integrated strain. FIG. 50B: Hemicellulases from other fungi.

[0104] FIG. 51: A graphical presentation of the saccharification performance across pretreatment conditions. The X-axis corresponds to the experimental results listed in Table 12. Yields are calculated based on the theoretical amounts of glucan or xylan available in the raw switchgrass. All yields are based on monomeric sugars released after 3 days of saccharification with the enzyme cocktail.

[0105] FIG. 52: Amino acid sequence alignment of a number of GH39 .beta.-xylosidases. Underlined residues in bold face are the predicted catalytic general acid-base residue (marked with "A" above the alignment) and catalytic nucleophile residue (marked with "N" above the alignment). Underlined residues in normal face in the bottom two sequences are within 4 angstroms of the substrate in the active sites of the respective 3D structures (pdb: 1uhv and 2bs9, respectively). Underlined residues in the Fv39A sequence are predicted to be within 4 angstroms of a bound substrate in the active site.

[0106] FIG. 53: Amino acid sequence alignment of a number of GH43 family hydrolases. Amino acid residues highly conserved among members of the family are shown underlined and in bold face.

[0107] FIG. 54: Amino acid sequence alignment of a number of GH51 family enzymes. Amino acid residues highly conserved among members of the family are shown underlined and in bold type.

[0108] FIGS. 55A-55B: Amino acid sequence alignments of a number of GH10 and GH11 family endoxylanases. FIG. 55A; Alignment of GH10 family xylanases. Underlined residues in bold face are the catalytic nucleophile residues (marked with "N" above the alignment). FIG. 55B; Alignment of GH11 family xylanases. Underlined residues in bold face are the catalytic nucleophile residues and general acid base residues (marked with "N" and "A", respectively, above the alignment).

[0109] FIGS. 56A-56B: Saccharification of dilute ammonia pretreated switchgrass with various enzyme blends/compositions; FIG. 56A: glucan conversion; FIG. 56B: xylan conversion. The numbers below the figures on the x-axis refer to the amount of total mg of each protein in a given blend/composition per g of glucan or xylan, as described in Example 13.

[0110] FIGS. 57A-57B: Saccharification of dilute ammonia pretreated switchgrass with an enzyme composition produced by integrated strain H3A; FIG. 57A: glucan conversion; FIG. 57B: xylan conversion. Experimental conditions are described in Example 14.

[0111] FIGS. 58A-58C: Saccharification of steam-expanded sugarcane bagasse with an enzyme composition produced by integrated strain H3A at different enzyme doses; FIG. 58A: glucan conversion; FIG. 58B: xylan conversion; FIG. 58C: 3-day glucan and xylan conversions. Experimental conditions are described in Example 17.

[0112] FIGS. 59A-59C: Saccharification of dilute-acid pretreated corn fiber with various enzymes or enzyme blends; FIG. 59A: glucan conversion; FIG. 59B: xylan conversion: FIG. 59C: 5-day glucan and xylan conversions. The adjusted sugar (glucose or xylose) reflected the sugar being produced from the enzymatic step minus the starting sugar levels. Ratios shown along the x-axis represent the enzyme dose in mg of total protein per gram of cellulose. Experimental conditions are described in Example 18.

[0113] FIGS. 60A-60B: FIG. 60A: Deduced cDNA for Pa51A (SEQ ID NO:46). FIG. 60B: Codon optimized cDNA for Pa51A (SEQ ID NO:47).

[0114] FIG. 61: Coding sequence for a construct comprising a CBH1 signal sequence (underlined) upstream of genomic DNA encoding mature Gz43A (SEQ ID NO:48).

[0115] FIG. 62: Coding sequence for a construct comprising a CBH1 signal sequence (underlined) upstream of genomic DNA encoding mature Fo43A (SEQ ID NO:49).

[0116] FIG. 63: Codon optimized coding sequence for a construct comprising a CBH1 signal sequence (underlined) upstream of codon optimized DNA encoding mature Pf51A (SEQ ID NO:50).

[0117] FIG. 64: Amino acid sequence alignment of a number of GH3 family hydrolases. Amino acid residues highly conserved among members of the family are shown underlined and in bold face type. The box marks the predicted catalytic residue with flanking residues predicted to be involved in substrate binding.

[0118] FIG. 65: Amino acid sequence alignment of two representative Fusarium GH30 family hydrolases. Amino acid residues that are conserved among members of the family are shown underlined and in bold face type.

6. DETAILED DESCRIPTION

[0119] Enzymes have traditionally been classified by substrate specificity and reaction products. In the pre-genomic era, function was regarded as the most amenable (and perhaps most useful) basis for comparing enzymes and assays for various enzymatic activities have been well-developed for many years, resulting in the familiar EC classification scheme. Cellulases and other glycosyl hydrolases, which act upon glycosidic bonds between two carbohydrate moieties (or a carbohydrate and non-carbohydrate moiety--as occurs in nitrophenol-glycoside derivatives) are, under this classification scheme, designated as EC 3.2.1.-, with the final number indicating the exact type of bond cleaved. For example, according to this scheme an endo-acting cellulase (1,4-.beta.-endoglucanase) is designated EC 3.2.1.4.

[0120] With the advent of widespread genome sequencing projects, sequencing data have facilitated analyses and comparison of related genes and proteins. Additionally, a growing number of enzymes capable of acting on carbohydrate moieties (i.e., carbohydrases) have been crystallized and their 3-D structures solved. Such analyses have identified discreet families of enzymes with related sequence, which contain conserved three-dimensional folds that can be predicted based on their amino acid sequence. Further, it has been shown that enzymes with the same or similar three-dimensional folds exhibit the same or similar stereospecificity of hydrolysis, even when catalyzing different reactions (Henrissat et al., 1998, FEBS Lett 425(2): 352-4; Coutinho and Henrissat, 1999, in Genetics, biochemistry and ecology of cellulose degradation. T. Kimura. Tokyo, Uni Publishers Co: 15-23.).

[0121] These findings form the basis of a sequence-based classification of carbohydrase modules, which is available in the form of an internet database, the Carbohydrate-Active enZYme server (CAZy), available at http://afmb.cnrs-mrs.fr/CAZY/index.html (Carbohydrate-active enzymes: an integrated database approach. See Cantarel et al., 2009, Nucleic Acids Res. 37 (Database issue):D233-38).

[0122] CAZy defines four major classes of carbohydrases distinguishable by the type of reaction catalyzed: Glycosyl Hydrolases (GH's), Glycosyltransferases (GT's), Polysaccharide Lyases (PL's), and Carbohydrate Esterases (CE's). The enzymes of the disclosure are glycosyl hydrolases. GH's are a group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. A classification system for glycosyl hydrolases, grouped by sequence similarity, has led to the definition of over 85 different families. This classification is available on the CAZy web site.

[0123] The enzymes of the disclosure belong, inter alia, to the glycosyl hydrolase families 3, 10, 11, 30, 39, 43, and/or 51.

[0124] Glycoside hydrolase family 3 ("GH3") enzymes include, e.g., .beta.-glucosidase (EC:3.2.1.21); .beta.-xylosidase (EC:3.2.1.37); N-acetyl .beta.-glucosaminidase (EC:3.2.1.52); glucan .beta.-1,3-glucosidase (EC:3.2.1.58); cellodextrinase (EC:3.2.1.74); exo-1,3-1,4-glucanase (EC:3.2.1); and .beta.-galactosidase (EC 3.2.1.23). For example, GH3 enzymes can be those that have .beta.-glucosidase, .beta.-xylosidase, N-acetyl .beta.-glucosaminidase, glucan .beta.-1,3-glucosidase, cellodextrinase, exo-1,3-1,4-glucanase, and/or .beta.-galactosidase activity. Generally, GH3 enzymes are globular proteins and can consist of two or more subdomains. A catalytic residue has been identified as an aspartate residue that, in .beta.-glucosidases, located in the N-terminal third of the peptide and sits within the amino acid fragment SDW (Li et al. 2001, Biochem. J. 355:835-840). The corresponding sequence in Bgl1 from T. reesei is T266D267W268 (counting from the methionine at the starting position), with the catalytic residue aspartate being the D267. The hydroxyl/aspartate sequence is also conserved in the GH3 .beta.-xylosidases tested. For example, the corresponding sequence in T. reesei Bxl1 is S310D311 and the corresponding sequence in Fv3A is S290D291.

[0125] Glycoside hydrolase family 39 ("GH39") enzymes have .alpha.-L-iduronidase (EC:3.2.1.76) or .beta.-xylosidase (EC:3.2.1.37) activity. The three-dimensional structure of two GH39 .beta.-xylosidases, from Thermoanaerobacterium saccharolyticum (Uniprot Accession No. P36906) and Geobacillus stearothermophilus (Uniprot Accession No. Q9ZFM2), have been solved (see Yang et al. J. Mol. Biol. 2004, 335(1):155-65 and Czjzek et al., J. Mol. Biol. 2005, 353(4):838-46). The most highly conserved regions in these enzymes are located in their N-terminal sections, which have a classic (.alpha./.beta.)8 TIM barrel fold with the two key active site glutamic acids located at the C-terminal ends of .beta.-strands 4 (acid/base) and 7 (nucleophile). Fv39A residues E168 and E272 are predicted to function as catalytic acid-base and nucleophile, respectively, based on a sequence alignment of the abovementioned GH39 .beta.-xylosidases from Thermoanaerobacterium saccharolyticum and Geobacillus stearothermophilus with Fv39A.

[0126] Glycoside hydrolase family 43 ("GH43") enzymes include, e.g., L-.alpha.-arabinofuranosidase (EC 3.2.1.55); .beta.-xylosidase (EC 3.2.1.37); endo-arabinanase (EC 3.2.1.99); and/or galactan 1,3-.beta.-galactosidase (EC 3.2.1.145). For example, GH43 enzymes can have L-.alpha.-arabinofuranosidase activity, .beta.-xylosidase activity, endo-arabinanase activity, and/or galactan 1,3-.beta.-galactosidase activity. GH43 family enzymes display a five-bladed-.beta.-propeller-like structure. The propeller-like structure is based upon a five-fold repeat of blades composed of four-stranded .beta.-sheets. The catalytic general base, an aspartate, the catalytic general acid, a glutamate, and an aspartate that modulates the pKa of the general base were identified through the crystal structure of Cellvibrio japonicus CjAbn43A, and confirmed by site-directed mutagenesis (see Nurizzo et al. Nat. Struct. Biol. 2002, 9(9) 665-8). The catalytic residues are arranged in three conserved blocks spread widely through the amino acid sequence (Pons et al. Proteins: Structure, Function and Bioinformatics, 2004, 54:424-432). Among the GH43 family enzymes tested for useful activities in biomass hydrolysis, the predicted catalytic residues are shown as the bold and underlined residues in the sequences of FIG. 53. The crystal structure of the Geobacillus stearothermophylus xylosidase (Brux et al. J. Mol. Bio., 2006, 359:97-109) suggests several additional residues that may be important for substrate binding in this enzyme. Because the GH43 family enzymes tested for biomass hydrolysis had differing substrate preferences, these residues are not fully conserved in the sequences aligned in FIG. 53. However among the xylosidases tested, several conserved residues that contribute to substrate binding, either through hydrophobic interaction or through hydrogen bonding, are conserved and are noted by single underlines in FIG. 53.

[0127] Glycoside hydrolase family 51 ("GH51") enzymes have L-.alpha.-arabinofuranosidase (EC 3.2.1.55) and/or endoglucanase (EC 3.2.1.4) activity. High-resolution crystal structure of a GH51 L-.alpha.-arabinofuranosidase from Geobacillus stearothermophilus T-6 shows that the enzyme is a hexamer, with each monomer organized into two domains: an 8-barrel (.beta./.alpha.) and a 12-stranded .beta. sandwich with jelly-roll topology (see Hovel et al. EMBO J. 2003, 22(19):4922-4932). It can be expected that the catalytic residues will be acidic and conserved across enzyme sequences in the family. When the amino acid sequences of Fv51A, Pf51A, and Pa51A are aligned with GH51 enzymes of more diverse sequence, 8 acidic residues remain conserved. Those are shown bold and underlined in FIG. 54.

[0128] Glycoside hydrolase family 10 ("GH10") enzymes also have an 8-barrel (.beta./.alpha.) structure. They hydrolyze in an endo fashion with a retaining mechanism that uses at least one acidic catalytic residue in a generally acid/base catalysis process (Pell et al., J. Biol. Chem., 2004, 279(10): 9597-9605). Crystal structures of the GH10 xylanases of Penicillium simplicissimum (Uniprot P56588) and Thermoascus aurantiacus (Uniprot P23360) complexed with substrates in the active sites have been solved (see Schmidt et al. Biochem., 1999, 38:2403-2412; and Lo Leggio et al. FEBS Lett. 2001, 509: 303-308). Trichoderma reesei Xyn3 residues that are important for substrate binding and catalysis can be derived from an alignment with the sequences of abovementioned GH10 xylanases from Penicillium simplicissimum and Thermoascus aurantiacus (FIG. 55A). Trichoderma reesei Xyn3 residue E282 is predicted to be the catalytic nucleophilic residue, whereas residues E91, N92, K95, Q97, S98, H128, W132, Q135, N175, E176, Y219, Q252, H254, W312, and/or W320 are predicted to be involved in substrate binding and/or catalysis.

[0129] Glycoside hydrolase family 11 ("GH11") enzymes have a .beta.-jelly roll structure. They hydrolyze in an endo fashion with a retaining mechanism that uses at least one acidic catalytic residue in a generally acid/base catalysis process. Several other residues spread throughout their structure may contribute to stabilizing the xylose units in the substrate neighboring the pair of xylose monomers that are cleaved by hydrolysis. Three GH11 family endoxylanases were tested and their sequences are aligned in FIG. 55B. E118 (or E86 in mature T. reesei Xyn2) and E209 (or E177 in mature T. reesei Xyn2) have been identified as catalytic nucleophile and general/acid base residues in Trichoderma reesei Xyn2, respectively (see Havukainen et al. Biochem., 1996, 35:9617-24).

[0130] Glycoside hydrolase family 30 ("GH30") enzymes are retaining enzymes having glucosylceramidase (EC 3.2.1.45); .beta.-1,6-glucanase (EC 3.2.1.75); .beta.-xylosidase (EC 3.2.1.37); .beta.-glucosidase (3.2.1.21) activity. The first GH30 crystal structure was the Gaucher disease-related human .beta.-glucocerebrosidase solved by Grabowski, Gatt and Horowitz (Crit. Rev Biochem Mol Biol 1990; 25(6) 385-414). GH30 have an (.alpha./.beta.).sub.8 TIM barrel fold with the two key active site glutamic acids located at the C-terminal ends of .beta.-strands 4 (acid/base) and 7 (nucleophile) (Henrissat B, et al. Proc Natl Acad Sci USA, 92(15):7090-4, 1995; Jordan et al., Applied Microbiol Biotechnol, 86:1647, 2010). Glutamate 162 of Fv30A is conserved in 14 of 14 aligned GH30 proteins (13 bacterial proteins and one endo-b-xylanase from the fungi Biospora accession no. ADG62369) and glutamate 250 of Fv30A is conserved in 10 of the same 14, is an aspartate in another three and non-acidic in one. There are other moderately conserved acidic residues but no others are as widely conserved.

[0131] 6.1 Polypeptides of the Disclosure

[0132] The disclosure provides isolated, synthetic or recombinant polypeptides comprising an amino acid sequence having at least about 80%, e.g., at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete (100%) sequence identity to a polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, 44, or 45, over a region of at least about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 residues, or over the full length of the immature polypeptide, the full length mature polypeptide, the full length of the conserved domain, and/or the full length CBM. The conserved domain can be a predicted catalytic domain ("CD"). Exemplary polypeptides also include fragments of at least about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 residues in length. The fragments can comprise a conserved domain and/or a CBM. Where a fragment comprises a conserved domain and a CBM of an enzyme, the fragment optionally includes a linker separating the two. The linker can be a native linker or a heterologous linker It is contemplated that the polypeptides of the disclosure can be encoded by a nucleic acid sequence having at least about 85%, about 86%, about 87%, about 88%, about 89%, or about 90% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 41, or by a nucleic acid sequence capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 41, or to a fragment thereof. Exemplary nucleic acids of the disclosure are described in Section 6.2 below.

[0133] The polypeptides of the disclosure include proteins having an amino acid sequence with at least 85%, e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least 50 contiguous amino acid residues of the glycosyl hydrolase sequences of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, 44, or 45. For example, a polypeptide of the disclosure can include amino acid sequences having at least 85%, e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least 10, e.g., at least 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, or 350 contiguous amino acid residues of the glycosyl hydrolase sequences of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, 44, or 45. The contiguous amino acid sequence corresponds to the conserved domain and/or the CBM and/or the signal sequence.

[0134] Any of the amino acid sequences described herein can be produced together or in conjunction with at least 1, e.g., at least 2, 3, 5, 10, or 20 heterologous amino acids flanking each of the C- and/or N-terminal ends of the specified amino acid sequence, and or deletions of at least 1, e.g., at least 2, 3, 5, 10, or 20 amino acids from the C- and/or N-terminal ends of an enzyme of the disclosure.

[0135] Other variations also are within the scope of this disclosure. For example, one or more amino acid residues can be modified to increase or decrease the pI of an enzyme. The change of pI value can be achieved by removing a glutamate residue or substituting it with another amino acid residue.

[0136] The disclosure specifically provides an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, a Fv43D, a Pf43B, Fv43B, a Fv51A, a Trichoderma reesei Xyn3, a Trichoderma reesei Xyn2, a Trichoderma reesei Bxl1, and/or a Trichoderma reesei Bgl1 polypeptide. A combination of one or more of these enzymes is suitably present in the enzyme blend/composition of the invention, for example, one that is non-naturally occurring.

[0137] Fv3A:

[0138] The amino acid sequence of Fv3A (SEQ ID NO:2) is shown in FIGS. 1B, 38, and 64. SEQ ID NO:2 is the sequence of the immature Fv3A. Fv3A has a predicted signal sequence corresponding to residues 1 to 23 of SEQ ID NO:2 (underlined); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 24 to 766 of SEQ ID NO:2. The predicted conserved domains are in boldface type in FIG. 1B. Fv3A was shown to have .beta.-xylosidase activity, for example, in an enzymatic assay using p-nitrophenyl-.beta.-xylopyranoside, xylobiose, mixed linear xylo-oligomers, branched arabinoxylan oligomers from hemicellulose, or dilute ammonia pretreated corncob as substrates. The predicted catalytic residue is D291, while the flanking residues, S290 and C292, are predicted to be involved in substrate binding (FIG. 64). E175 and E213 are conserved across other GH3 enzymes and are predicted to have catalytic functions. As used herein, "an Fv3A polypeptide" refers to a polypeptide and/or to a variant thereof comprising a sequence having at least 85%, e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, e.g., at least 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 contiguous amino acid residues among residues 24 to 766 of SEQ ID NO:2. An Fv3A polypeptide preferably is unaltered as compared to native Fv3A in residues D291, S290, C292, E175, and E213. An Fv3A polypeptide is preferably unaltered in at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among Fv3A, Trichoderma reesei Bxl1 and/or Trichoderma reesei Bgl1, as shown in the alignment of FIG. 64. An Fv3A polypeptide suitably comprises the entire predicted conserved domain of native Fv3A as shown in FIG. 1B. An exemplary Fv3A polypeptide of the invention comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Fv3A sequence as shown in FIG. 1B. The Fv3A polypeptide of the invention preferably has .beta.-xylosidase activity.

[0139] Accordingly an Fv3A polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2, or to residues (i) 24-766, (ii) 73-321, (iii) 73-394, (iv) 395-622, (v) 24-622, or (vi) 73-622 of SEQ ID NO:2. The polypeptide suitably has .beta.-xylosidase activity.

[0140] Pf43A:

[0141] The amino acid sequence of Pf43A (SEQ ID NO:4) is shown in FIGS. 2B and 53. SEQ ID NO:4 is the sequence of the immature Pf43A. Pf43A has a predicted signal sequence corresponding to residues 1 to 20 of SEQ ID NO:4 (underlined in FIG. 2B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 21 to 445 of SEQ ID NO:4. The predicted conserved domain is in boldface type, the predicted CBM is in uppercase type, and the predicted linker separating the CD and CBM is in italics in FIG. 2B. Pf43A has been shown to have .beta.-xylosidase activity, in, for example, an enzymatic assay using p-nitophenyl-.beta.-xylopyranoside, xylobiose, mixed linear xylo-oligomers, or ammonia pretreated corncob as substrates. The predicted catalytic residues include either D32 or D60, D145, and E206. The C-terminal region underlined in FIG. 53 is the predicted CBM. As used herein, "a Pf43A polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 contiguous amino acid residues among residues 21 to 445 of SEQ ID NO:4. A Pf43A polypeptide preferably is unaltered as compared to the native Pf43A in residues D32 or D60, D145, and E206. A Pf43A is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are found conserved across a family of proteins including Pf43A and 1, 2, 3, 4, 5, 6, 7, or all 8 of other amino acid sequences in the alignment of FIG. 53. A Pf43A polypeptide of the invention suitably comprises two or more or all of the following domains: (1) the predicted CBM, (2) the predicted conserved domain, and (3) the linker of Pf43A as shown in FIG. 2B. An exemplary Pf43A polypeptide of the invention comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Pf43A sequence as shown in FIG. 2B. The Pf43A polypeptide of the invention preferably has .beta.-xylosidase activity.

[0142] Accordingly a Pf43A polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4, or to residues (i) 21-445, (ii) 21-301, (iii) 21-323, (iv) 21-444, (v) 302-444, (vi) 302-445, (vii) 324-444, or (viii) 324-445 of SEQ ID NO:4. The polypeptide suitably has .beta.-xylosidase activity.

[0143] Fv43E:

[0144] The amino acid sequence of Fv43E (SEQ ID NO:6) is shown in FIGS. 3B and 53. SEQ ID NO:6 is the sequence of the immature Fv43E. Fv43E has a predicted signal sequence corresponding to residues 1 to 18 of SEQ ID NO:6 (underlined in FIG. 3B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 19 to 530 of SEQ ID NO:6. The predicted conserved domain is marked in boldface type in FIG. 3B. Fv43E was shown to have .beta.-xylosidase activity, in, for example, enzymatic assay using 4-nitophenyl-.beta.-D-xylopyranoside, xylobiose, and mixed, linear xylo-oligomers, or ammonia pretreated corncob as substrates. The predicted catalytic residues include either D40 or D71, D155, and E241. As used herein, "an Fv43E polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 contiguous amino acid residues among residues 19 to 530 of SEQ ID NO:6. An Fv43E polypeptide preferably is unaltered as compared to the native Fv43E in residues D40 or D71, D155, and E241. An Fv43E polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are found to be conserved among a family of enzymes including Fv43E, and 1, 2, 3, 4, 5, 6, 7, or all other 8 amino acid sequences in the alignment of FIG. 53. An Fv43E polypeptide suitably comprises the entire predicted conserved domain of native Fv43E as shown in FIG. 3B. An exemplary Fv43E polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to mature Fv43E sequence as shown in FIG. 3B. The Fv43E polypeptide of the invention preferably has .beta.-xylosidase activity.

[0145] Accordingly, an Fv43E polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:6, or to residues (i) 19-530, (ii) 29-530, (iii) 19-300, or (iv) 29-300 of SEQ ID NO:6. The polypeptide suitably has .beta.-xylosidase activity.

[0146] Fv39A:

[0147] The amino acid sequence of Fv39A (SEQ ID NO:8) is shown in FIGS. 4B and 52. SEQ ID NO:8 is the sequence of the immature Fv39A. Fv39A has a predicted signal sequence corresponding to residues 1 to 19 of SEQ ID NO:8 (underlined in FIG. 4B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 20 to 439 of SEQ ID NO:8. The predicted conserved domain is shown in boldface type in FIG. 4B. Fv39A was shown to have .beta.-xylosidase activity in, for example, an enzymatic assay using p-nitophenyl-.beta.-xylopyranoside, xylobiose or mixed, linear xylo-oligomers as substrates. Fv39A residues E168 and E272 are predicted to function as catalytic acid-base and nucleophile, respectively, based on a sequence alignment of the above-mentioned GH39 xylosidases from Thermoanaerobacterium saccharolyticum (Uniprot Accession No. P36906) and Geobacillus stearothermophilus (Uniprot Accession No. Q9ZFM2) with Fv39A. As used herein, "an Fv39A polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 contiguous amino acid residues among residues 20 to 439 of SEQ ID NO:8. An Fv39A polypeptide preferably is unaltered as compared to native Fv39A in residues E168 and E272. An Fv39A polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among a family or enzymes including Fv39A and xylosidases from Thermoanaerobacterium saccharolyticum and Geobacillus stearothermophilus (see above). An Fv39A polypeptide suitably comprises the entire predicted conserved domain of native Fv39A as shown in FIG. 4B. An exemplary Fv39A polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Fv39A sequence as shown in FIG. 4B. The Fv39A polypeptide of the invention preferably has .beta.-xylosidase activity.

[0148] Accordingly, an Fv39A polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:8, or to residues (i) 20-439, (ii) 20-291, (iii) 145-291, or (iv) 145-439 of SEQ ID NO:8. The polypeptide suitably has .beta.-xylosidase activity.

[0149] Fv43A:

[0150] The amino acid sequence of Fv43A (SEQ ID NO:10) is provided in FIGS. 5B and 53. SEQ ID NO:10 is the sequence of the immature Fv43A. Fv43A has a predicted signal sequence corresponding to residues 1 to 22 of SEQ ID NO:10 (underlined in FIG. 5B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 23 to 449 of SEQ ID NO:10. In FIG. 5B, the predicted conserved domain is in boldface type, the predicted CBM is in uppercase type, and the predicted linker separating the CD and CBM is in italics. Fv43A was shown to have .beta.-xylosidase activity in, for example, an enzymatic assay using 4-nitophenyl-.beta.-D-xylopyranoside, xylobiose, mixed, linear xylo-oligomers, branched arabinoxylan oligomers from hemicellulose, and/or linear xylo-oligomers as substrates. The predicted catalytic residues including either D34 or D62, D148, and E209. As used herein, "an Fv43A polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 contiguous amino acid residues among residues 23 to 449 of SEQ ID NO:10. An Fv43A polypeptide preferably is unaltered, as compared to native Fv43A, at residues D34 or D62, D148, and E209, An Fv43A polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among a family of enzymes including Fv43A and 1, 2, 3, 4, 5, 6, 7, 8, or all 9 other amino acid sequences in the alignment of FIG. 53. An Fv43A polypeptide suitably comprises the entire predicted CBM of native Fv43A, and/or the entire predicted conserved domain of native Fv43A, and/or the linker of Fv43A as shown in FIG. 5B. An exemplary Fv43A polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Fv43A sequence as shown in FIG. 5B. The Fv45A polypeptide of the invention preferably has .beta.-xylosidase activity.

[0151] Accordingly an Fv43A polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:10, or to residues (i) 23-449, (ii) 23-302, (iii) 23-320, (iv) 23-448, (v) 303-448, (vi) 303-449, (vii) 321-448, or (viii) 321-449 of SEQ ID NO:10. The polypeptide suitably has .beta.-xylosidase activity.

[0152] Fv43B:

[0153] The amino acid sequence of Fv43B (SEQ ID NO:12) is shown in FIGS. 6B and 53. SEQ ID NO:12 is the sequence of the immature Fv43B. Fv43B has a predicted signal sequence corresponding to residues 1 to 16 of SEQ ID NO:12 (underlined in FIG. 6B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 17 to 574 of SEQ ID NO:12. The predicted conserved domain is in boldface type in FIG. 6B. Fv43B was shown to have both .beta.-xylosidase and L-.alpha.-arabinofuranosidase activities, in, for example, a first enzymatic assay using 4-nitophenyl-.beta.-D-xylopyranoside and p-nitrophenyl-.alpha.-L-arabinofuranoside as substrates. It was shown, in a second enzymatic assay, to catalyze the release of arabinose from branched arabino-xylooligomers and to catalyze the increased xylose release from oligomer mixtures in the presence of other xylosidase enzymes. The predicted catalytic residues include either D38 or D68, D151, and E236. As used herein, "an Fv43B polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, or 550 contiguous amino acid residues among residues 17 to 574 of SEQ ID NO:12. An Fv43B polypeptide preferably is unaltered, as compared to native Fv43B, at residues D38 or D68, D151, and E236. An Fv43B polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among a family of enzymes including Fv43B and 1, 2, 3, 4, 5, 6, 7, 8, or all 9 other amino acid sequences in the alignment of FIG. 53. An Fv43B polypeptide suitably comprises the entire predicted conserved domain of native Fv43B as shown in FIGS. 6B and 53. An exemplary Fv43B polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Fv43B sequence as shown in FIG. 6B. The Fv43B polypeptide of the present invention preferably has .beta.-xylosidase activity, L-.alpha.-arabinofuranosidase activity, or both .beta.-xylosidase and L-.alpha.-arabinofuranosidase activities.

[0154] Accordingly, an Fv43B polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:12, or to residues (i) 17-574, (ii) 27-574, (iii) 17-303, or (iv) 27-303 of SEQ ID NO:12. The polypeptide suitably has .beta.-xylosidase activity, L-.alpha.-arabinofuranosidase activity, or both .beta.-xylosidase and L-.alpha.-arabinofuranosidase activities.

[0155] Pa51A:

[0156] The amino acid sequence of Pa51A (SEQ ID NO:14) is shown in FIGS. 7B and 54. SEQ ID NO:14 is the sequence of the immature Pa51A. Pa51A has a predicted signal sequence corresponding to residues 1 to 20 of SEQ ID NO:14 (underlined in FIG. 7B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 21 to 676 of SEQ ID NO:14. The predicted L-.alpha.-arabinofuranosidase conserved domain is in boldface type in FIG. 7B. Pa51A was shown to have both .beta.-xylosidase activity and L-.alpha.-arabinofuranosidase activity in, for example, enzymatic assays using artificial substrates p-nitrophenyl-.beta.-xylopyranoside and p-nitophenyl-.alpha.-L-arabinofuranoside. It was shown to catalyze the release of arabinose from branched arabino-xylo oligomers and to catalyze the increased xylose release from oligomer mixtures in the presence of other xylosidase enzymes. Conserved acidic residues include E43, D50, E257, E296, E340, E370, E485, and E493. As used herein, "a Pa51A polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 contiguous amino acid residues among residues 21 to 676 of SEQ ID NO:14. A Pa51A polypeptide preferably is unaltered, as compared to native Pa51A, at residues E43, D50, E257, E296, E340, E370, E485, and E493. A Pa51A polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among a group of enzymes including Pa51A, Fv51A, and Pf51A, as shown in the alignment of FIG. 54. A Pa51A polypeptide suitably comprises the predicted conserved domain of native Pa51A as shown in FIG. 7B. An exemplary Pa51A polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Pa51A sequence as shown in FIG. 7B. The Pa51A polypeptide of the invention preferably has .beta.-xylosidase activity, L-.alpha.-arabinofuranosidase activity, or both .beta.-xylosidase and L-.alpha.-arabinofuranosidase activities.

[0157] Accordingly, a Pa51A polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:14, or to residues (i) 21-676, (ii) 21-652, (iii) 469-652, or (iv) 469-676 of SEQ ID NO:14. The polypeptide suitably has .beta.-xylosidase activity, L-.alpha.-arabinofuranosidase activity, or both .beta.-xylosidase and L-.alpha.-arabinofuranosidase activities.

[0158] Gz43A:

[0159] The amino acid sequence of Gz43A (SEQ ID NO:16) is shown in FIGS. 8B and 53. SEQ ID NO:16 is the sequence of the immature Gz43A. Gz43A has a predicted signal sequence corresponding to residues 1 to 18 of SEQ ID NO:16 (underlined in FIG. 8B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 19 to 340 of SEQ ID NO:16. The predicted conserved domain is in boldface type in FIG. 8B. Gz43A was shown to have .beta.-xylosidase activity in, for example, an enzymatic assay using p-nitophenyl-.beta.-xylopyranoside, xylobiose or mixed, and/or linear xylo-oligomers as substrates. The predicted catalytic residues include either D33 or D68, D154, and E243. As used herein, "a Gz43A polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, or 300 contiguous amino acid residues among residues 19 to 340 of SEQ ID NO:16. A Gz43A polypeptide preferably is unaltered, as compared to native Gz43A, at residues D33 or D68, D154, and E243. A Gz43A polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among a group of enzymes including Gz43A and 1, 2, 3, 4, 5, 6, 7, 8 or all 9 other amino acid sequences in the alignment of FIG. 53. A Gz43A polypeptide suitably comprises the predicted conserved domain of native Gz43A as shown in FIG. 8B. An exemplary Gz43A polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Gz43A sequence as shown in FIG. 8B. The Gz43A polypeptide of the invention preferably has .beta.-xylosidase activity.

[0160] Accordingly a Gz43A polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:16, or to residues (i) 19-340, (ii) 53-340, (iii) 19-383, or (iv) 53-383 of SEQ ID NO:16. The polypeptide suitably has .beta.-xylosidase activity.

[0161] Fo43A:

[0162] The amino acid sequence of Fo43A (SEQ ID NO:18) is shown in FIGS. 9B and 53. SEQ ID NO:18 is the sequence of the immature Fo43A. Fo43A has a predicted signal sequence corresponding to residues 1 to 20 of SEQ ID NO:18 (underlined in FIG. 9B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 21 to 348 of SEQ ID NO:18. The predicted conserved domain is in boldface type in FIG. 9B. Fo43A was shown to have .beta.-xylosidase activity in, for example, an enzymatic assay using p-nitophenyl-.beta.-xylopyranoside, xylobiose and/or mixed, linear xylo-oligomers as substrates. The predicted catalytic residues include either D37 or D72, D159, and E251. As used herein, "an Fo43A polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, or 300 contiguous amino acid residues among residues 18 to 344 of SEQ ID NO:18. An Fo43A polypeptide preferably is unaltered, as compared to native Fo43A, at residues D37 or D72, D159, and E251. An Fo43A polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among a group of enzymes including Fo43A and 1, 2, 3, 4, 5, 6, 7, 8 or all 9 other amino acid sequences in the alignment of FIG. 53. An Fo43A polypeptide suitably comprises the predicted conserved domain of native Fo43A as shown in FIG. 9B. An exemplary Fo43A polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Fo43A sequence as shown in FIG. 9B. The Fo43A polypeptide of the invention preferably has .beta.-xylosidase activity.

[0163] Accordingly an Fo43A polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:18, or to residues (i) 21-341, (ii) 107-341, (iii) 21-348, or (iv) 107-348 of SEQ ID NO:18. The polypeptide suitably has .beta.-xylosidase activity.

[0164] Af43A:

[0165] The amino acid sequence of Af43A (SEQ ID NO:20) is shown in FIGS. 10B and 53. SEQ ID NO:20 is the sequence of the immature Af43A. The predicted conserved domain is in boldface type in FIG. 10B. Af43A was shown to have L-.alpha.-arabinofuranosidase activity in, for example, an enzymatic assay using p-nitophenyl-.alpha.-L-arabinofuranoside as a substrate. Af43A was shown to catalyze the release of arabinose from the set of oligomers released from hemicellulose via the action of endoxylanase. The predicted catalytic residues include either D26 or D58, D139, and E227. As used herein, "an Af43A polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, or 300 contiguous amino acid residues of SEQ ID NO:20. An Af43A polypeptide preferably is unaltered, as compared to native Af43A, at residues D26 or D58, D139, and E227. An Af43A polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among a group of enzymes including Af43A and 1, 2, 3, 4, 5, 6, 7, 8, or all 9 other amino acid sequences in the alignment of FIG. 53. An Af43A polypeptide suitably comprises the predicted conserved domain of native Af43A as shown in FIG. 10B. An exemplary Af43A polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:20. The Af43A polypeptide of the invention preferably has L-.alpha.-arabinofuranosidase activity.

[0166] Accordingly an Af43A polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:20, or to residues (i) 15-558, or (ii) 15-295 of SEQ ID NO:20. The polypeptide suitably has L-.alpha.-arabinofuranosidase activity.

[0167] Pf51A:

[0168] The amino acid sequence of Pf51A (SEQ ID NO:22) is shown in FIGS. 11B and 54. SEQ ID NO:22 is the sequence of the immature Pf51A. Pf51A has a predicted signal sequence corresponding to residues 1 to 20 of SEQ ID NO:22 (underlined in FIG. 11B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 21 to 642 of SEQ ID NO:22. The predicted L-.alpha.-arabinofuranosidase conserved domain is in boldface type in FIG. 11B. Pf51A was shown to have L-.alpha.-arabinofuranosidase activity in, for example, an enzymatic assay using 4-nitrophenyl-.alpha.-L-arabinofuranoside as a substrate. Pf51A was shown to catalyze the release of arabinose from the set of oligomers released from hemicellulose via the action of endoxylanase. The predicted conserved acidic residues include E43, D50, E248, E287, E331, E360, E472, and E480. As used herein, "a Pf51A polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous amino acid residues among residues 21 to 642 of SEQ ID NO:22. A Pf51A polypeptide preferably is unaltered, as compared to native Pf51A, at residues E43, D50, E248, E287, E331, E360, E472, and E480. A Pf51A polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among Pf51A, Pa51A, and Fv51A, as shown in in the alignment of FIG. 54. A Pf51A polypeptide suitably comprises the predicted conserved domain of native Pf51A shown in FIG. 11B. An exemplary Pf51A polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Pf51A sequence shown in FIG. 11B. The Pf51A polypeptide of the invention preferably has L-.alpha.-arabinofuranosidase activity.

[0169] Accordingly a Pf51A polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:22, or to residues (i) 21-632, (ii) 461-632, (iii) 21-642, or (iv) 461-642 of SEQ ID NO:22. The polypeptide has L-.alpha.-arabinofuranosidase activity.

[0170] AfuXyn2:

[0171] The amino acid sequence of AfuXyn2 (SEQ ID NO:24) is shown in FIGS. 12B and 55B. SEQ ID NO:24 is the sequence of the immature AfuXyn2. AfuXyn2 has a predicted signal sequence corresponding to residues 1 to 18 of SEQ ID NO:24 (underlined in FIG. 12B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 19 to 228 of SEQ ID NO:24. The predicted GH11 conserved domain is in boldface type in FIG. 12B. AfuXyn2 was shown to have endoxylanase activity indirectly by observing its ability to catalyze the increased xylose monomer production in the presence of xylobiosidase when the enzymes act on pretreated biomass or on isolated hemicellulose. The conserved catalytic residues include E124, E129, and E215. As used herein, "an AfuXyn2 polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, or 200 contiguous amino acid residues among residues 19 to 228 of SEQ ID NO:24. An AfuXyn2 polypeptide preferably is unaltered, as compared to native AfuXyn2, at residues E124, E129 and E215. An AfuXyn2 polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among AfuXyn2, AfuXyn5, and Trichoderma reesei Xyn2, as shown in the alignment of FIG. 55B. An AfuXyn2 polypeptide suitably comprises the entire predicted conserved domain of native AfuXyn2 shown in FIG. 12B. An exemplary AfuXyn2 polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature AfuXyn2 sequence shown in FIG. 12B. The AfuXyn2 polypeptide of the invention preferably has xylanase activity.

[0172] AfuXyn5:

[0173] The amino acid sequence of AfuXyn5 (SEQ ID NO:26) is shown in FIGS. 13B and 55B. SEQ ID NO:26 is the sequence of the immature AfuXyn5. AfuXyn5 has a predicted signal sequence corresponding to residues 1 to 19 of SEQ ID NO:26 (underlined in FIG. 13B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 20 to 313 of SEQ ID NO:26. The predicted GH11 conserved domains are in boldface type in FIG. 13B. AfuXyn5 was shown to have endoxylanase activity indirectly by observing its ability to catalyze increased xylose monomer production in the presence of xylobiosidase when the enzymes act on pretreated biomass or on isolated hemicellulose. The conserved catalytic residues include E119, E124, and E210. The predicted CBM is near the C-terminal end, characterized by numerous hydrophobic residues and follows the long serine-, threonine-rich series of amino acids. The region is shown underlined in FIG. 55B. As used herein, "an AfuXyn5 polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, or 275 contiguous amino acid residues among residues 20 to 313 of SEQ ID NO:26. An AfuXyn5 polypeptide preferably is unaltered, as compared to native AfuXyn5, at residues E119, E120, and E210. An AfuXyn5 polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among AfuXyn5, AfuXyn2, and Trichoderma reesei Xyn2, as shown in the alignment of FIG. 55B. An AfuXyn5 polypeptide suitably comprises the entire predicted CBM of native AfuXyn5 and/or the entire predicted conserved domain of native AfuXyn5 (underlined) shown in FIG. 13B. An exemplary AfuXyn5 polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature AfuXyn5 sequence shown in FIG. 13B. The AfuXyn5 polypeptide of the invention preferably has xylanase activity.

[0174] Fv43D:

[0175] The amino acid sequence of Fv43D (SEQ ID NO:28) is shown in FIGS. 14B and 53. SEQ ID NO:28 is the sequence of the immature Fv43D. Fv43D has a predicted signal sequence corresponding to residues 1 to 20 of SEQ ID NO:28 (underlined in FIG. 14B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 21 to 350 of SEQ ID NO:28. The predicted conserved domain is in boldface type in FIG. 14B. Fv43D was shown to have .beta.-xylosidase activity in, for example, an enzymatic assay using p-nitophenyl-.beta.-xylopyranoside, xylobiose, and/or mixed, linear xylo-oligomers as substrates. The predicted catalytic residues include either D37 or D72, D159, and E251. As used herein, "an Fv43D polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, or 320 contiguous amino acid residues among residues 21 to 350 of SEQ ID NO:28. An Fv43D polypeptide preferably is unaltered, as compared to native Fv43D, at residues D37 or D72, D159, and E251. An Fv43D polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among a group of enzymes including Fv43D and 1, 2, 3, 4, 5, 6, 7, 8, or all 9 other amino acid sequences in the alignment of FIG. 53. An Fv43D polypeptide suitably comprises the entire predicted CD of native Fv43D shown in FIG. 14B. An exemplary Fv43D polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Fv43D sequence shown in FIG. 14B. The Fv43D polypeptide of the invention preferably has .beta.-xylosidase activity.

[0176] Accordingly an Fv43D polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:28, or to residues (i) 20-341, (ii) 21-350, (iii) 107-341, or (iv) 107-350 of SEQ ID NO:28. The polypeptide suitably has .beta.-xylosidase activity.

[0177] Pf43B:

[0178] The amino acid sequence of Pf43B (SEQ ID NO:30) is shown in FIGS. 15B and 53. SEQ ID NO:30 is the sequence of the immature Pf43B. Pf43B has a predicted signal sequence corresponding to residues 1 to 20 of SEQ ID NO:30 (underlined in FIG. 15B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 21 to 321 of SEQ ID NO:30. The predicted conserved domain is in boldface type in FIG. 15B. Conserved acidic residues within the conserved domain include D32, D61, D148, and E212. Pf43B was shown to have .beta.-xylosidase activity in, for example, an enzymatic assay using p-nitrophenyl-p-xylopyranaside, xylobiose, and/or mixed, linear xylo-oligomers as substrates. As used herein, "a Pf43B polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, or 280 contiguous amino acid residues among residues 21 to 321 of SEQ ID NO:30. A Pf43B polypeptide preferably is unaltered, as compared to native Pf43B, at residues D32, D61, D148, and E212. A Pf43B polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among a group of enzymes including Pf43B and 1, 2, 3, 4, 5, 6, 7, 8, or all 9 other amino acid sequences in the alignment of FIG. 53. A Pf43B polypeptide suitably comprises the predicted conserved domain of native Pf43B shown in FIG. 15B. An exemplary Pf43B polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Pf43B sequence shown in FIG. 15B. The Pf43B polypeptide of the invention preferably has .beta.-xylosidase activity.

[0179] Accordingly a Pf43B polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:30. The polypeptide suitably has .beta.-xylosidase activity.

[0180] Fv51A:

[0181] The amino acid sequence of Fv51A (SEQ ID NO:32) is shown in FIGS. 16B and 54. SEQ ID NO:32 is the sequence of the immature Fv51A. Fv51A has a predicted signal sequence corresponding to residues 1 to 19 of SEQ ID NO:32 (underlined in FIG. 16B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 20 to 660 of SEQ ID NO:32. The predicted L-.alpha.-arabinofuranosidase conserved domain is in boldface type in FIG. 16B. Fv51A was shown to have L-.alpha.-arabinofuranosidase activity in, for example, an enzymatic assay using 4-nitrophenyl-.alpha.-L-arabinofuranoside as a substrate. Fv51A was shown to catalyze the release of arabinose from the set of oligomers released from hemicellulose via the action of endoxylanase. Conserved residues include E42, D49, E247, E286, E330, E359, E479, and E487. As used herein, "an Fv51A polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 625 contiguous amino acid residues among residues 20 to 660 of SEQ ID NO:32. An Fv51A polypeptide preferably is unaltered, as compared to native Fv51A, at residues E42, D49, E247, E286, E330, E359, E479, and E487. An Fv51A polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among Fv51A, Pa51A, and Pf51A, as shown in the alignment of FIG. 54. An Fv51A polypeptide suitably comprises the predicted conserved domain of native Fv51A shown in FIG. 16B. An exemplary Fv51A polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Fv51A sequence shown in FIG. 16B. The Fv51A polypeptide of the invention preferably has L-.alpha.-arabinofuranosidase activity.

[0182] Accordingly an Fv51A polypeptide of the invention suitably comprise an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:32, or to residues (i) 21-660, (ii) 21-645, (iii) 450-645, or (iv) 450-660 of SEQ ID NO:32. The polypeptide suitably has L-.alpha.-arabinofuranosidase activity.

[0183] Xyn3: The amino acid sequence of Trichoderma reesei Xyn3 (SEQ ID NO:42) is shown in FIG. 21B. SEQ ID NO:42 is the sequence of the immature Trichoderma reesei Xyn3. Trichoderma reesei Xyn3 has a predicted signal sequence corresponding to residues 1 to 16 of SEQ ID NO:42 (underlined in FIG. 21B); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 17 to 347 of SEQ ID NO:42. The predicted conserved domain is in boldface type in FIG. 21B. Trichoderma reesei Xyn3 was shown to have endoxylanase activity indirectly by observation of its ability to catalyze increased xylose monomer production in the presence of xylobiosidase when the enzymes act on pretreated biomass or on isolated hemicellulose. The conserved catalytic residues include E91, E176, E180, E195, and E282, as determined by alignment with another GH10 family enzyme, the Xys1 delta from Streptomyces halstedii (Canals et al., 2003, Act Crystalogr. D Biol. 59:1447-53), which has 33% sequence identity to Trichoderma reesei Xyn3. As used herein, "a Trichoderma reesei Xyn3 polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, or 300 contiguous amino acid residues among residues 17 to 347 of SEQ ID NO:42. A Trichoderma reesei Xyn3 polypeptide preferably is unaltered, as compared to native Trichoderma reesei Xyn3, at residues E91, E176, E180, E195, and E282. A Trichoderma reesei Xyn3 polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved between Trichoderma reesei Xyn3 and Xys1 delta. A Trichoderma reesei Xyn3 polypeptide suitably comprises the entire predicted conserved domain of native Trichoderma reesei Xyn3 shown in FIG. 21B. An exemplary Trichoderma reesei Xyn3 polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Trichoderma reesei Xyn3 sequence shown in FIG. 21B. The Trichoderma reesei Xyn3 polypetpide of the invention preferably has xylanase activity.

[0184] Xyn2:

[0185] The amino acid sequence of Trichoderma reesei Xyn2 (SEQ ID NO:43) is shown in FIGS. 22 and 55B. SEQ ID NO:43 is the sequence of the immature Trichoderma reesei Xyn2. Trichoderma reesei Xyn2 has a predicted prepropeptide sequence corresponding to residues 1 to 33 of SEQ ID NO:43 (underlined in FIG. 22); cleavage of the predicted signal sequence between positions 16 and 17 is predicted to yield a propeptide, which is processed by a kexin-like protease between positions 32 and 33, generating the mature protein having a sequence corresponding to residues 33 to 222 of SEQ ID NO:43. The predicted conserved domain is in boldface type in FIG. 22. Trichoderma reesei Xyn2 was shown to have endoxylanase activity indirectly by observation of its ability to catalyze an increased xylose monomer production in the presence of xylobiosidase when the enzymes act on pretreated biomass or on isolated hemicellulose. The conserved acidic residues include E118, E123, and E209. As used herein, "a Trichoderma reesei Xyn2 polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, or 175 contiguous amino acid residues among residues 33 to 222 of SEQ ID NO:43. A Trichoderma reesei Xyn2 polypeptide preferably is unaltered, as compared to a native Trichoderma reesei Xyn2, at residues E118, E123, and E209. A Trichoderma reesei Xyn2 polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among Trichoderma reesei Xyn2, AfuXyn2, and AfuXyn5, as shown in the alignment of FIG. 55B. A Trichoderma reesei Xyn2 polypeptide suitably comprises the entire predicted conserved domain of native Trichoderma reesei Xyn2 shown in FIG. 22. An exemplary Trichoderma reesei Xyn2 polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Trichoderma reesei Xyn2 sequence shown in FIG. 22. The Trichoderma reesei Xyn2 polypeptide of the invention preferably has xylanase activity.

[0186] Bxl1:

[0187] The amino acid sequence of Trichoderma reesei Bxl1 (SEQ ID NO:44) is shown in FIGS. 23 and 64. SEQ ID NO:44 is the sequence of the immature Trichoderma reesei Bxl1. Trichoderma reesei Bxl1 has a predicted signal sequence corresponding to residues 1 to 18 of SEQ ID NO:44 (underlined in FIG. 23); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 19 to 797 of SEQ ID NO:44. The predicted conserved domains are in boldface type in FIG. 23. Trichoderma reesei Bxl1 was shown to have .beta.-xylosidase activity in, for example, an enzymatic assay using p-nitophenyl-.beta.-xylopyranoside, xylobiose and/or mixed, linear xylo-oligomers as substrates. The conserved acidic residues include E193, E234, and D310. As used herein, "a Trichoderma reesei Bxl1 polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 contiguous amino acid residues among residues 17 to 797 of SEQ ID NO:44. A Trichoderma reesei Bxl1 polypeptide preferably is unaltered, as compared to a native Trichoderma reesei Bxl1, at residues E193, E234, and D310. A Trichoderma reesei Bxl1 polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among Trichoderma reesei Bxl1, Fv3A, and Trichoderma reesei Bgl1, as shown in the alignment of FIG. 64. A Trichoderma reesei Bxl1 polypeptide suitably comprises the entire predicted conserved domains of native Trichoderma reesei Bxl1 shown in FIG. 23. An exemplary Trichoderma reesei Bxl1 polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Trichoderma reesei Bxl1 sequence shown in FIG. 23. The Trichoderma reesei Bxl1 polypeptide of the invention preferably has .beta.-xylosidase activity.

[0188] Accordingly a Trichoderma reesei Bxl1 polypeptide of the invention suitably comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 44. The polypeptide suitably has .beta.-xylosidase activity.

[0189] Bgl1:

[0190] The amino acid sequence of Trichoderma reesei Bgl1 (SEQ ID NO:45) is shown in FIGS. 24 and 64. Trichoderma reesei Bgl1 has a predicted signal sequence corresponding to residues 1 to 19 of SEQ ID NO:45 (underlined in FIG. 24); cleavage of the signal sequence is predicted to yield a mature protein having a sequence corresponding to residues 20 to 744 of SEQ ID NO:45. The predicted conserved domain is in boldface type in FIG. 24. Trichoderma reesei Bgl1 has been shown to have .beta.-glucosidase activity by observation of a capacity to catalyze the hydrolysis of para-nitrophenyl-.beta.-D-glucopyranoside to produce para-nitrophenol, and a capacity to catalyze the hydrolysis of cellobiose. The conserved acidic residues include D164, E197, and D267. As used herein, "a Trichoderma reesei Bgl1 polypeptide" refers to a polypeptide and/or a variant thereof comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 780 contiguous amino acid residues among residues 20 to 744 of SEQ ID NO:45. A Trichoderma reesei Bgl1 polypeptide preferably is unaltered, as compared to a native Bgl1, at residues D164, E197, and D267. A Trichoderma reesei Bgl1 polypeptide is preferably unaltered in at least 70%, 80%, 90%, 95%, 98%, or 99% of the amino acid residues that are conserved among Trichoderma reesei Bgl1, Fv3A, and Trichoderma reesei Bxl1, as shown in the alignment of FIG. 64. A Trichoderma reesei Bgl1 polypeptide suitably comprises the entire predicted conserved domain of native Trichoderma reesei Bgl1 shown in FIG. 24. An exemplary Trichoderma reesei Bgl1 polypeptide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature Trichoderma reesei Bgl1 sequence shown in FIG. 24. The Trichoderma reesei Bgl1 polypeptide of the invention preferably has 3-glucosidase activity.

[0191] Accordingly, the present disclosure provides a number of isolated, synthetic, or recombinant hemicelluloytic polypeptides or variants as described below:

(1) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 24 to 766 of SEQ ID NO:2; (ii) 73 to 321 of SEQ ID NO:2; (iii) 73 to 394 of SEQ ID NO:2; (iv) 395 to 622 of SEQ ID NO:2; (v) 24 to 622 of SEQ ID NO:2; or (iv) 73 to 622 of SEQ ID NO:2; the polypeptide preferably has .beta.-xylosidase activity; or (2) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 445 of SEQ ID NO:4; (ii) 21 to 301 of SEQ ID NO:4; (iii) 21 to 323 of SEQ ID NO:4; (iv) 21 to 444 of SEQ ID NO:4; (v) 302 to 444 of SEQ ID NO:4; (vi) 302 to 445 of SEQ ID NO:4; (vii) 324 to 444 of SEQ ID NO:4; or (viii) 324 to 445 of SEQ ID NO:4; the polypeptide preferably has .beta.-xylosidase activity; or (3) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 19 to 530 of SEQ ID NO:6; (ii) 29 to 530 of SEQ ID NO:6; (iii) 19 to 300 of SEQ ID NO:6; or (iv) 29 to 300 of SEQ ID NO:6; the polypeptide preferably has .beta.-xylosidase activity; or (4) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 20 to 439 of SEQ ID NO:8; (ii) 20 to 291 of SEQ ID NO:8; (iii) 145 to 291 of SEQ ID NO:8; or (iv) 145 to 439 of SEQ ID NO:8; the polypeptide preferably has .beta.-xylosidase activity; or (5) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 23 to 449 of SEQ ID NO:10; (ii) 23 to 302 of SEQ ID NO:10; (iii) 23 to 320 of SEQ ID NO:10; (iv) 23 to 448 of SEQ ID NO:10; (v) 303 to 448 of SEQ ID NO:10; (vi) 303 to 449 of SEQ ID NO:10; (vii) 321 to 448 of SEQ ID NO:10; or (viii) 321 to 449 of SEQ ID NO:10; the polypeptide preferably has .beta.-xylosidase activity; or (6) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 17 to 574 of SEQ ID NO:12; (ii) 27 to 574 of SEQ ID NO:12; (iii) 17 to 303 of SEQ ID NO:12; or (iv) 27 to 303 of SEQ ID NO:12; the polypeptide preferably has both .beta.-xylosidase activity and L-.alpha.-arabinofuranosidase activity; or (7) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 676 of SEQ ID NO:14; (ii) 21 to 652 of SEQ ID NO:14; (iii) 469 to 652 of SEQ ID NO:14; or (iv) 469 to 676 of SEQ ID NO:14; the polypeptide preferably has both .beta.-xylosidase activity and L-.alpha.-arabinofuranosidase activity; or (8) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 19 to 340 of SEQ ID NO:16; (ii) 53 to 340 of SEQ ID NO:16; (iii) 19 to 383 of SEQ ID NO:16; or (iv) 53 to 383 of SEQ ID NO:16; the polypeptide preferably has .beta.-xylosidase activity; or (9) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 341 of SEQ ID NO:18; (ii) 107 to 341 of SEQ ID NO:18; (iii) 21 to 348 of SEQ ID NO:18; or (iv) 107 to 348 of SEQ ID NO:18; the polypeptide preferably has .beta.-xylosidase activity; or (10) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 15 to 558 of SEQ ID NO:20; or (ii) 15 to 295 of SEQ ID NO:20; the polypeptide preferably has L-.alpha.-arabinofuranosidase activity; or (11) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 632 of SEQ ID NO:22; (ii) 461 to 632 of SEQ ID NO:22; (iii) 21 to 642 of SEQ ID NO:22; or (iv) 461 to 642 of SEQ ID NO:22; the polypeptide preferably has L-.alpha.-arabinofuranosidase activity; or (12) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 20 to 341 of SEQ ID NO:28; (ii) 21 to 350 of SEQ ID NO:28; (iii) 107 to 341 of SEQ ID NO:28; or (iv) 107 to 350 of SEQ ID NO:28; the polypeptide has .beta.-xylosidase activity; or (13) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 660 of SEQ ID NO:32; (ii) 21 to 645 of SEQ ID NO:32; (iii) 450 to 645 of SEQ ID NO:32; or (iv) 450 to 660 of SEQ ID NO:32; the polypeptide preferably has L-.alpha.-arabinofuranosidase activity.

[0192] The present disclosure provides also compositions (e.g., cellulase compositions, or enzyme blends/compositions) or fermentation broths enriched with one or more of the above-described polypeptides. The enzyme blend/composition is thus a non-naturally-occurring composition. The cellulase composition can be, for example, a filamentous fungal cellulase composition, such as a Trichoderma cellulase composition. The fermentation broth can be a fermentation broth of a filamentous fungus, for example, a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, or Chrysosporium fermentation broth. In particular, the fermentation broth can be, for example, one of Trichoderma spp. such as a Trichoderma reesei, or Penicillium spp., such as a Penicillium funiculosum. The fermentation broth can also suitably be a cell-free fermentation broth.

[0193] Additionally the instant disclosure provides host cells that are recombiantly engineered to express a polypeptide described above. The host cells can be, for example, filamentous fungal host cells, such as Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, cochliobolus, Pyricularia, or Chrysosporium cells. In particular, the host cells can be, for example, a Trichoderma spp. cell (such as a Trichoderma reesei cell), or a Penicillium cell (such as a Penicillium funiculosum cell), an Aspergillus cell (such as an Aspergillus oryzae or Aspergillus nidulans cell), or a Fusarium cell (such as a Fusarium verticilloides or Fusarium oxysporum cell).

[0194] 6.1.1 Fusion Proteins

[0195] The present disclosure also provides a fusion protein that includes a domain of a protein of the present disclosure attached to one or more fusion segments, which are typically heterologous to the protein (i.e., derived from a different source than the protein of the disclosure). Suitable fusion segments include, without limitation, segments that can enhance a protein's stability, provide other desirable biological activity, and/or facilitate purification of the protein (e.g., by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, solubility, action or biological activity; and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the domain(s) of a protein of the present disclosure. The fusion segments can be susceptible to cleavage. There may be some advantage in having this susceptibility, for example, it may enable straight-forward recovery of the protein of interest. Fusion proteins are preferably produced by culturing a recombinant cell transfected with a fusion nucleic acid that encodes a protein, which includes a fusion segment attached to either the carboxyl or amino terminal end, or fusion segments attached to both the carboxyl and amino terminal ends, of a protein, or a domain thereof.

[0196] Accordingly, proteins of the present disclosure also include expression products of gene fusions (e.g., an overexpressed, soluble, and active form of a recombinant protein), of mutagenized genes (e.g., genes having codon modifications to enhance gene transcription and translation), and of truncated genes (e.g., genes having signal sequences removed or substituted with a heterologous signal sequence).

[0197] Glycosyl hydrolases that utilize insoluble substrates are often modular enzymes. They usually comprise catalytic modules appended to one or more non-catalytic carbohydrate-binding domains (CBMs). In nature, CBMs are thought to promote the glycosyl hydrolase's interaction with its target substrate polysaccharide. Thus, the disclosure provides chimeric enzymes having altered substrate specificity; including, for example, chimeric enzymes having multiple substrates as a result of "spliced-in" heterologous CBMs. The heterologous CBMs of the chimeric enzymes of the disclosure can also be designed to be modular, such that they are appended to a catalytic module or catalytic domain (a "CD", e.g., at an active site), which can likewise be heterologous or homologous to the glycosyl hydrolase.

[0198] Thus, the disclosure provides peptides and polypeptides consisting of, or comprising, CBM/CD modules, which can be homologously paired or joined to form chimeric (heterologous) CBM/CD pairs. Thus, these chimeric polypeptides/peptides can be used to improve or alter the performance of an enzyme of interest. Accordingly, the disclosure provides chimeric enzymes comprising, e.g., at least one CBM of an enzyme of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, 44, or 45. A polypeptide of the disclosure, for example, includes an amino acid sequence comprising the CD and/or CBM of the glycosyl hydrolase sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, 44, or 45. The polypeptide of the disclosure can thus suitably be a fusion protein comprising functional domains from two or more different proteins (e.g., a CBM from one protein linked to a CD from another protein).

[0199] The polypeptides of the disclosure can suitably be obtained and/or used in "substantially pure" form. For example, a polypeptide of the disclosure constitutes at least about 80 wt. % (e.g., at least about 85 wt. %, 90 wt. %, 91 wt. %, 92 wt. %, 93 wt. %, 94 wt. %, 95 wt. %, 96 wt. %, 97 wt. %, 98 wt. %, or 99 wt. %) of the total protein in a given composition, which also includes other ingredients such as a buffer or solution.

[0200] Also, the polypeptides of the disclosure can suitably be obtained and/or used in recombinant culture broths (e.g., a filamentous fungal culture broth). The recombinant culture broths can be non-naturally occurring; for example, the culture broth can be produced by a recombinant host cell that is engineered to express a heterologous polypeptide of the disclosure, or by a recombinant host cell that is engineered to express an endogenous polypeptide of the disclosure in greater or lesser amounts than the endogenous expression levels (e.g., in an amount that is 1-, 2-, 3-, 4-, 5-, or more-fold greater or less than the endogenous expression levels). Furthermore, the polypeptides of the disclosure can suitably be obtained and/or used as recombinant culture broths produced by "integrated" host cell strains that have been engineered to express a plurality of polypeptides of the disclosure in desired ratios. Exemplary desired ratios are described herein, for example, in Section 6.3.4 below.

[0201] 6.2 Nucleic Acids and Host Cells

[0202] The present disclosure provides nucleic acids encoding a polypeptide of the disclosure, for example one described in Section 6.1 above.

[0203] The disclosure provides isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence having at least about 70%, e.g., at least about 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%, or 99%, or complete (100%) sequence identity to a nucleic acid of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 46, 47, 48, 49, or 50, over a region of at least about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or 2000 nucleotides. The present disclosure also provides nucleic acids encoding at least one polypeptide having a hemicellulolytic activity (e.g., a xylanase, .beta.-xylosidase, and/or L-.alpha.-arabinofuranosidase activity).

[0204] Nucleic acids of the disclosure also include isolated, synthetic or recombinant nucleic acids encoding an enzyme having the sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, 44, or 45, and subsequences thereof (e.g., a conserved domain or carbohydrate binding domain ("CBM"), and variants thereof. A nucleic acid of the disclosure can, for example, encode the mature portion of a protein of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 43, 44, or 45.

[0205] The disclosure specifically provides a nucleic acid encoding an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, a Fv43D, a Pf43B, an Fv43B, an Fv51A, a Trichoderma reesei Xyn3, a Trichoderma reesei Xyn2, a Trichoderma reesei Bxl1, or a Trichoderma reesei Bgl1 polypeptide.

[0206] For example, the disclosure provides an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes:

(1) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 24 to 766 of SEQ ID NO:2; (ii) 73 to 321 of SEQ ID NO:2; (iii) 73 to 394 of SEQ ID NO:2; (iv) 395 to 622 of SEQ ID NO:2; (v) 24 to 622 of SEQ ID NO:2; or (iv) 73 to 622 of SEQ ID NO:2; or (2) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 445 of SEQ ID NO:4; (ii) 21 to 301 of SEQ ID NO:4; (iii) 21 to 323 of SEQ ID NO:4; (iv) 21 to 444 of SEQ ID NO:4; (v) 302 to 444 of SEQ ID NO:4; (vi) 302 to 445 of SEQ ID NO:4; (vii) 324 to 444 of SEQ ID NO:4; or (viii) 324 to 445 of SEQ ID NO:4; or (3) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 19 to 530 of SEQ ID NO:6; (ii) 29 to 530 of SEQ ID NO:6; (iii) 19 to 300 of SEQ ID NO:6; or (iv) 29 to 300 of SEQ ID NO:6; or (4) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 20 to 439 of SEQ ID NO:8; (ii) 20 to 291 of SEQ ID NO:8; (iii) 145 to 291 of SEQ ID NO:8; or (iv) 145 to 439 of SEQ ID NO:8; or (5) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 23 to 449 of SEQ ID NO:10; (ii) 23 to 302 of SEQ ID NO:10; (iii) 23 to 320 of SEQ ID NO:10; (iv) 23 to 448 of SEQ ID NO:10; (v) 303 to 448 of SEQ ID NO:10; (vi) 303 to 449 of SEQ ID NO:10; (vii) 321 to 448 of SEQ ID NO:10; or (viii) 321 to 449 of SEQ ID NO:10; or (6) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 17 to 574 of SEQ ID NO:12; (ii) 27 to 574 of SEQ ID NO:12; (iii) 17 to 303 of SEQ ID NO:12; or (iv) 27 to 303 of SEQ ID NO:12; or (7) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 676 of SEQ ID NO:14; (ii) 21 to 652 of SEQ ID NO:14; (iii) 469 to 652 of SEQ ID NO:14; or (iv) 469 to 676 of SEQ ID NO:14; or (8) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 19 to 340 of SEQ ID NO:16; (ii) 53 to 340 of SEQ ID NO:16; (iii) 19 to 383 of SEQ ID NO:16; or (iv) 53 to 383 of SEQ ID NO:16; or (9) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 341 of SEQ ID NO:18; (ii) 107 to 341 of SEQ ID NO:18; (iii) 21 to 348 of SEQ ID NO:18; or (iv) 107 to 348 of SEQ ID NO:18; or (10) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 15 to 558 of SEQ ID NO:20; or (ii) 15 to 295 of SEQ ID NO:20; or (11) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 632 of SEQ ID NO:22; (ii) 461 to 632 of SEQ ID NO:22; (iii) 21 to 642 of SEQ ID NO:22; or (iv) 461 to 642 of SEQ ID NO:22; or (12) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 20 to 341 of SEQ ID NO:28; (ii) 21 to 350 of SEQ ID NO:28; (iii) 107 to 341 of SEQ ID NO:28; or (iv) 107 to 350 of SEQ ID NO:28; or (13) a polypeptide comprising an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence corresponding to positions (i) 21 to 660 of SEQ ID NO:32; (ii) 21 to 645 of SEQ ID NO:32; (iii) 450 to 645 of SEQ ID NO:32; or (iv) 450 to 660 of SEQ ID NO:32.

[0207] The instant disclosure also provides:

(1) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:1, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:1, or to a fragment thereof; or (2) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:3, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:3, or to a fragment thereof; or (3) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:5, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:5, or to a fragment thereof; or (4) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:7, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:7, or to a fragment thereof; or (5) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:9, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:9, or to a fragment thereof; or (6) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:11, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:11, or to a fragment thereof; or (7) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:13, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:13, or to a fragment thereof; or (8) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:15, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:15, or to a fragment thereof; or (9) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:17, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:17, or to a fragment thereof; or (10) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:19, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:19, or to a fragment thereof; or (11) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:21, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:21, or to a fragment thereof; or (12) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:27, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:27, or to a fragment thereof; or (13) a nucleic acid having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:31, or a nucleic acid that is capable of hybridizing under high stringency conditions to a complement of SEQ ID NO:31, or to a fragment thereof.

[0208] The disclosure also provides expression cassettes and/or vectors comprising the above-described nucleic acids.

[0209] Suitably, the nucleic acid encoding an enzyme of the disclosure is operably linked to a promoter. Specifically, where recombinant expression in a filamentous fungal host is desired, the promoter can be a filamentous fungal promoter. The nucleic acids can be, for example, under the control of heterologous promoters. The nucleic acids can also be expressed under the control of constitutive or inducible promoters. Examples of promoters that can be used include, but are not limited to, a cellulase promoter, a xylanase promoter, the 1818 promoter (previously identified as a highly expressed protein by EST mapping Trichoderma). For example, the promoter can suitably be a cellobiohydrolase, endoglucanase, or .beta.-glucosidase promoter. A particularly suitable promoter can be, for example, a T. reesei cellobiohydrolase, endoglucanase, or 6-glucosidase promoter. For example, the promoter is a cellobiohydrolase I (cbh1) promoter. Non-limiting examples of promoters include a cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, pki1, gpd1, xyn1, or xyn2 promoter. Additional non-limiting examples of promoters include a T. reesei cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, pki1, gpd1, xyn1, or xyn2 promoter.

[0210] The present disclosure provides host cells that are engineered to express one or more enzymes of the disclosure. Suitable host cells include cells of any microorganism (e.g., cells of a bacterium, a protist, an alga, a fungus (e.g., a yeast or filamentous fungus), or other microbe), and are preferably cells of a bacterium, a yeast, or a filamentous fungus.

[0211] Suitable host cells of the bacterial genera include, but are not limited to, cells of Escherichia, Bacillus, Lactobacillus, Pseudomonas, and Streptomyces. Suitable cells of bacterial species include, but are not limited to, cells of Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Lactobacillus brevis, Pseudomonas aeruginosa, and Streptomyces lividans.

[0212] Suitable host cells of the genera of yeast include, but are not limited to, cells of Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, Kluyveromyces, and Phaffia. Suitable cells of yeast species include, but are not limited to, cells of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha, Pichia pastoris, P. canadensis, Kluyveromyces marxianus, and Phaffia rhodozyma.

[0213] Suitable host cells of filamentous fungi include all filamentous forms of the subdivision Eumycotina. Suitable cells of filamentous fungal genera include, but are not limited to, cells of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysoporium, Coprinus, Coriolus, Corynascus, Chaetomium, Cryptococcus, Filobasidium, Fusarium, Gibberella, Humicola, Hypocrea, Magnaporthe, Mucor, Myceliophthora, Mucor, Neocaffimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Scytaldium, Schizophyllum, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma. Suitable cells can also include cells of various anamorph and teleomorph forms fo these filamentous fungal genera.

[0214] Suitable cells of filamentous fungal species include, but are not limited to, cells of Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0215] The disclosure further provides a recombinant host cell that is engineered to express one or more, two or more, three or more, four or more, or five or more of an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5 an Fv43D, a Pf43B, and an Fv51A polypeptide. The recombinant host cell is, for example, a recombinant Trichoderma reesei host cell. In a particular example, the disclosure provides a recombinant fungus, such as a recombinant Trichoderma reesei, that is engineered to express one or more, two or more, three or more, four or more, or five or more of an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, an Fv43D, a Pf43B, and an Fv51A polypeptide. The disclosure provides a recombinant Trichoderma reesei host cell engineered to express 1, 2, 3, 4, 5, or more of an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, an Fv43D, a Pf43B, and an Fv51A polypeptide.

[0216] The disclosure provides a host cell, for example, a recombinant fungal host cell or a recombinant filamentous fungus, engineered to recombinantly express at least one xylanase, at least one .beta.-xylosidase, and one L-.alpha.-arabinofuranosidase. The disclosure also provides a recombinant host cell, e.g., a recombinant fungal host cell or a recombinant filamentous fungus such as a recombinant Trichoderma reesei, that is engineered to express 1, 2, 3, 4, 5, or more of an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, an Fv43D, a Pf43B, and an Fv51A polypeptide, in addition to one or more of Trichoderma reesei Xyn2, Trichoderma reesei Xyn3, Trichoderma reesei Bxl1 and/or Trichoderma reesei Bgl1. The recombinant host cell is, for example, a Trichoderma reesei host cell. The recombinant fungus is, for example, a recombinant Trichoderma reesei. The disclosure provides a Trichoderma reesei host cell, or a recombinant Trichoderma reesei fungus, that is engineered to recombinantly express 1, 2, 3, 4, 5, or more of an Fv3A, a Pf43A, an Fv43E, an Fv39A, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, an Af43A, a Pf51A, an AfuXyn2, an AfuXyn5, an Fv43D, a Pf43B, and an Fv51A polypeptide, in addition to recombiantly express one or more of Trichoderma reesei Xyn2, Trichoderma reesei Xyn3, Trichoderma reesei Bxl1 and/or Trichoderma reesei Bgl1.

[0217] The present disclosure also provides a recombinant host cell e.g., a recombinant fungal host cell or a recombinant organism, e.g., a filamentous fungus, such as a recombinant Trichoderma reesei, that is engineered to recombinantly express Trichoderma reesei Xyn3, Trichoderma reesei Bgl1, Fv3A, Fv43D, and Fv51A polypeptides. For example, the recombinant host cell is suitably a Trichoderma reesei host cell. The recombinant fungus is suitably a recombinant Trichoderma reesei. The disclosure provides, for example, a Trichoderma reesei host cell engineered to recombinantly express Trichoderma reesei Xyn3, Trichoderma reesei Bgl1, Fv3A, Fv43D, and Fv51A polypeptides.

[0218] Additionally the disclosure provides a recombinant host cell or recombinant fungus that is engineered to express an enzyme blend comprising suitable enzymes in ratios suitable for saccharification. The recombinant host cell is, for example, a fungal host cell. The recombinant fungus is, for example, a recombinant Trichoderma reesei. Exemplary enzyme ratios/amounts present in suitable enzyme blends are described in Section 6.3.4 below.

[0219] The disclosure further provides transgenic plants comprising a nucleic acid of the disclosure or an expression cassette of the disclosure. The transgenic plant can be, for example, a cereal plant, a corn plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant, or a tobacco plant.

[0220] 6.3 Enzyme Blends for Saccharification

[0221] The present disclosure provides a composition comprising an enzyme blend/composition that is capable of breaking down lignocellulose material. Such a multi-enzyme blend/composition comprises at least one polypeptide of the present disclosure, in combination with one or more additional polypeptides of the present disclosure, or one or more enzymes from other microorganisms, plants, or organisms. Synergistic enzyme combinations and related methods are contemplated. The disclosure includes methods for identifying the optimum ratios of the enzymes included in the blends/compositions for degrading a particular lignocellulosic material. These methods include, e.g., tests to identify the optimum enzyme blend/composition and ratios for efficient conversion of a given lignocellulosic substrate to its constituent sugars. The Examples below include assays that may be used to identify optimum ratios and blends/compositions of enzymes with which to degrade lignocellulosic materials.

[0222] 6.3.1 Background

[0223] The cell walls of higher plants are comprised of a variety of carbohydrate polymer (CP) components. These CP interact through covalent and non-covalent means, providing the structural integrity required to form rigid cell walls and resist turgor pressure in plants. The major CP found in plants is cellulose, which forms the structural backbone of the cell wall. During cellulose biosynthesis, chains of poly-.beta.-1,4-D-glucose self associate through hydrogen bonding and hydrophobic interactions to form cellulose microfibrils, which further self-associate to form larger fibrils. Cellulose microfibrils are often irregular structurally and contain regions of varying crystallinity. The degree of crystallinity of cellulose fibrils depends on how tightly ordered the hydrogen bonding is between and among its component cellulose chains. Areas with less-ordered bonding, and therefore more accessible glucose chains, are referred to as amorphous regions.

[0224] The general model for cellulose depolymerization to glucose involves a minimum of three distinct enzymatic activities. Endoglucanases cleave cellulose chains internally to shorter chains in a process that increases the number of accessible ends, which are more susceptible to exoglucanase activity than the intact cellulose chains. These exoglucanases (e.g., cellobiohydrolases) are specific for either reducing ends or non-reducing ends, liberating, in most cases, cellobiose, the dimer of glucose. The accumulating cellobiose is then subject to cleavage by cellobiases (e.g., .beta.-1,4-glucosidases) to glucose.

[0225] Cellulose contains only anhydro-glucose. In contrast, hemicellulose contains a number of different sugar monomers. For instance, aside from glucose, sugar monomers in hemicellulose can also include xylose, mannose, galactose, rhamnose, and arabinose. Hemicelluloses mostly contain D-pentose sugars and occasionally small amounts of L-sugars. Xylose is typically present in the largest amount, but mannuronic acid and galacturonic acid also tend to be present. Hemicelluloses include xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan.

[0226] The enzymes and multi-enzyme compositions of the disclosure are useful for saccharification of hemicellulose materials, including, e.g., xylan, arabinoxylan, and xylan- or arabinoxylan-containing substrates. Arabinoxylan is a polysaccharide composed of xylose and arabinose, wherein L-.alpha.-arabinofuranose residues are attached as branch-points to a .beta.-(1,4)-linked xylose polymeric backbone.

[0227] Most biomass sources are rather complex, containing cellulose, hemicellulose, pectin, lignin, protein, and ash, among other components. Accordingly, in certain aspects, the present disclosure provides enzyme blends/compositions containing enzymes that impart a range or variety of substrate specificities when working together to degrade biomass into fermentable sugars in the most efficient manner. One example of a multi-enzyme blend/composition of the present invention is a mixture of cellobiohydrolase(s), xylanase(s), endoglucanase(s), .beta.-glucosidase(s), .beta.-xylosidase(s), and, optionally, accessory proteins. The enzyme blend/composition is suitably a non-naturally occurring composition.

[0228] Accordingly, the disclosure provides enzyme blends/compositions (including products of manufacture) comprising a mixture of xylan-hydrolyzing, hemicellulose- and/or cellulose-hydrolyzing enzymes, which include at least one, several, or all of a cellulase, including a glucanase; a cellobiohydrolase; an L-.alpha.-arabinofuranosidase; a xylanase; a .beta.-glucosidase; and a .beta.-xylosidase. Preferably each of the enzyme blends/compositions of the disclosure comprises at least one enzyme of the disclosure. The present disclosure also provides enzyme blends/compositions that are non-naturally occurring compositions. As used herein, the term "enzyme blends/compositions" refers to: [0229] (1) a composition made by combining component enzymes, whether in the form of a fermentation broth or partially or completely isolated or purified; [0230] (2) a composition produced by an organism modified to express one or more component enzymes; in certain embodiments, the organism used to express one or more component enzymes can be modified to delete one or more genes; in certain other embodiments, the organism used to express one or more component enzymes can further comprise proteins affecting xylan hydrolysis, hemicellulose hydrolysis, and/or cellulose hydrolysis; [0231] (3) a composition made by combining component enzymes simultaneously, separately, or sequentially during a saccharification or fermentation reaction; and [0232] (4) an enzyme mixture produced in situ, e.g., during a saccharification or fermentation reaction; [0233] (5) a composition produced in accordance with any or all of the above (1)-(4).

[0234] The term "fermentation broth" as used herein refers to an enzyme preparation produced by fermentation that undergoes no or minimal recovery and/or purification subsequent to fermentation. For example, microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes). Then, once the enzyme(s) are secreted into the cell culture media, the fermentation broths can be used. The fermentation broths of the disclosure can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. For example, the fermentation broths of the invention are unfractionated and comprise the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) undergo a fermentation process. The fermentation broth can suitably contain the spent cell culture media, extracellular enzymes, and live or killed microbial cells. Alternatively, the fermentation broths can be fractionated to remove the microbial cells. In those cases, the fermentation broths can, for example, comprise the spent cell culture media and the extracellular enzymes.

[0235] Any of the enzymes described specifically herein can be combined with any one or more of the enzymes described herein or with any other available and suitable enzymes, to produce a suitable multi-enzyme blend/composition. The disclosure is not restricted or limited to the specific exemplary combinations listed below.

[0236] 6.3.2 Biomass

[0237] The disclosure provides methods and processes for biomass saccharification, using enzymes, enzyme blends/compositions of the disclosure. The term "biomass," as used herein, refers to any composition comprising cellulose and/or hemicellulose (optionally also lignin in lignocellulosic biomass materials). As used herein, biomass includes, without limitation, seeds, grains, tubers, plant waste or byproducts of food processing or industrial processing (e.g., stalks), corn (including, e.g., cobs, stover, and the like), grasses (including, e.g., Indian grass, such as Sorghastrum nutans; or, switchgrass, e.g., Panicum species, such as Panicum virgatum), wood (including, e.g., wood chips, processing waste), paper, pulp, and recycled paper (including, e.g., newspaper, printer paper, and the like). Other biomass materials include, without limitation, potatoes, soybean (e.g., rapeseed), barley, rye, oats, wheat, beets, and sugar cane bagasse.

[0238] The disclosure provides methods of saccharification comprising contacting a composition comprising a biomass material, for example, a material comprising xylan, hemicellulose, cellulose, and/or a fermentable sugar, with a polypeptide of the disclosure, or a polypeptide encoded by a nucleic acid of the disclosure, or any one of the enzyme blends/compositions, or products of manufacture of the disclosure.

[0239] The saccharified biomass (e.g., lignocellulosic material processed by enzymes of the disclosure) can be made into a number of bio-based products, via processes such as, e.g., microbial fermentation and/or chemical synthesis. As used herein, "microbial fermentation" refers to a process of growing and harvesting fermenting microorganisms under suitable conditions. The fermenting microorganism can be any microorganism suitable for use in a desired fermentation process for the production of bio-based products. Suitable fermenting microorganisms include, without limitation, filamentous fungi, yeast, and bacteria. The saccharified biomass can, for example, be made it into a fuel (e.g., a biofuel such as a bioethanol, biobutanol, biomethanol, a biopropanol, a biodiesel, a jet fuel, or the like) via fermentation and/or chemical synthesis. The saccharified biomass can, for example, also be made into a commodity chemical (e.g., ascorbic acid, isoprene, 1,3-propanediol), lipids, amino acids, proteins, and enzymes, via fermentation and/or chemical synthesis.

[0240] 6.3.3. Pretreatment

[0241] Prior to saccharification, biomass (e.g., lignocellulosic material) is preferably subject to one or more pretreatment step(s) in order to render xylan, hemicellulose, cellulose and/or lignin material more accessible or susceptable to enzymes and thus more amenable to hydrolysis by the enzyme(s) and/or enzyme blends/compositions of the disclosure.

[0242] In an exemplary embodiment, the pretreatment entails subjecting biomass material to a catalyst comprising a dilute solution of a strong acid and a metal salt in a reactor. The biomass material can, e.g., be a raw material or a dried material. This pretreatment can lower the activation energy, or the temperature, of cellulose hydrolysis, ultimately allowing higher yields of fermentable sugars. See, e.g., U.S. Pat. Nos. 6,660,506; 6,423,145.

[0243] Another exemplary pretreatment method entails hydrolyzing biomass by subjecting the biomass material to a first hydrolysis step in an aqueous medium at a temperature and a pressure chosen to effectuate primarily depolymerization of hemicellulose without achieving significant depolymerization of cellulose into glucose. This step yields a slurry in which the liquid aqueous phase contains dissolved monosaccharides resulting from depolymerization of hemicellulose, and a solid phase containing cellulose and lignin. The slurry is then subject to a second hydrolysis step under conditions that allow a major portion of the cellulose to be depolymerized, yielding a liquid aqueous phase containing dissolved/soluble depolymerization products of cellulose. See, e.g., U.S. Pat. No. 5,536,325.

[0244] A further exemplary method involves processing a biomass material by one or more stages of dilute acid hydrolysis using about 0.4% to about 2% of a strong acid; followed by treating the unreacted solid lignocellulosic component of the acid hydrolyzed material with alkaline delignification. See, e.g., U.S. Pat. No. 6,409,841.

[0245] Another exemplary pretreatment method comprises prehydrolyzing biomass (e.g., lignocellulosic materials) in a prehydrolysis reactor; adding an acidic liquid to the solid lignocellulosic material to make a mixture; heating the mixture to reaction temperature; maintaining reaction temperature for a period of time sufficient to fractionate the lignocellulosic material into a solubilized portion containing at least about 20% of the lignin from the lignocellulosic material, and a solid fraction containing cellulose; separating the solubilized portion from the solid fraction, and removing the solubilized portion while at or near reaction temperature; and recovering the solubilized portion. The cellulose in the solid fraction is rendered more amenable to enzymatic digestion. See, e.g., U.S. Pat. No. 5,705,369.

[0246] Further pretreatment methods can involve the use of hydrogen peroxide H.sub.2O.sub.2. See Gould, 1984, Biotech, and Bioengr. 26:46-52.

[0247] Pretreatment can also comprise contacting a biomass material with stoichiometric amounts of sodium hydroxide and ammonium hydroxide at a very low concentration. See Teixeira et al., 1999, Appl. Biochem. and Biotech. 77-79:19-34.

[0248] Pretreatment can also comprise contacting a lignocellulose with a chemical (e.g., a base, such as sodium carbonate or potassium hydroxide) at a pH of about 9 to about 14 at moderate temperature, pressure, and pH. See PCT Publication WO2004/081185.

[0249] Ammonia is used, for example, in a preferred pretreatment method. Such a pretreatment method comprises subjecting a biomass material to low ammonia concentration under conditions of high solids. See, e.g., U.S. Patent Publication No. 20070031918 and PCT publication WO 06110901.

[0250] 6.3.4 Exemplary Enzyme Blends

[0251] The present disclosure provides enzyme blends/compositions comprising one or more enzymes of the disclosure. One or more enzymes of the enzyme blends/compositions can be produced by a recombinant host cell or a recombinant organism. The enzyme blends/compositions are suitably non-naturally occurring compositions.

[0252] An enzyme blend/composition of the disclosure can suitably comprise a first polypeptide having .beta.-xylosidase activity, and further comprises 1, 2, 3, or 4 of a second polypeptide having .beta.-xylosidase activity, one or more polypeptides having L-.alpha.-arabinofuranosidase activity, one or more polypeptides having xylanase activity, and one or more polypeptides having cellulase activity. The first polypeptide having .beta.-xylosidase activity is, for example, an Fv3A, a Pf43A, an Fv43E, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, or an Fv39A polypeptide. The second polypeptide having R-xylosidase activity, if present, is, for example, different from the first polypeptide having .beta.-xylosidase activity, and is suitably an Fv3A, a Pf43A, an Fv43E, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fv43D, an Fo43A, an Fv39A, or a Trichoderma reesei Bxl1 polypeptide. Each of the one or more polypeptides having L-.alpha.-arabinofuranosidase activity, if present, is, for example, an Af43A, a Pf51A, a Pa51A, an Fv43B, or an Fv51A polypeptide. Each of the one or more polypeptides having xylanase activity is, for example, a Trichoderma reesei Xyn, a Trichoderma reesei Xyn2, an AfuXyn2, or an AfuXyn5. Each of the one or more polypeptides having cellulase activity, if present, is, for example, an endoglucanase, for example, a Trichoderma reesei EG1 or EG2, a cellobiohydrolase, for example, a Trichoderma reesei CBH1 or CBH2, or a .beta.-glucosidase, for example, a Trichoderma reesei Bgl1.

[0253] Another enzyme blend/composition of the disclosure can suitably comprise a first polypeptide having L-.alpha.-arabinofuranosidase activity, and further comprises 1, 2, 3, or 4 of a second polypeptide having L-.alpha.-arabinofuranosidase activity, one or more polypeptides having .beta.-xylosidase activity, one or more polypeptides having xylanase activity, and/or one or more polypeptides having cellulase activity. The first L-.alpha.-arabinofuranosidase is an Af43A, a Pf51A, or an Fv51A polypeptide. The second L-.alpha.-arabinofuranosidase is different from the first L-.alpha.-arabinofuranosidase, and is, for example, an Af43A, a Pf51A, a Pa51A, an Fv43B, or an Fv51A polypeptide. Each of the one or more .beta.-xylosidases is, for example, an Fv3A, a Pf43A, an Fv43E, an Fv43A, an Fv43B, a Pa51A, an Fv43D, a Gz43A, an Fo43A, an Fv39A, or a Trichoderma reesei Bxl1 polypeptide. In certain embodiments, each of the one or more xylanases is a Trichoderma reesei Xyn3, a Trichoderma reesei Xyn2, an AfuXyn2, or an AfuXyn5 polypeptide. In certain embodiments, each of the one or more cellulases is independently an endoglucanase, for example, a Trichoderma reesei EG1 or EG2 polypeptide, a cellobiohydrolase, for example, a Trichoderma reesei CBH1 or CBH2 polypeptide, or a .beta.-glucosidase, for example, a Trichoderma reesei Bgl1 polypeptide.

[0254] Xylanases:

[0255] The xylanase(s) suitably constitutes about 0.05 wt. % to about 75 wt. % of the enzymes in an enzyme blend/composition of the disclosure (i.e., the percentage xylanase(s) is a weight percentage relative to the weight of all proteins in the composition) or a relative weight basis (i.e., wherein the percentage xylanase(s) is a weight percentage relative to the combined weight of xylanases, .beta.-xylosidases, cellulases, L-.alpha.-arabinofuranosidases, and accessory proteins). The ratio of any pair of proteins relative to each other can be readily calculated in the enzyme blends/compositions of the disclosure. Blends/compositions comprising enzymes in any weight ratio derivable from the weight percentages disclosed herein are contemplated. The xylanase content can be in a range wherein the lower limit is 0.05 wt. %, 1 wt. %, 2 wt. %, 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10 wt. %, 12 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 40 wt. %, 45 wt. %, or 50 wt. % of the total weight of enzymes in the enzyme blend/composition, and the upper limit is 10 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 35 wt. %, 40 wt. %, 50 wt. %, 55 wt. %, 60 wt. %, 65 wt. %, 70 wt. %, or 75 wt. % of the total weight of enzymes in the enzyme blend/composition. The one or more xylanases in an enzyme blend or composition can represent, for example, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %, or 15 wt. % to 25 wt. % of the total enzymes in the enzyme blend/composition. Exemplary suitable xylanases for inclusion in the enzyme blends/compositions of the disclosure are described in Section 6.3.6 below.

[0256] L-.alpha.-Arabinofuranosidases:

[0257] The L-.alpha.-arabinofuranosidase(s) suitably constitutes about 0.05 wt. % to about 75 wt. % of the total weight of all enzymes in a given enzyme blend/composition (i.e., wherein the percentage L-.alpha.-arabinofuranosidase(s) is a weight percentage relative to the weight of all proteins in the blend/composition) or a relative weight basis (i.e., wherein the percentage L-.alpha.-arabinofuranosidase(s) is a weight percentage relative to the combined weight of xylanases, .beta.-xylosidases, cellulases, L-.alpha.-arabinofuranosidases, and accessory proteins). The ratio of any pair of proteins relative to each other can be readily calculated based on the disclosure. Blends/compositions comprising enzymes in any weight ratio derivable from the weight percentages disclosed herein are contemplated. The L-.alpha.-arabinofuranosidase content can be in a range wherein the lower limit is 0.05 wt. %, 1 wt. %, 2 wt. %, 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. % 7 wt. %, 8 wt. %, 9 wt. %, 10 wt. %, 12 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 40 wt. %, 45 wt. %, or 50 wt. % of the total weight of enzymes in the blend/composition, and the upper limit is 10 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 35 wt. %, 40 wt. %, 50 wt. %, 55 wt. %, 60 wt. %, 65 wt. %, 70 wt. % or 75 wt. % of the total weight of enzymes in the blend/composition. For example, the one or more L-.alpha.-arabinofuranosidase(s) can suitably represent 2 wt. % to 25 wt. %; 5 wt. % to 20 wt. %; or 5 wt. % to 10 wt. % of the total weight of enzymes in the blend/composition. Exemplary suitable L-.alpha.-arabinofuranosidase(s) for inclusion in the enzyme blends/compositions of the disclosure are described in Section 6.3.8 below.

[0258] .beta.-Xylosidases:

[0259] The .beta.-xylosidase(s) suitably constitutes about 0.05 wt. % to about 75 wt. % of the total weight of enzymes in an enzyme blend/composition. The ratio of any pair of proteins relative to each other can be readily calculated based on the disclosure herein. Blends/compositions comprising enzymes in any weight ratio derivable from the weight percentages disclosed herein are contemplated. The 6-xylosidase content can be in a range wherein the lower limit is about 0.05 wt. %, 1 wt. %, 2 wt. %, 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. % 7 wt. %, 8 wt. %, 9 wt. %, 10 wt. %, 12 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 40 wt. %, 45 wt. %, or 50 wt. % of the total weight of enzymes in the blend/composition, and the upper limit is about 10 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 35 wt. %, 40 wt. %, 50 wt. %, 55 wt. %, 60 wt. %, 65 wt. %, 70 wt. %, or 75 wt. % of the total weight of enzymes in the blend/composition. For example, the .beta.-xylosidase(s) can represent about 0.05 wt. % to about 75 wt. % of the total weight of enzymes in the blend/composition. Also, the .beta.-xylosidase(s) can represent 0.05 wt. % to about 70 wt. %, about 1 wt. % to about 65 wt. %, about 1 wt. % to about 60 wt. %, about 2 wt. % to about 55 wt %, about 3 wt. % to about 50 wt. %, about 4 wt. % to about 45 wt. %, or about 5 wt. % to about 40 wt. % of the total weight of enzymes in the blend/composition. In yet a further example, the .beta.-xylosidase(s) suitably represent 2 wt. % to 30 wt. %; 10 wt. % to 20 wt. %; or 5 wt. % to 10 wt. % of the total weight of enzymes in the blend/composition. Exemplary suitable .beta.-xylosidase(s) are described in Section 6.3.7 below.

[0260] Cellulases:

[0261] The cellulase(s) suitably constitutes about 0.05 wt. % to about 90 wt. % of the total weight of enzymes in an enzyme blend/composition. Ratio of any pair of proteins relative to each other can be readily calculated based on the disclosure herein. Blends/compositions comprising enzymes in any weight ratio derivable from the weight percentages disclosed herein are contemplated. The cellulase content can be in a range wherein the lower limit is about 0.05 wt. %, 5 wt. %, 10 wt. %, 20 wt. %, 30 wt. %, 40 wt. %, 50 wt. %, 60 wt. %, 70 wt. % of the total weight of enzymes in the blend/composition, and the upper limit is about 20 wt. %, 30 wt. %, 40 wt. %, 50 wt. %, 60 wt. %, 70 wt. %, 80 wt. %, or 90 wt. % of the total weight of enzymes in the blend/composition. For example, the cellulase(s) suitably represents 30 wt. % to 80 wt. %, 50 wt. % to 70 wt. %, or 40 wt. % to 60 wt. % of the total weight of enzymes in the blend/composition. Exemplary suitable cellulases are described in Section 6.3.5 below. The cellulase components in an enzyme blend/composition of the disclosure are suitably capable of achieving at least about 0.005 fraction product per mg protein per gram of phosphoric acid swollen cellulose (PASC) as determined by a calcofluor assay. For example, the cellulase components in a blend/composition of the disclosure are capable of achieving a range of fraction product per mg protein per gram of PASC, wherein the lower limit of the range is about 0.005, 0.01, 0.015, 0.02, 0.03, 0.04, 0.05, 0.06, 0.075, or 0.1, and wherein the upper limit of the range is, 0.03, 0.04, 0.05, 0.06, 0.075, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7. The cellulase components in a blend/composition of the disclosure can, for example, achieve 0.00005-0.0001, 0.0005-0.001, 0.001-0.005, 0.005-0.03, 0.01-0.06, 0.02-0.04, 0.01-0.03, 0.02-0.05, 0.02-0.04, 0.01-0.05, 0.015-0.035, or 0.015-0.075 product fraction product per mg protein per gram PASC as determined by a calcofluor assay. The cellulase can be, for example, a whole cellulase. The cellulase can also, for example, suitably be enriched with a .beta.-glucosidase.

[0262] Accessory Proteins:

[0263] The enzyme blend/composition may suitably further comprise one or more accessory proteins. The accessory protein content of an enzyme blend/composition can range from about 0 wt. % to about 60 wt. % of the total weight of proteins in an enzyme blend/composition. Ratio of any pair of proteins relative to each other can be readily determined based on the disclosure herein. Blends/compositions comprising enzymes in any weight ratio derivable from the weight percentages disclosed herein are contemplated. The accessory protein content can be in a range wherein the lower limit is 0 wt. %, 1 wt. %, 2 wt. %, 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 10 wt. %, 12 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, or 35 wt. % of the total weight of proteins in the enzyme blend/composition, and the upper limit is 2 wt. %, 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10 wt. %, 11 wt. %, 12 wt. %, 13 wt. %, 14 wt. % 15 wt. %, 20 wt. %, 30 wt. %, 40 wt. %, 50 wt. %, or 60 wt. % of the total weight of proteins in the enzyme blend/composition. For example, the accessory protein(s) can suitably represent 0 wt. % to 2 wt. %, 5 wt. % to 10 wt. %, 20 wt. % to 50 wt. %, or 2 wt. % to 5 wt. % in the enzyme blend/composition. Exemplary suitable accessory proteins for inclusion in the enzyme blends/compositions of the disclosure are described in Section 6.3.9 below.

[0264] The present disclosure provides a first enzyme blend/composition for lignocellulose saccharification comprising: [0265] (1) about 30 wt. % to about 80 wt. % (e.g., 30 wt. % to 80 wt. %, 35 wt. % to 75 wt. %, 40 wt. % to 70 wt. %, 40 wt. % to 60 wt. %, 50 wt. % to 70 wt. %, etc.) cellulase(s), e.g., whole cellulase or .beta.-glucosidase enriched whole cellulase; [0266] (2) about 3 wt. % to about 50 wt. % (e.g., 5 wt. % to 40 wt. %, 10 wt. % to 30 wt. %, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %, 15 wt. % to 25 wt. %, etc.) xylanase(s), e.g., a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5, or a mixture of two or more of the foregoing enzymes; [0267] (3) about 2 wt. % to about 40 wt. % (e.g., 2 wt. % to 35 wt. %, 5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 30 wt. %, 10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.) .beta.-xylosidase(s), e.g., an Fv3A, a Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fv43A, an Fv43B, a Pa51A, an Fo43A, a Gz43A, a Trichoderma reesei Bxl1, or a mixture of two or more of the foregoing enzymes; [0268] (4) about 2 wt. % to about 40 wt. % (e.g., 2 wt. % to 35 wt. %, 5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 25 wt. %, 5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc) L-.alpha.-arabinofuranosidase(s), e.g., an Af43A, an Fv43B, a Pa51A, a Pf51A, an Fv51A, or a mixture of two or more of the foregoing enzymes; and [0269] (5) about 0 wt. % to about 50 wt. % (2 wt. % to 40 wt. %, 5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 0 wt. % to 2 wt. %, 5 wt. % to 10 wt. %, 20 wt. % to 50 wt. %, 2 wt. % to 5 wt. %, etc) accessory protein(s).

[0270] The present disclosure provides a second enzyme blend/composition for lignocellulose saccharification comprising: [0271] (1) about 30 wt. % to about 80 wt. % (e.g., 30 wt. % to 80 wt. %, 35 wt. % to 75 wt. %, 40 wt. % to 70 wt. %, 40 wt. % to 60 wt. %, 50 wt. % to 70 wt. %, etc.) cellulase(s), e.g., whole cellulase or .beta.-glucosidase enriched whole cellulase, or about 2 wt. % to about 10 wt. % (e.g., 2 wt. % to 8 wt. %, 4 wt. % to 6 wt. %, 2 wt. % to 4 wt. %, 6 wt. % to 8 wt. %, 8 wt. % to 10 wt. %, 2 wt. % to 10 wt. %, etc) .beta.-glucosidase(s), e.g., a Trichoderma reesei Bgl1; [0272] (2) about 3 wt. % to about 50 wt. % (e.g., 5 wt. % to 40 wt. %, 10 wt. % to 30 wt. %, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %, 15 wt. % to 25 wt. %, etc.) xylanase(s), e.g., a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5, or a mixture of two or more of the foregoing enzymes; [0273] (3) about 2 wt. % to about 40 wt. % (e.g., 2 wt. % to 35 wt. %, 5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 30 wt. %, 10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.) of at least two .beta.-xylosidase(s), wherein at least one .beta.-xylosidase is selected from Group 1 and at least one .beta.-xylosidase is selected from Group 2; [0274] wherein: [0275] Group 1: an Fv3A, an Fv43A, or a mixture thereof; [0276] Group 2: an Fv43D, a Pa51A, a Gz43A, a Trichoderma reesei Bxl1, a Pf43A, an Fv43E, an Fv39A, an Fo43A, an Fv43B, or a mixture of two or more of the foregoing enzymes; [0277] (4) about 2 wt. % to about 25 wt. % (e.g., 2 wt. % to 25 wt. %, 5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc) L-.alpha.-arabinofuranosidase(s), e.g., an Af43A, an Fv43B, a Pa51A, a Pf51A, Fv51A, or a mixture of two or more of the foregoing enzymes; and [0278] (5) about 0 wt. % to about 50 wt. % (2 wt. % to 40 wt. %, 5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 0 wt. % to 2 wt. %, 5 wt. % to 10 wt. %, 20 wt. % to 50 wt. %, 2 wt. % to 5 wt. %, etc) accessory protein(s).

[0279] The present disclosure provides a third enzyme blend/composition for lignocellulose saccharification comprising: [0280] (1) about 3 wt. % to about 50 wt. % (e.g., 5 wt. % to 40 wt. %, 10 wt. % to 30 wt. %, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %, 15 wt. % to 25 wt. %, etc.) xylanase(s), e.g., a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5, or a mixture of two or more of the foregoing enzymes; and [0281] (2) about 2 wt. % to 40 wt. % (e.g., 2 wt. % to 35 wt. %, 5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 30 wt. %, 10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.) of at least two .beta.-xylosidase(s), wherein at least one .beta.-xylosidase is selected from Group 1 and at least one .beta.-xylosidase is selected from Group 2; [0282] wherein: [0283] Group 1: an Fv3A, an Fv43A, or a mixture thereof; [0284] Group 2: an Fv43D, a Pa51A, a Gz43A, a Trichoderma reesei Bxl1, a Pf43A, an Fv43E, an Fv39A, an Fo43A, an Fv43B, or a mixture of two or more of the foregoing enzymes.

[0285] The present disclosure provides a fourth enzyme blend/composition for lignocellulose saccharification comprising: [0286] (1) about 5 wt. % to about 25 wt. % (e.g., 2 wt. % to 25 wt. %, 5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc) xylanase(s), e.g., a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5, or a mixture of two or more of the foregoing enzymes; [0287] (2) about 2 wt. % to about 30 wt. % (e.g., 2 wt. % to 30 wt. %, 10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.) .beta.-xylosidase(s), e.g., an Fv3A, a Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, a Trichoderma reesei Bxl1, or a mixture of two or more of the foregoing enzymes; and [0288] (3) about 2 wt. % to about 50 wt. % (e.g., 2 wt. % to 5 wt. %, 5 wt. % to 45 wt. %, 10 wt. % to 40 wt. %, 15 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 5 wt. % to 20 wt. %, 15 wt. % to 40 wt. %, etc) .beta.-glucosidase(s), e.g., a Trichoderma reesei Bgl1.

[0289] The present disclosure provides a fifth enzyme blend/composition for lignocellulose saccharification comprising: [0290] (1) about 5 wt. % to about 25 wt. % (e.g., 2 wt. % to 25 wt. %, 5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc) xylanase(s), e.g., a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5, or a mixture of two or more of the foregoing enzymes; and [0291] (2) about 2 wt. % to about 30 wt. % (e.g., 2 wt. % to 30 wt. %, 10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.) .beta.-xylosidase(s), e.g., an Fv3A, a Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, a Trichoderma reesei Bxl1, or a mixture of two or more of the foregoing enzymes.

[0292] The present disclosure provides a sixth enzyme blend/composition for lignocellulose saccharification comprising: [0293] (1) about 2 wt. % to about 50 wt. % (e.g., 2 wt. % to 5 wt. %, 5 wt. % to 45 wt. %, 10 wt. % to 40 wt. %, 15 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 5 wt. % to 20 wt. %, 15 wt. % to 40 wt. %, etc) .beta.-glucosidase(s), e.g., a Bgl1; [0294] (2) about 3 wt. % to about 50 wt. % (e.g., 5 wt. % to 40 wt. %, 10 wt. % to 30 wt. %, 5 wt. % to 20 wt. %, 10 wt. % to 15 wt. %, 15 wt. % to 25 wt. %, etc.) xylanase(s), e.g., a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, an AfuXyn5, or a mixture of two or more of the foregoing enzymes; [0295] (3) about 2 wt. % to 40 wt. % (e.g., 2 wt. % to 35 wt. %, 5 wt. % to 30 wt. %, 10 wt. % to 25 wt. %, 2 wt. % to 30 wt. %, 10 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc.) .beta.-xylosidase(s), e.g., an Fv3A, a Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fv43A, an Fv43B, a Pa51A, a Gz43A, an Fo43A, a Trichoderma reesei Bxl1, or a mixture of two or more of the foregoing enzymes; and [0296] (4) about 2 wt. % to about 25 wt. % (e.g., 2 wt. % to 25 wt. %, 5 wt. % to 20 wt. %, 5 wt. % to 10 wt. %, etc) L-.alpha.-arabinofuranosidase(s), e.g., an Af43A, an Fv43B, a Pa51A, a Pf51A, Fv51A, or a mixture of two or more of the foregoing enzymes.

[0297] The sixth enzyme blend/composition for lignocellulose saccharification above can, for example, comprise about 2 wt. % to about 40 wt. % of at least two .beta.-xylosidase, wherein at least one .beta.-xylosidase is selected from Group 1 and at least one .beta.-xylosidase is selected from Group 2; wherein: [0298] Group 1: an Fv3A, an Fv43A, or a mixture thereof; [0299] Group 2: an Fv43D, a Pa51A, a Gz43A, a Trichoderma reesei Bxl1, a Pf43A, an Fv43E, an Fv39A, an Fo43A, an Fv43B, or a mixture of two or more of the foregoing enzymes.

[0300] Where an enzyme blend/composition of the disclosure contains both Group 1 and a Group 2 .beta.-xylosidases, the ratio of Group 1 to Group 2 .beta.-xylosidases is preferably 1:10 to 10:1. For example, the ratio is suitably 1:2 to 2:1, 2:5 to 5:2, 3:8 to 8:3, 1:4 to 4:1, 1:5 to 5:1, 1:7 to 7:1, or any range between any pair the foregoing endpoints (e.g., 1:10 to 2:1, 4:1 to 2:5, 3:8 to 5:1, etc.). A particular example of a suitable ratio is approximately 1:1.

[0301] Where an enzyme blend/composition of the disclosure contains an Fv43A as a .beta.-xylosidase, the blend/composition can further contain Fv43B as an L-.alpha.-arabinofuranosidase.

[0302] An enzyme blend/composition of the disclosure is, for example, suitably part of a saccharification reaction mixture containing biomass in addition to the components of the enzyme blend/composition. For example, the saccharification reaction mixture can be characterized by 1, 2, 3 or all 4 of the following features: [0303] (1) the total weight of xylanase(s) per kg of hemicellulase in said saccharification reaction mixture is in a range in which the lower limit is about 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 7 g, or 10 g, and the upper limit is independently about 5 g, 7 g, 10 g, 15 g, 20 g, 30 g, or 40 g; for example, the total weight of xylanase(s) per kg of hemicellulase in the reaction mixture can be 0.5 g to 40 g, 0.5 g to 30 g, 0.5 g to 20 g, 0.5 to 10 g, 0.5 to 5 g, 1 g to 40 g, 2 g to 40 g, 3 g to 40 g, 5 g to 40 g, 7 g to 30 g, 10 g to 30 g, 5 g to 20 g, or 5 g to 30 g. [0304] (ii) the total weight of .beta.-xylosidase(s) per kg of hemicellulase in said saccharification reaction mixture is in a range in which the lower limit is about 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 7 g, or 10 g and the upper limit is independently about 5 g, 7 g, 10 g, 15 g, 20 g, 30 g, 40 g, or 50 g; for example, the total weight of .beta.-xylosidase(s) per kg of hemicellulase in the reaction mixture can be 0.5 g to 40 g, 0.5 to 50 g, 0.5 g to 30 g, 0.5 g to 20 g, 0.5 g to 10 g, 0.5 g to 5 g, 1 g to 40 g, 2 g to 40 g, 3 g to 40 g, 5 g to 40 g, 7 g to 30 g, 10 g, to 30 g, 5 g to 30 g, 5 g to 20 g. [0305] (iii) the total weight of L-.alpha.-arabinofuranosidase(s) per kg of hemicellulase in said saccharification reaction mixture is in a range in which the lower limit is about 0.2 g, 0.5 g, 1 g, 1.5 g, 2 g, 2.5 g, 3 g, 4 g, or 5 g and the upper limit is independently about 2 g, 3 g, 4 g, 5 g, 7 g, 10 g, 15 g, or 20 g; for example, the total weight of L-.alpha.-arabinofuranosidase(s) per kg of hemicellulase in the reaction mixture can be 0.2 g to 20 g, 0.5 g to 20 g, 1 g to 20 g, 2 g to 20 g, 2.5 g to 20 g, 3 g to 15 g, 4 g to 20 g, 5 g to 15 g, 5 g to 10 g, 5 g to 20 g, or 2.5 g to 15 g. [0306] (iv) the total weight of cellulase(s) per kg of cellulase in said saccharification reaction mixture is in a range in which the lower limit is about 1 g, 3 g, 5 g, 7 g, 10 g, 12 g, 15 g, 18 g, or 20 g, and the upper limit is independently about 10 g, 15 g, 18 g, 20 g, 25 g, 30 g, 50 g, 75 g, or 100 g; for example, the total weight of cellulase(s) per kg of cellulase in said reaction mixture can be 1 g to 100 g, 3 g to 100 g, 5 g to 100 g, 7 g to 100 g, 12 g to 100 g, 15 g to 100 g, 18 g to 100 g, 3 g to 75 g, 5 g to 50 g, 7 g to 75 g, 10 g to 75 g, 10 g to 50 g, 12 g to 75 g, 12 g to 50 g, 15 g to 75 g, 15 g to 50 g, 18 g to 30 g, 18 g to 75 g.

[0307] 6.3.5. Cellulases

[0308] The enzyme blends/compositions of the disclosure can comprise one or more cellulases. Cellulases are enzymes that hydrolyze cellulose (.beta.-1,4-glucan or .beta. D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC 3.2.1.91) ("CBH") and .beta.-glucosidases (.beta.-D-glucoside glucohydrolase; EC 3.2.1.21) ("BG") (Knowles et al., 1987, Trends in Biotechnology 5(9):255-261; Shulein, 1988, Methods in Enzymology, 160:234-242). Endoglucanases act mainly on the amorphous parts of the cellulose fiber, whereas cellobiohydrolases are also able to degrade crystalline cellulose.

[0309] Cellulases for use in accordance with the methods and compositions of the disclosure can be obtained from, or produced recombinantly from, inter alia, one or more of the following organisms: Crinipellis scapella, Macrophomina phaseolina, Myceliophthora thermophila, Sordaria fimicola, Volutella colletotrichoides, Thielavia terrestris, Acremonium sp., Exidia glandulosa, Fomes fomentarius, Spongipeffis sp., Rhizophlyctis rosea, Rhizomucor pusillus, Phycomyces niteus, Chaetostylum fresenii, Diplodia gossypina, Ulospora bilgramii, Saccobolus dilutellus, Penicillium verruculosum, Penicillium chrysogenum, Thermomyces verrucosus, Diaporthe syngenesia, Colletotrichum lagenarium, Nigrospora sp., Xylaria hypoxylon, Nectria pinea, Sordaria macrospora, Thielavia thermophila, Chaetomium mororum, Chaetomium virscens, Chaetomium brasiliensis, Chaetomium cunicolorum, Syspastospora boninensis, Cladorrhinum foecundissimum, Scytalidium thermophila, Gliocladium catenulatum, Fusarium oxysporum ssp. lycopersici, Fusarium oxysporum ssp. passiflora, Fusarium solani, Fusarium anguioides, Fusarium poae, Humicola nigrescens, Humicola grisea, Panaeolus retirugis, Trametes sanguinea, Schizophyllum commune, Trichothecium roseum, Microsphaeropsis sp., Acsobolus stictoideus spej., Poronia punctata, Nodulisporum sp., Trichoderma sp. (e.g., Trichoderma reesei) and Cylindrocarpon sp.

[0310] For example, a cellulase for use in the method and/or composition of the disclosure is a whole cellulase and/or is capable of achieving at least 0.1 (e.g. 0.1 to 0.4) fraction product as determined by the calcofluor assay described in Section 7.1.10 below.

[0311] 6.3.5.1 .beta.-Glucosidase

[0312] The enzyme blends/compositions of the disclosure can optionally comprise one or more .beta.-glucosidases. The term ".beta.-glucosidase" as used herein refers to a .beta.-D-glucoside glucohydrolase classified as EC 3.2.1.21, and/or members of certain GH families, including, without limitation, members of GH families 1, 3, 9 or 48, which catalyze the hydrolysis of cellobiose to release .beta.-D-glucose.

[0313] Suitable .beta.-glucosidase can be obtained from a number of microorganisms, by recombinant means, or be purchased from commercial sources. Examples of .beta.-glucosidases from microorganisms include, without limitation, ones from bacteria and fungi. For example, a .beta.-glucosidase of the present disclosure is suitably obtained from a filamentous fungus.

[0314] The .beta.-glucosidases can be obtained, or produced recombinantly, from, inter alia, Aspergillus aculeatus (Kawaguchi et al. Gene 1996, 173: 287-288), Aspergillus kawachi (Iwashita et al. Appl. Environ. Microbiol. 1999, 65: 5546-5553), Aspergillus oryzae (WO 2002/095014), Cellulomonas biazotea (Wong et al. Gene, 1998, 207:79-86), Penicillium funiculosum (WO 2004/078919), Saccharomycopsis fibuligera (Machida et al. Appl. Environ. Microbiol. 1988, 54: 3147-3155), Schizosaccharomyces pombe (Wood et al. Nature 2002, 415: 871-880), or Trichoderma reesei (e.g., .beta.-glucosidase 1 (U.S. Pat. No. 6,022,725), .beta.-glucosidase 3 (U.S. Pat. No. 6,982,159), .beta.-glucosidase 4 (U.S. Pat. No. 7,045,332), .beta.-glucosidase 5 (U.S. Pat. No. 7,005,289), .beta.-glucosidase 6 (U.S. Publication No. 20060258554), .beta.-glucosidase 7 (U.S. Publication No. 20060258554)).

[0315] The .beta.-glucosidase can be produced by expressing an endogenous or exogenous gene encoding a .beta.-glucosidase. For example, .beta.-glucosidase can be secreted into the extracellular space e.g., by Gram-positive organisms (e.g., Bacillus or Actinomycetes), or eukaryotic hosts (e.g., Trichoderma, Aspergillus, Saccharomyces, or Pichia). The .beta.-glucosidase can be, in some circumstances, overexpressed or underexpressed.

[0316] The .beta.-glucosidase can also be obtained from commercial sources. Examples of commercial .beta.-glucosidase preparation suitable for use in the present disclosure include, for example, Trichoderma reesei .beta.-glucosidase in Accellerase.RTM. BG (Danisco US Inc., Genencor); NOVOZYM.TM. 188 (a .beta.-glucosidase from Aspergillus niger); Agrobacterium sp. .beta.-glucosidase, and Thermatoga maritima .beta.-glucosidase from Megazyme (Megazyme International Ireland Ltd., Ireland.).

[0317] Moreover, the .beta.-glucosidase can be a component of a whole cellulase, as described in Section 6.3.5.4 below.

[0318] .beta.-glucosidase activity can be determined by a number of suitable means known in the art, such as the assay described by Chen et al., in Biochimica et Biophysica Acta 1992, 121:54-60, wherein 1 pNPG denotes 1 .mu.mol. of Nitrophenol liberated from 4-nitrophenyl-.beta.-D-glucopyranoside in 10 min at 50.degree. C. (122.degree. F.) and pH 4.8.

[0319] 6.3.5.2 Endoglucanases

[0320] The enzyme blends/compositions of the disclosure optionally comprise one or more endoglucanase. Any endoglucanase (EC 3.2.1.4) can be used in the methods and compositions of the present disclosure. An endoglucanse can be produced by expressing an endogenous or exogenous endoglucanase gene. The endoglucanase can be, in some circumstances, overexpressed or underexpressed.

[0321] For example, Trichoderma reesei EG1 (Penttila et al., Gene 1986, 63:103-112) and/or EG2 (Saloheimo et al., Gene 1988, 63:11-21) are suitably used in the methods and compositions of the present disclosure.

[0322] A thermostable Thielavia terrestris endoglucanase (Kvesitadaze et al., Applied Biochem. Biotech. 1995, 50:137-143) is, in another example, used in the methods and compositions of the present disclosure. Moreover, a Trichoderma reesei EG3 (Okada et al. Appl. Environ. Microbiol. 1988, 64:555-563), EG4 (Saloheimo et al. Eur. J. Biochem. 1997, 249:584-591), EG5 (Saloheimo et al. Molecular Microbiology 1994, 13:219-228), EG6 (U.S. Patent Publication No. 20070213249), or EG7 (U.S. Patent Publication No. 20090170181), an Acidothermus cellulolyticus EI endoglucanase (U.S. Pat. No. 5,536,655), a Humicola insolens endoglucanase V (EGV) (Protein Data Bank entry 4ENG), a Staphylotrichum coccosporum endoglucanase (U.S. Patent Publication No. 20070111278), an Aspergillus aculeatus endoglucanase F1-CMC (Ooi et al. Nucleic Acid Res. 1990, 18:5884), an Aspergillus kawachii IFO 4308 endoglucanase CMCase-1 (Sakamoto et al. Curr. Genet. 1995, 27:435-439), an Erwinia carotovara (Saarilahti et al. Gene 1990, 90:9-14); or an Acremonium thermophilum ALKO4245 endoglucanase (U.S. Patent Publication No. 20070148732) can also be used. Additional suitable endoglucanases are described in, e.g., WO 91/17243, WO 91/17244, WO 91/10732, U.S. Pat. No. 6,001,639.

[0323] 6.3.5.3 Cellobiohydrolases

[0324] Any cellobiohydrolase (EC 3.2.1.91) ("CBH") can be optionally used in the methods and blends/compositions of the present disclosure. The cellobiohydrolase can be produced by expressing an endogeneous or exogeneous cellobiohydrolase gene. The cellobiohydrolase can be, in some circumstances, overexpressed or under expressed.

[0325] For example, Trichoderma reesei CBHI (Shoemaker et al. Bio/Technology 1983, 1:691-696) and/or GBHII (Teeri et al. Bio/Technology 1983, 1:696-699) can be suitably used in the methods and blends/compositions of the present disclosure.

[0326] Suitable CBHs can be selected from an Agaricus bisporus CBH1 (Swiss Prot Accession No. Q92400), an Aspergillus aculeatus CBH1 (Swiss Prot Accession No. O59843), an Aspergillus nidulans CBHA (GenBank Accession No. AF420019) or CBHB (GenBank Accession No. AF420020), an Aspergillus niger CBHA (GenBank Accession No. AF156268) or CBHB (GenBank Accession No. AF156269), a Claviceps purpurea CBH1 (Swiss Prot Accession No. O00082), a Cochliobolus carbonarum CBH1 (Swiss Prot Accession No. Q00328), a Cryphonectria parasitica CBH1 (Swiss Prot Accession No. Q00548), a Fusarium oxysporum CBH1 (Cel7A) (Swiss Prot Accession No. P46238), a Humicola grisea CBH1.2 (GenBank Accession No. U50594), a Humicola grisea var. thermoidea CBH1 (GenBank Accession No. D63515) a CBHI.2 (GenBank Accession No. AF123441), or an exo1 (GenBank Accession No. AB003105), a Melanocarpus albomyces Cel7B (GenBank Accession No. AJ515705), a Neurospora crassa CBHI (GenBank Accession No. X77778), a Penicillium funiculosum CBHI (Cel7A) (U.S. Patent Publication No. 20070148730), a Penicillium janthinellum CBHI (GenBank Accession No. S56178), a Phanerochaete chrysosporium CBH (GenBank Accession No. M22220), or a CBHI-2 (Cel7D) (GenBank Accession No. L22656), a Talaromyces emersonii CBH1A (GenBank Accession No. AF439935), a Trichoderma viride CBH1 (GenBank Accession No. X53931), or a Volvariella volvacea V14 CBH1 (GenBank Accession No. AF156693).

[0327] 6.3.5.4 Whole Cellulases

[0328] An enzyme blend/composition of the disclosure can further comprise a whole cellulase. As used herein, a "whole cellulase" refers to either a naturally occurring or a non-naturally occurring cellulase-containing composition comprising at least 3 different enzyme types: (1) an endoglucanase, (2) a cellobiohydrolase, and (3) a .beta.-glucosidase, or comprising at least 3 different enzymatic activities: (1) an endoglucanase activity, which catalyzes the cleavage of internal .beta.-1,4 linkages, resulting in shorter glucooligosaccharides, (2) a cellobiohydrolase activity, which catalyzes an "exo"-type release of cellobiose units .beta.-1,4 glucose-glucose disaccharide), and (3) a .beta.-glucosidase activity, which catalyzes the release of glucose monomer from short cellooligosaccharides (e.g., cellobiose).

[0329] A "naturally occurring cellulase-containing" composition is one produced by a naturally occurring source, which comprises one or more cellobiohydrolase-type, one or more endoglucanase-type, and one or more .beta.-glucosidase-type components or activities, wherein each of these components or activities is found at the ratio and level produced in nature, untouched by the human hand. Accordingly, a naturally occurring cellulase-containing composition is, for example, one that is produced by an organism unmodified with respect to the cellulolytic enzymes such that the ratio or levels of the component enzymes are unaltered from that produced by the native organism in nature. A "non-naturally occurring cellulase-containing composition" refers to a composition produced by: (1) combining component cellulolytic enzymes either in a naturally occurring ratio or a non-naturally occurring, i.e., altered, ratio; or (2) modifying an organism to overexpress or underexpress one or more cellulolytic enzymes; or (3) modifying an organism such that at least one cellulolytic enzyme is deleted. A "non-naturally occurring cellulase containing" composition can also refer to a composition resulting from adjusting the culture conditions for a naturally-occurring organism, such that the naturally-occurring organism grows under a non-native condition, and produces an altered level or ratio of enzymes. Accordingly, in some embodiments, the whole cellulase preparation of the present disclosure can have one or more EGs and/or CBHs and/or .beta.-glucosidases deleted and/or overexpressed.

[0330] In the present disclosure, a whole cellulase preparation can be from any microorganism that is capable of hydrolyzing a cellulosic material. In some embodiments, the whole cellulase preparation is a filamentous fungal whole cellulase. For example, the whole cellulase preparation can be from an Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma species. The whole cellulase preparation is, for example, an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae whole cellulase. Moreover, the whole cellulase preparation can be a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum whole cellulase preparation. The whole cellulase preparation can also be a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Penicillium funiculosum, Scytalidium thermophilum, or Thielavia terrestris whole cellulase preparation. Moreover, the whole cellulase preparation can be a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei (e.g., RL-P37 (Sheir-Neiss G et al. Appl. Microbiol. Biotechnology, 1984, 20, pp. 46-53), QM9414 (ATCC No. 26921), NRRL 15709, ATCC 13631, 56764, 56466, 56767), or a Trichoderma viride (e.g., ATCC 32098 and 32086) whole cellulase preparation.

[0331] The whole cellulase preparation can, in particular, suitably be a Trichoderma reesei RutC30 whole cellulase preparation, which is available from the American Type Culture Collection as Trichoderma reesei ATCC 56765. For example, the whole cellulase preparation can also suitably be a whole cellulase of Penicillium funiculosum, which is available from the American Type Culture Collection as Penicillium funiculosum ATCC Number: 10446.

[0332] The whole cellulase preparation can also be obtained from commercial sources. Examples of commercial cellulase preparations suitable for use in the methods and compositions of the present disclosure include, for example, CELLUCLAST.TM. and Cellic.TM. (Novozymes A/S) and LAMINEX.TM. BG, IndiAge.TM. 44L, Primafast.TM. 100, Primafast.TM. 200, Spezyme.TM. CP, Accellerase.RTM. 1000 and Accellerase.RTM. 1500 (Danisco US. Inc., Genencor).

[0333] Suitable whole cellulase preparations can be made using any microorganism cultivation methods known in the art, especially fermentation, resulting in the expression of enzymes capable of hydrolyzing a cellulosic material. As used herein, "fermentation" refers to shake flask cultivation, small- or large-scale fermentation, such as continuous, batch, fed-batch, or solid state fermentations in laboratory or industrial fermenters performed in a suitable medium and under conditions that allow the cellulase and/or enzymes of interest to be expressed and/or isolated.

[0334] Generally, the microorganism is cultivated in a cell culture medium suitable for production of enzymes capable of hydrolyzing a cellulosic material. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures and variations known in the art. Suitable culture media, temperature ranges and other conditions for growth and cellulase production are known in the art. As a non-limiting example, a typical temperature range for the production of cellulases by Trichoderma reesei is 24.degree. C. to 28.degree. C.

[0335] The whole cellulase preparation can be used as it is produced by fermentation with no or minimal recovery and/or purification. For example, once cellulases are secreted into the cell culture medium, the cell culture medium containing the cellulases can be used directly. The whole cellulase preparation can comprise the unfractionated contents of fermentation material, including the spent cell culture medium, extracellular enzymes and cells. On the other hand, the whole cellulase preparation can also be subject to further processing in a number of routine steps, e.g., precipitation, centrifugation, affinity chromatography, filtration, or the like. For example, the whole cellulase preparation can be concentrated, and then used without further purification. The whole cellulase preparation can, for example, be formulated to comprise certain chemical agents that decrease cell viability or kills the cells after fermentation. The cells can, for example, be lysed or permeabilized using methods known in the art.

[0336] The endoglucanase activity of the whole cellulase preparation can be determined using carboxymethyl cellulose (CMC) as a substrate. A suitable assay measures the production of reducing ends created by the enzyme mixture acting on CMC wherein 1 unit is the amount of enzyme that liberates 1 .mu.mol. of product/min (Ghose, T. K., Pure & Appl. Chem. 1987, 59, pp. 257-268).

[0337] The whole cellulase can be a .beta.-glucosidase-enriched cellulase. The .beta.-glucosidase-enriched whole cellulase generally comprises a .beta.-glucosidase and a whole cellulase preparation. The .beta.-glucosidase-enriched whole cellulase compositions can be produced by recombinant means. For example, such a whole cellulase preparation can be achieved by expressing a .beta.-glucosidase in a microorganism capable of producing a whole cellulase The .beta.-glucosidase-enriched whole cellulase composition can also, for example, comprise a whole cellulase preparation and a .beta.-glucosidase. For instance, the .beta.-glucosidase-enriched whole cellulase composition can suitably comprise at least 5 wt. %, 7 wt. %, 10 wt. %, 15 wt. % or 20 wt. %, and up to 25 wt. %, 30 wt. %, 35 wt. %, 40 wt. %, or 50 wt. % .beta.-glucosidase based on the total weight of proteins in that blend/composition.

[0338] 6.3.6 Xylanases

[0339] The enzyme blends/compositions of the disclosure, for example, can, comprise one or more Group A xylanases, which may be a Trichoderma reesei Xyn2, a Trichoderma reesei Xyn3, an AfuXyn2, or an AfuXyn5. Suitable Trichoderma reesei Xyn2, Trichoderma reesei Xyn3, AfuXyn2, or AfuXyn5 polypeptides are described in Section 6.1 above.

[0340] The enzyme blends/compositions of the disclosure optionally comprise one or more xylanases in addition to or in place of the one or more Group A xylanases. Any xylanase (EC 3.2.1.8) can be used as the additional one or more xylanases. Suitable xylanases include, e.g., a Caldocellum saccharolyticum xylanase (Luthi et al. 1990, Appl. Environ. Microbiol. 56(9):2677-2683), a Thermatoga maritima xylanase (Winterhalter & Liebel, 1995, Appl. Environ. Microbiol. 61(5):1810-1815), a Thermatoga Sp. Strain FJSS-B.1 xylanase (Simpson et al. 1991, Biochem. J. 277, 413-417), a Bacillus circulans xylanase (BcX) (U.S. Pat. No. 5,405,769), an Aspergillus niger xylanase (Kinoshita et al. 1995, Journal of Fermentation and Bioengineering 79(5):422-428), a Streptomyces lividans xylanase (Shareck et al. 1991, Gene 107:75-82; Morosoli et al. 1986 Biochem. J. 239:587-592; Kluepfel et al. 1990, Biochem. J. 287:45-50), a Bacillus subtilis xylanase (Bernier et al. 1983, Gene 26(1):59-65), a Cellulomonas fimi xylanase (Clarke et al., 1996, FEMS Microbiology Letters 139:27-35), a Pseudomonas fluorescens xylanase (Gilbert et al. 1988, Journal of General Microbiology 134:3239-3247), a Clostridium thermocellum xylanase (Dominguez et al., 1995, Nature Structural Biology 2:569-576), a Bacillus pumilus xylanase (Nuyens et al. Applied Microbiology and Biotechnology 2001, 56:431-434; Yang et al. 1998, Nucleic Acids Res. 16(14B):7187), a Clostridium acetobutylicum P262 xylanase (Zappe et al. 1990, Nucleic Acids Res. 18(8):2179), or a Trichoderma harzianum xylanase (Rose et al. 1987, J. Mol. Biol. 194(4):755-756).

[0341] The xylanase can be produced by expressing an endogenous or exogenous gene encoding a xylanase. The xylanase can be, in some circumstances, overexpressed or underexpressed.

[0342] 6.3.7 .beta.-Xylosidases

[0343] The enzyme blends/compositions of the disclosure, for example, can suitablycomprise one or more .beta.-xylosidases. For example, the .beta.-xylosidase is a Group 1 .beta.-xylosidase enzyme (e.g., an Fv3A or an Fv43A) or a Group 2 .beta.-xylosidase enzyme (e.g., a Pf43A, an Fv43D, an Fv39A, an Fv43E, an Fo43A, an Fv43B, a Pa51A, a Gz43A, or a Trichoderma reesei Bxl1). These polypeptides are described in Section 0 above. For example, an enzyme blend/composition of the disclosure can suitably comprise one or more Group 1 .beta.-xylosidases and one or more Group 2 .beta.-xylosidases.

[0344] The enzyme blends/compositions of the disclosure can optionally comprise one or more .beta.-xylosidases, in addition to or in place of the Group 1 and/or Group 2 .beta.-xylosidases above. Any .beta.-xylosidase (EC 3.2.1.37) can be used as the additional .beta.-xylosidases. Suitable .beta.-xylosidases include, for example, a Talaromyces emersonii Bxl1 (Reen et al. 2003, Biochem Biophys Res Commun. 305(3):579-85), a Geobacillus stearothermophilus .beta.-xylosidases (Shallom et al. 2005, Biochemistry 44:387-397), a Scytalidium thermophilum .beta.-xylosidases (Zanoelo et al. 2004, J. Ind. Microbiol. Biotechnol. 31:170-176), a Trichoderma lignorum .beta.-xylosidases (Schmidt, 1998, Methods Enzymol. 160:662-671), an Aspergillus awamori .beta.-xylosidases (Kurakake et al. 2005, Biochim. Biophys. Acta 1726:272-279), an Aspergillus versicolor .beta.-xylosidases (Andrade et al. 2004, Process Biochem. 39:1931-1938), a Streptomyces sp. .beta.-xylosidases (Pinphanichakarn et al. 2004, World J. Microbiol. Biotechnol. 20:727-733), a Thermotoga maritima .beta.-xylosidases (Xue and Shao, 2004, Biotechnol. Lett. 26:1511-1515), a Trichoderma sp. SY .beta.-xylosidases (Kim et al. 2004, J. Microbiol. Biotechnol. 14:643-645), an Aspergillus niger .beta.-xylosidases (Oguntimein and Reilly, 1980, Biotechnol. Bioeng. 22:1143-1154), or a Penicillium wortmanni .beta.-xylosidases (Matsuo et al. 1987, Agric. Biol. Chem. 51:2367-2379).

[0345] The .beta.-xylosidase can be produced by expressing an endogenous or exogenous gene encoding a .beta.-xylosidase. The .beta.-xylosidase can be, in some circumstances, overexpressed or underexpressed.

[0346] 6.3.8 L-.alpha.-Arabinofuranosidases

[0347] The enzyme blends/compositions of the disclosure can, for example, suitably comprise one or more L-.alpha.-arabinofuranosidases. The L-.alpha.-arabinofuranosidase is, for example, an Af43A, an Fv43B, a Pf51A, a Pa51A, or an Fv51A. Af43A, Fv43B, Pf51A, Pa51A, and Fv51A polypeptides are described in Section 6.1 above.

[0348] The enzyme blends/compositions of the disclosure optionally comprise one or more L-.alpha.-arabinofuranosidases in addition to or in place of the foregoing L-.alpha.-arabinofuranosidases. L-.alpha.-arabinofuranosidases (EC 3.2.1.55) from any suitable organism can be used as the additional L-.alpha.-arabinofuranosidases. Suitable L-.alpha.-arabinofuranosidases include, e.g., an L-.alpha.-arabinofuranosidases of Aspergillus oryzae (Numan & Bhosle, J. Ind. Microbiol. Biotechnol. 2006, 33:247-260), Aspergillus sojae (Oshima et al. J. Appl. Glycosci. 2005, 52:261-265), Bacillus brevis (Numan & Bhosle, J. Ind. Microbiol. Biotechnol. 2006, 33:247-260), Bacillus stearothermophilus (Kim et al., J. Microbiol. Biotechnol. 2004, 14:474-482), Bifidobacterium breve (Shin et al., Appl. Environ. Microbiol. 2003, 69:7116-7123), Bifidobacterium longum (Margolies et al., Appl. Environ. Microbiol. 2003, 69:5096-5103), Clostridium thermocellum (Taylor et al., Biochem. J. 2006, 395:31-37), Fusarium oxysporum (Panagiotou et al., Can. J. Microbiol. 2003, 49:639-644), Fusarium oxysporum f. sp. dianthi (Numan & Bhosle, J. Ind. Microbiol. Biotechnol. 2006, 33:247-260), Geobacillus stearothermophilus T-6 (Shallom et al., J. Biol. Chem. 2002, 277:43667-43673), Hordeum vulgare (Lee et al., J. Biol. Chem. 2003, 278:5377-5387), Penicillium chrysogenum (Sakamoto et al., Biophys. Acta 2003, 1621:204-210), Penicillium sp. (Rahman et al., Can. J. Microbiol. 2003, 49:58-64), Pseudomonas cellulosa (Numan & Bhosle, J. Ind. Microbiol. Biotechnol. 2006, 33:247-260), Rhizomucor pusillus (Rahman et al., Carbohydr. Res. 2003, 338:1469-1476), Streptomyces chartreusis, Streptomyces thermoviolacus, Thermoanaerobacter ethanolicus, Thermobacillus xylanilyticus (Numan & Bhosle, J. Ind. Microbiol. Biotechnol. 2006, 33:247-260), Thermomonospora fusca (Tuncer and Ball, Folia Microbiol. 2003, (Praha) 48:168-172), Thermotoga maritima (Miyazaki, Extremophiles 2005, 9:399-406), Trichoderma sp. SY (Jung et al. Agric. Chem. Biotechnol. 2005, 48:7-10), Aspergillus kawachii (Koseki et al., Biochim. Biophys. Acta 2006, 1760:1458-1464), Fusarium oxysporum f. sp. dianthi (Chacon-Martinez et al., Physiol. Mol. Plant. Pathol. 2004, 64:201-208), Thermobacillus xylanilyticus (Debeche et al., Protein Eng. 2002, 15:21-28), Humicola insolens, Meripilus giganteus (Sorensen et al., Biotechnol. Prog. 2007, 23:100-107), or Raphanus sativus (Kotake et al. J. Exp. Bot. 2006, 57:2353-2362).

[0349] The L-.alpha.-arabinofuranosidase can be produced by expressing an endogenous or exogenous gene encoding an L-.alpha.-arabinofuranosidase. The L-.alpha.-arabinofuranosidase can be, in some circumstances, overexpressed or underexpressed.

[0350] 6.3.9 Accessory Proteins

[0351] The enzyme blends/compositions of the disclosure can, for example, suitably further comprise one or more accessory proteins. Examples of accessory proteins include, without limitation, mannanases (e.g., endomannanases, exomannanases, and .beta.-mannosidases), galactanases (e.g., endo- and exo-galactanases), arabinases (e.g., endo-arabinases and exo-arabinases), ligninases, amylases, glucuronidases, proteases, esterases (e.g., ferulic acid esterases, acetyl xylan esterases, coumaric acid esterases or pectin methyl esterases), lipases, glycoside hydrolase Family 61 polypeptides, xyloglucanases, CIP1, CIP2, swollenin, expansins, and cellulose disrupting proteins. Examples of accessory proteins can also include CIP1-like proteins, CIP2-like proteins, cellobiose dehydrogenases and manganese peroxidases. In particular embodiments, the cellulose disrupting proteins are cellulose binding modules.

[0352] 6.4 Further Applications

[0353] In addition to saccharification of biomass, the enzymes and/or enzyme blends/compositions of the disclosure can be used in industrial, agricultural, food and feed, as well as food and feed supplement processing processes. Exemplary applications are described below.

[0354] 6.4.1 Wood, Paper and Pulp Treatments

[0355] The enzymes, enzyme blends/compositions, and methods of the disclosure can be used in wood, wood product, wood waste or by-product, paper, paper product, paper or wood pulp, Kraft pulp, or wood or paper recycling treatment or industrial process. These processes include, e.g., treatments of wood, wood pulp, paper waste, paper, or pulp, or deinking of wood or paper. The enzymes, enzyme blends/compositions of the disclosure can be, for example, used to treat/pretreat paper pulp, or recycled paper or paper pulp, and the like. The enzymes, enzyme blends/compositions of the disclosure can be used to increase the "brightness" of the paper when they are included in the paper, pulp, recycled paper or paper pulp treatment/pretreatment. It can be appreciated that the higher the grade of paper, the greater the brightness; the brightness can impact the scan capability of optical scanning equipment. As such, the enzymes, enzyme blends/compositions, and methods/processes can be used to make high grade, "bright" papers, including inkjet, laser and photo printing quality paper.

[0356] The enzymes, enzyme blends/compositions of the disclosure can be used to process or treat a number of other cellulosic material, including, e.g., fibers from wood, cotton, hemp, flax or linen.

[0357] Accordingly, the disclosure provides wood, wood pulp, paper, paper pulp, paper waste or wood or paper recycling treatment processes using an enzyme, enzyme blend/composition of the disclosure.

[0358] The enzymes, enzyme blends/compositions of the disclosure can be used for deinking printed wastepaper, such as newspaper, or for deinking noncontact-printed wastepaper, e.g., xerographic and laser-printed paper, and mixtures of contact and noncontact-printed wastepaper, as described in U.S. Pat. No. 6,767,728 or 6,426,200; Neo, J. Wood Chem. Tech. 1986, 6(2):147. They can also be used to produce xylose from a paper-grade hardwood pulp in a process involving extracting xylan contained in pulp into a liquid phase, subjecting the xylan contained in the obtained liquid phase to conditions sufficient to hydrolyze xylan to xylose, and recovering the xylose. The extracting step, for example, can include at least one treatment of an aqueous suspension of pulp or an alkali-soluble material by an enzyme or an enzyme blend/composition (see, U.S. Pat. No. 6,512,110). The enzymes, enzyme blends/compositions of the disclosure can be used to dissolve pulp from cellulosic fibers such as recycled paper products made from hardwood fiber, a mixture of hardwood fiber and softwood fiber, waste paper, e.g., from unprinted envelopes, de-inked envelopes, unprinted ledger paper, de-inked ledger paper, and the like, as described in, e.g., U.S. Pat. No. 6,254,722.

[0359] 6.4.2 Treating Fibers and Textiles

[0360] The disclosure provides methods of treating fibers and fabrics using one or more enzymes, enzyme blends/compositions of the disclosure. The enzymes, enzyme blends/compositions can be used in any fiber- or fabric-treating method, which are known in the art. See, e.g., U.S. Pat. Nos. 6,261,828; 6,077,316; 6,024,766; 6,021,536; 6,017,751; 5,980,581; U.S. Patent Publication No. 20020142438 A1. For example, enzymes, enzyme blends/compositions of the disclosure can be used in fiber and/or fabric desizing. The feel and appearance of a fabric can be, for example, improved by a method comprising contacting the fabric with an enzyme or enzyme blend/composition of the disclosure in a solution. Optionally, the fabric is treated with the solution under pressure. The enzymes, enzyme blends/composition of the disclosure can also be used to remove stains.

[0361] The enzymes, enzyme blends/compositions of the disclosure can be used to treat a number of other cellulosic material, including fibers (e.g., fibers from cotton, hemp, flax or linen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yarns, and toweling, made from cotton, cotton blends or natural or manmade cellulosics or blends thereof. The textile treating processes can be used in conjunction with other textile treatments, e.g., scouring and/or bleaching. Scouring, for example, is the removal of non-cellulosic material from the cotton fiber, e.g., the cuticle (mainly consisting of waxes) and primary cell wall (mainly consisting of pectin, protein and xyloglucan).

[0362] 6.4.3 Treating Foods and Food Processing

[0363] The enzymes, enzyme blends/compositions of the disclosure have numerous applications in food processing industry. They can, for example, be used to improve extraction of oil from oil-rich plant material, e.g., oil-rich seeds. The enzymes, enzyme blends/compositions of the disclosure can be used to extract soybean oil from soybeans, olive oil from olives, rapeseed oil from rapeseed, or sunflower oil from sunflower seeds.

[0364] The enzymes, enzyme blends/compositions of the disclosure can also be used to separate components of plant cell materials. For example, they can be used to separate plant cells into components. The enzymes, enzyme blends/compositions of the disclosure can also be used to separate crops into protein, oil, and hull fractions. The separation process can be performed using known methods.

[0365] The enzymes, enzyme blends/compositions of the disclosure can, in addition to the uses above, be used to increase yield in the preparation of fruit or vegetable juices, syrups, extracts and the like. They can also be used in the enzymatic treatment of various plant cell wall-derived materials or waste materials from, e.g., cereals, grains, wine or juice production, or agricultural residues such as, e.g., vegetable hulls, bean hulls, sugar beet pulp, olive pulp, potato pulp, and the like. Further, they can be used to modify the consistency and/or appearance of processed fruits or vegetables. Additionally, they can be used to treat plant material so as to facilitate processing of the plant material (including foods), purification or extraction of plant components. The enzymes, enzyme blends/compositions of the disclosure can be used to improve feed value, decrease the water binding capacity, improve the degradability in waste water plants and/or improve the conversion of plant material to ensilage, and the like.

[0366] The enzymes, enzyme blends/compositions of the disclosure can also be used in baking applications. In some embodiments, they are used to create non-sticky doughs that are not difficult to machines and to reduce biscuit sizes. They can also be used to hydrolyze arabinoxylans to prevent rapid rehydration of the baked product that can lead to loss of crispiness and reduced shelf-life. For example, they are used as additives in dough processing.

[0367] 6.4.4 Animal Feeds and Food or Feed or Food Additives

[0368] The disclosure provides methods for treating animal feeds and foods and food or feed additives (supplements) using enzymes, enzyme blends/compositions of the disclosure. Animals including mammals (e.g., humans), birds, fish, and the like. The disclosure provides animal feeds, foods, and additives (supplements) comprising enzymes, enzyme blends/compositions of the disclosure. Treating animal feeds, foods and additives using enzymes of the disclosure can help in the availability of nutrients, e.g., starch, protein, and the like, in the animal feed or additive (supplements). By breaking down difficult to digest proteins or indirectly or directly unmasking starch (or other nutrients), the enzymes, enzyme blends/compositions can make nutrients more accessible to other endogenous or exogenous enzymes. They can also simply cause the release of readily digestible and easily absorbed nutrients and sugars.

[0369] When added to animal feed, enzymes, enzyme blends/compositions of the disclosure improve the in vivo break-down of plant cell wall material partly by reducing the intestinal viscosity (see, e.g., Bedford et al., Proceedings of the 1st Symposium on Enzymes in Animal Nutrition, 1993, pp. 73-77), whereby a better utilization of the plant nutrients by the animal is achieved. Thus, by using enzymes, enzyme blends/compositions of the disclosure in feeds, the growth rate and/or feed conversion ratio (i.e., the weight of ingested feed relative to weight gain) of the animal can be improved.

[0370] The animal feed additive of the disclosure may be a granulated enzyme product which can be readily mixed with feed components. Alternatively, feed additives of the disclosure can form a component of a pre-mix. The granulated enzyme product of the disclosure may be coated or uncoated. The particle size of the enzyme granulates can be compatible with that of the feed and/or the pre-mix components. This provides a safe and convenient mean of incorporating enzymes into feeds. Alternatively, the animal feed additive of the disclosure can be a stabilized liquid composition. This may be an aqueous- or oil-based slurry. See, e.g., U.S. Pat. No. 6,245,546.

[0371] An enzyme, enzyme blend/composition of the disclosure can be supplied by expressing the enzymes directly in transgenic feed crops (e.g., as transgenic plants, seeds and the like), such as grains, cereals, corn, soy bean, rape seed, lupin and the like. As discussed above, the disclosure provides transgenic plants, plant parts and plant cells comprising a nucleic acid sequence encoding a polypeptide of the disclosure. The nucleic acid is expressed such that the enzyme of the disclosure is produced in recoverable quantities. The xylanase can be recovered from any plant or plant part. Alternatively, the plant or plant part containing the recombinant polypeptide can be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.

[0372] The disclosure provides methods for removing oligosaccharides from feed prior to consumption by an animal subject using an enzyme, enzyme blend/composition of the disclosure. In this process a feed is formed to have an increased metabolizable energy value. In addition to enzymes, enzyme blends/compositions of the disclosure, galactosidases, cellulases, and combinations thereof can be used.

[0373] The disclosure provides methods for utilizing an enzyme, an enzyme blend/composition of the disclosure as a nutritional supplement in the diets of animals by preparing a nutritional supplement containing a recombinant enzyme of the disclosure, and administering the nutritional supplement to an animal to increase the utilization of hemicellulase contained in food ingested by the animal.

[0374] 6.4.5 Waste Treatment

[0375] The enzymes, enzyme blends/compositions of the disclosure can be used in a variety of other industrial applications, e.g., in waste treatment. For example, in one aspect, the disclosure provides a solid waste digestion process using the enzymes, enzyme blends/compositions of the disclosure. The methods can comprise reducing the mass and volume of substantially untreated solid waste. Solid waste can be treated with an enzymatic digestive process in the presence of an enzymatic solution (including the enzymes, enzyme blends/compositions of the disclosure) at a controlled temperature. This results in a reaction without appreciable bacterial fermentation from added microorganisms. The solid waste is converted into a liquefied waste and any residual solid waste. The resulting liquefied waste can be separated from said any residual solidified waste. See, e.g., U.S. Pat. No. 5,709,796.

[0376] 6.4.6 Detergent, Disinfectant and Cleaning Compositions

[0377] The disclosure provides detergent, disinfectant or cleanser (cleaning or cleansing) compositions comprising one or more enzymes, enzyme blends/compositions of the disclosure, and methods of making and using these compositions. The disclosure incorporates all known methods of making and using detergent, disinfectant or cleanser compositions. See, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561; 6,365,561; 6,380,147.

[0378] In specific embodiments, the detergent, disinfectant or cleanser compositions can be a one- and two-part aqueous composition, a non-aqueous liquid composition, a cast solid, a granular form, a particulate form, a compressed tablet, a gel and/or a paste and a slurry form. The enzymes, enzyme blends/compositions of the disclosure can also be used as a detergent, disinfectant, or cleanser additive product in a solid or a liquid form. Such additive products are intended to supplement or boost the performance of conventional detergent compositions, and can be added at any stage of the cleaning process.

[0379] The present disclosure provides cleaning compositions including detergent compositions for cleaning hard surfaces, detergent compositions for cleaning fabrics, dishwashing compositions, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning solutions.

[0380] When the enzymes of the disclosure are components of compositions suitable for use in a laundry machine washing method, the compositions can comprise, in addition to an enzyme, enzyme blend/composition of the disclosure, both a surfactant and a builder compound. They can additionally comprise one or more detergent components, e.g., organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents, and corrosion inhibitors.

[0381] Laundry compositions of the disclosure can also contain softening agents, as additional detergent components. Such compositions containing carbohydrase can provide fabric cleaning, stain removal, whiteness maintenance, softening, color appearance, dye transfer inhibition and sanitization when formulated as laundry detergent compositions.

[0382] The disclosure thus further provides a process of saccharification a biomass material comprising hemicellulose. Such a biomass material can optionally comprise cellulose. Exemplary biomass materials include, without limitation, corcob, switchgrass, sorghum, and/or bagasse. Accordingly the disclosure provides a process of saccharification, comprising treating a biomass material herein comprising hemicelluose and optionally cellose with an enzyme blend/composition as described herein. The enzyme blend/compositon used in such a process of the invention include 0.5 g to 40 g (e.g., 0.5 g to 20 g, 0.5 g to 30 g, 0.5 g to 40 g, 0.5 g to 15 g, 0.5 g to 10 g, 0.5 g to 5 g, 0.5 g to 7 g, etc) of polypeptides having xylanase activity per kg of hemicellulose in the biomass material. The enzyme blend/composition used in such a process of the invention can also include 1 g to 40 g (e.g., 2 g to 20 g, 3 g to 7 g, 1 g to 5 g, or 2 g to 5 g, etc.) of polypeptides having xylanase activity per kg of hemicellulose in the biomass material. The enzyme blend/composition used in such a process can include 0.5 g to 50 g (e.g., 0.5 g to 50 g, 0.5 g to 45 g, 0.5 g to 40 g, 0.5 g to 30 g, 0.5 g to 25 g, 0.5 g to 20 g, 0.5 g to 15 g, 0.5 g to 10 g, etc) of polypeptides having .beta.-xylosidase activity per kg of hemicellulose in the biomass material. The enzyme blend/composition used in such a process can also include 1 g to 50 g (e.g., 2 g to 40 g, 4 g to 20 g, 4 g to 10 g, 2 g to 10 g, 3 g to 7 g, etc.) of polypeptide having .beta.-xylosidase activity per kg of hemicellulose in the biomass material. The enzyme blend/compositon used in such a process of the invention can include 0.2 g to 20 g (e.g., 0.2 g to 18 g, 0.2 g to 15 g, 0.3 g to 10 g, 0.2 g to 8 g, 0.2 g to 5 g, etc) of polypeptides having L-.alpha.-arabinofuranosidase activity per kg of hemicellolose in the biomass material. The enzyme blend/composition used in such a process of the invention can include 0.5 g to 20 g (e.g., 1 g to 10 g, 1 g to 5 g, 2 g to 6 g, 0.5 g to 4 g, or 1 g to 3 g, etc) of polypeptides having L-.alpha.-arabinofuranosidase activity per kg of hemicellolose in the biomass material. The enzyme blend/composition can also include 1 g to 100 g (e.g., 1 g to 100 g, 2 g to 80 g, 3 g to 50 g, 5 g to 40 g, 2 g to 20 g, 10 g to 30 g, or 12 g to 18 g, etc) of polypeptides having cellulase activity per kg of cellulose in the biomass material. Optionally, the amount of polypeptides having .beta.-glucosidase activity can constitute up to 50% of the total weight of polypeptides having cellulase activity.

[0383] A suitable process of the invention preferably yields 60% to 90% xylose from the hemicellulose xylan of the biomass material treated. Suitable biomass materials include one or more of, for example, corncob, switchgrass, sorghum, and/or bagasse. As such, a process of the invention preferably yields at least 70% (e.g. at least 75%, at least 80%) xylose from hemicellulose xylan from one or more of these biomass materials. For example, the process yields 60% to 90% of xylose from hemicellulose xylan of a biomass material comprising hemicellulose, including, without limitation, corncob, switchgrass, sorghum, and/or bagasse.

[0384] The process of the invention optionally further comprises recovering monosaccharides.

[0385] The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.

7. EXAMPLE 1

Representative Experimental Methods

[0386] 7.1 Materials and Methods

[0387] The following assays/methods were used in Example 1 and subsequent examples. Any deviations from the protocols provided below are indicated.

[0388] 7.1.1 Preparation of Hemicellulose from Plant Tissues

[0389] Hemicellulose preparations were prepared using a modification of the NaOH/sonication procedure described by Erbringerova et al. (Carbohydrate Polymers 1998, 37:231). Dry plant material was ground to pass a 1 mm screen and 10 g of this material was suspended in 250 mL of 5% (wt/v) NaOH. The suspension was heated to 80.degree. C. without stirring for 30 min then sonicated for 15 min at ambient temperature using a probe sonicator at high setting. The suspension was returned to 80.degree. C. for an additional 30 min then allowed to cool to room temperature. Solids were removed from the suspension by centrifugation at 3000.times.g for 15 min and the resulting supernatant was decanted into 1 L of ethanol which was cooled on ice. After 30 min the resulting precipitate was recovered by first decanting the clear liquid above the precipitate then filtration without allowing the precipitate to fully air dry. The filter cake was first washed with 200 mL of cold, 80% ethanol then removed from the filter without allowing it to air dry. The filter cake was re-dissolved in 200 mL of water and the pH of the solution was adjusted to 5.5 with acetic acid. The extracted carbohydrate was re-precipitated by addition to 1 L of ethanol on ice and the resulting precipitate was again recovered by filtration as above. The filter cake was frozen and remaining solvent and water was removed by lyophylization. Yield of recovered carbohydrate ranged from 6 to 23% of the starting plant material depending on the tissue and the preparation.

[0390] 7.1.2 Dilute Ammonia Pretreatment of Biomass Substrates

[0391] Corncob and switchgrass were pretreated prior to enzymatic hydrolysis according to the methods and processing ranges described in WO06110901A (unless otherwise noted).

[0392] 7.1.3 Compositional Analysis of Biomass

[0393] The 2-step acid hydrolysis methods described in "Determination of structural carbohydrates and lignin in the biomass" (National Renewable Energy Laboratory, Golden, Colo. 2008, available at http://www.nrel.gov/biomass/pdfs/42618.pdf) were used to measure the composition of biomass substrates. Enzymatic hydrolysis results are reported in terms of percent conversion with respect to the theoretical yield from the starting glucan and xylan content of the substrate.

[0394] 7.1.4 Preparation of Crude Oligomers from Ammonia Pretreated Corncob

[0395] Crude oligomers for screening hemicellulases were prepared from corncob by the following procedure. Hammer-milled corncob (.about.1/4 in mean diameter) plus 6% ammonia (w/w) was heated to 145.degree. C. with direct injection of steam into a stirred pressure reactor. After 20 min excess ammonia was flashed out of the reactor at a final vacuum of .about.0.1 bar. The ammonia pretreated cob was then placed in a sterile stirred reactor for enzyme saccharification. Enough water was added to obtain a final total solids loading of 25% (w/w) after all additions are made. The pH of the reactor was maintained at pH 5.3 with 4 N sulfuric acid and the temperature controlled at 47.degree. C. Spezyme.RTM. CP, Multifect.RTM. Xylanase (Danisco US Inc., Genencor), and Novo 188 (Novozymes, Denmark) were added at loadings of 20, 10 and 5 mg/g of cellulose, respectively, and allowed to saccharify the pretreated cob to sugars and oligomers for 116 h. The material was then cooled to 33.degree. C. and the pH adjusted to 5.8 with 4 N NaOH. The glucose and xylose were then fermented to ethanol by adding a seed culture of a recombinant Zymomonas mobilis strain (10% total volume, ATCC accession no. PTA-1798) as described in U.S. Pat. No. 7,354,755. The fermentation progress was followed until all of the glucose and .about.95% of the xylose was consumed. A 0.5 L aliquot of the fermentation broth was clarified by centrifugation (21,000.times.g) for 20 min followed by filtration of the supernatant through a 0.2 micron filtering unit (Nalgene). The ethanol was removed from the filtered fermentation broth on a rotovap maintained at 35.degree. C. under house vacuum. The total volume of the final liquor was reduced by .about.4.times. by the latter procedure.

[0396] 7.1.5 Total Protein Assays

[0397] Different total protein determination methods were employed depending on the nature of the protein sample (i.e., purified, fermentation broth, commercial product, etc.). The BCA protein assay is an example of a colorimetric assay that measures protein concentration with a spectrophotometer.

[0398] Reagents:

[0399] BCA Protein Assay Kit (Pierce Chemical, Product #23227), 50 mM Sodium Acetate buffer pH 5.0, 15% trichloroacetic acid (TCA), 0.1 N NaOH, BSA stock solution, Reagent A, Reagent B (from protein assay kit)

[0400] Procedure:

[0401] Enzyme dilutions were prepared in test tubes using 50 mM Sodium Acetate buffer. Diluted enzyme solution (0.1 mL) was added to 2 mL Eppendorf centrifuge tubes containing 1 mL 15% TCA. The tubes were vortexed and placed in an ice bath for 10 min. The samples were then centrifuged at 14,000 rpm for 6 min. The supernatant was poured out, the pellet resuspended in 1 mL 0.1 N NaOH, and the tubes vortexed until the pellet dissolved. BSA standard solutions were prepared from a stock solution of 2 mg/mL. BCA working solution was prepared by mixing 0.5 mL Reagent B with 25 mL Reagent A. The resuspended protein (0.1 mL each) was added to 3 Eppendorf centrifuge tubes. Two mL Pierce BCA working solution was added to each of the sample and serially diluted BSA standard Eppendorf tubes. All tubes were incubated in a 37.degree. C. water bath for 30 min. The samples were then cooled to room temperature (15 min) and the absorbance measured at 562 nm in a spectrophotometer.

[0402] Calculations:

[0403] Average values for each BSA protein standard absorbance were calculated and plotted, absorbance on x-axis and concentration (mg/mL) on the y-axis. A linear curve fit was applied and the equation for the line calculated using the formula: y=mx+b

[0404] The raw concentration of the enzyme samples was calculated by substituting the absorbance for the x-value. The total protein concentration was calculated by multiplying with the dilution factor.

[0405] The total protein of purified samples was determined by A280 (see, e.g., Pace et al., Protein Science, 1995, 4:2411).

[0406] Some protein samples were measured using the Biuret method as modified by Weichselbaum and Gornall using Bovine Serum Albumin as a calibrator (modified Biuret) (Weichselbaum, Amer. J. Clin. Path. 1960, 16:40; Gornall et al., J. Biol. Chem. 1949, 177:752).

[0407] Total protein content of fermentation products was also sometimes measured as total nitrogen by combustion, capture and measurement of released nitrogen, either by Kjeldahl (rtech laboratories, www.rtechlabs.com) or in-house by the DUMAS method (TruSpec CN, www.leco.com) (SADER, et al. Archives of Veterinary Science, 9(2):73-79, 2004). For complex protein-containing samples, e.g. fermentation broths, an average 16% N content, and the conversion factor of 6.25 for nitrogen to protein was used. In some cases, total precipitable protein was measured to remove interfering non-protein nitrogen. A 12.5% final TCA concentration was used and the protein-containing TCA pellet was resuspended in 0.1 M NaOH.

[0408] In other cases, Coomassie Plus-the Better Bradford Assay (Thermo Scientific, Rockford, Ill. product #23238) was used according to manufacturer recommendation.

[0409] 7.1.6 Synthetic Substrate (Para-Nitrophenyl Substrate) Activity Assays

[0410] Active protein from T. reesei expression of cloned genes was confirmed with model substrate assays. Cellulase and hemicellulase activities on the synthetic substrates, such as 4-nitrophenyl .alpha.-L-arabinofuranoside (pNPA, Sigma N3641) and 4-nitrophenyl .beta.-D-glucopyranoside (pNPG, Sigma N7006), and 4-nitrophenyl .beta.-D-xylopyranoside (pNPX, Sigma N2132) were measured as follows: Substrate solution was prepared by dissolving 30 mg of synthetic substrate in 100 mL 50 mM Sodium Acetate buffer, pH 4.8. Sodium carbonate (1 M) was prepared for reaction quenching. Substrate solution (100 .mu.L) was dispensed into Costar 96 well plates (Cat no. 9017). 20 .mu.L of enzyme sample was dispensed into a microtiter plate well. The microtiter plate was incubated at 50.degree. C. for 10 min using a Thermomixer R heating and cooling shaker (Eppendorf). 50 .mu.L of 1 M sodium carbonate was added to each well to quench the reaction. Absorbance at 400 nm wavelength was read with SpectraMax 340C384 Microplate Spectrophotometer (Molecular Devices). Units per mL were determined by using a p-nitrophenol standard curve. A Quad-delete Trichoderma host, from which the cbh1, cbh2, egl1 and eg12 genes were deleted (see WO 05/001036), was analyzed with the enzyme samples as a control for the activity of enzymes expressed in this background.

[0411] 7.1.7 Cob Saccharification Assay

[0412] Typically, Corncob saccharification in a microtiter plate format was performed in accordance with the following procedures. The biomass substrate, dilute ammonia pretreated corncob, was diluted in water and pH-adjusted with sulfuric acid to create a pH 5, 7% cellulose slurry that was used directly in the assay. The enzymes tested included: commercial cellulase products, e.g. Accellerase.RTM. 1000, Accellerase.RTM. 1500 (Danisco US Inc., Genencor), T. reesei fermentation broths, and purified enzymes. The enzymes were loaded based on mg total protein per gram of cellulose (as determined by compositional analysis) in the corncob substrate. The enzymes were diluted in 50 mM Sodium Acetate pH 5.0 to obtain the desired loading concentration at the required volume. Forty microliters of enzyme solution was added to 70 mg of dilute-ammonia pretreated corncob at 7% cellulose per well (equivalent to 4.5% cellulose final). The assay plate was incubated at room temperature for 10 min. The assay plates were covered with aluminum plate sealers and the plates incubated at 50.degree. C., 200 rpm, for three days. At the end of the incubation period, the saccharification reaction was quenched by adding 100 .mu.L of 100 mM glycine buffer, pH10.0 per well and the plate was centrifuged for 5 min at 3,000 rpm. Ten microliters of the supernatant were added to 200 .mu.L of MilliQ water in a 96-well HPLC plate and the soluble sugars were measured by HPLC.

[0413] This describes a typical method that was used in multiple Examples herein. In certain Examples, corncob saccharification was measured using a modified protocol. The modifications are described with the individual examples.

[0414] 7.1.8 Sugar Analysis by HPLC

[0415] Typically, samples from cob saccharification hydrolysis were prepared by centrifugation to clear insoluble material, filtration through a 0.22 .mu.m nylon filter (Spin-X centrifuge tube filter, Corning Incorporated, Corning, N.Y.) and dilution to an appropriate concentration of soluble sugars with distilled water. Monomer sugars were determined on a Shodex Sugar SH-G SH1011, 8.times.300 mm with a 6.times.50 mm SH-1011P guard column (www.shodex.net). Solvent was 0.01 NH.sub.2SO.sub.4 run at 0.6 mL/min. Column temperature was 50.degree. C. and detection was made using a refractive index detector. External standards of glucose, xylose and arabinose were run with each sample set. Certain examples herein use a protocol to achieve the same end with a somewhat modified set of protocols. The specific modifications to the protocols are described with individual examples.

[0416] Oligomeric sugars were separated by size exclusion chromatography using a Tosoh Biosep G2000PW column 7.5 mm.times.60 cm (www.tosohbioscience.de). The solvent was distilled water at 0.6 mL/min and the column was run at room temperature. Six carbon sugar standards used for size calibration were: stachyose, raffinose, cellobiose and glucose; and 5 carbon sugars were: xylohexose, xylopentose, xylotetrose, xylotriose, xylobiose and xylose. Xylo-oligomers were obtained from Megazyme (www.megazyme.com). Detection was by refractive index and when reported quantitatively results are either as peak area units or relative peak areas by percent.

[0417] Total soluble sugars were determined by acid hydrolysis of the centrifuged and filter clarified samples described above. The clarified sample was diluted 1:1 with 0.8 N H.sub.2SO.sub.4 and the resulting solution was autoclaved in a capped vial for a total cycle time of 1 h at 121.degree. C. Results are reported without correction for loss of monomer sugar during the hydrolysis.

[0418] 7.1.9 Protein Analysis by HPLC

[0419] To separate and quantify the enzymes contained in broth from 14 L fermentations of the integrated expression strains, liquid chromatography (LC) and mass spectroscopy (MS) were performed. Enzyme samples were first treated with a recombinantly expressed endoH glycosidase from S. plicatus (e.g., NEB P0702L). EndoH was used at a ratio of 0.01-0.03 .mu.g endoH protein per .mu.g sample total protein and incubated for 3 h at 37.degree. C., pH 4.5-6.0 to enzymatically remove N-linked gycosylation prior to HPLC analysis. Approximately 50 .mu.g of protein was then injected for hydrophobic interaction chromatography using an Agilent 1100 HPLC system with an HIC-phenyl column and a high-to-low salt gradient over 35 min was performed on samples of concentrated fermentation broth. The gradient was achieved using high salt buffer A: 4 M ammonium sulphate containing 20 mM potassium phosphate pH 6.75 and low salt buffer B: 20 mM potassium phosphate pH 6.75. Peaks were detected with UV light at 222 nm and fractions were collected and identified by mass spectroscopy.

[0420] 7.1.10 Cellulase Activity Assay Using Calcofluor White

[0421] Cellulase activity was measured on PASC using a calcofluor white detection method (Appl. Biochem. Biotechnol. 161:313-317). All chemicals used were of analytical grade. Avicel PH-101 was purchased from FMC BioPolymer (Philadelphia, Pa.). Calcofluor white was purchased from Sigma (St. Louis, Mo.). Phosphoric acid swollen cellulose (PASC) was prepared from Avicel PH-101 using an adapted protocol of Walseth, TAPPI 1971, 35:228 and Wood, Biochem. J. 1971, 121:353-362. In short, Avicel was solubilized in concentrated phosphoric acid then precipitated using cold deionized water. After the cellulose was collected and washed with more water to neutralize the pH, it was diluted to 1% solids in 50 mM Sodium Acetate buffer, pH 5.0.

[0422] All enzyme dilutions were made into 50 mM Sodium Acetate buffer, pH 5.0. GC220 Cellulase (Danisco US Inc., Genencor) was diluted to 2.5, 5, 10, and 15 mg protein/g PASC, to produce a linear calibration curve. Samples to be tested were diluted to fall within the range of the calibration curve, i.e. to obtain a response of 0.1 to 0.4 fraction product. 150 .mu.L of cold 1% PASC was added to 20 .mu.L of enzyme solution in 96-well microtiter plates. The plate was covered and incubated for 2 h at 50.degree. C., 200 rpm in an Innova incubator/shaker. The reaction was quenched with 100 .mu.L of 50 .mu.g/mL Calcofluor in 100 mM Glycine, pH 10. Fluorescence was read on a fluorescence microplate reader (SpectraMax M5 by Molecular Devices) at excitation wavelength Ex=365 nm and emission wavelength Em=435 nm. The result (shown in FIG. 25) is expressed as the fraction product according to the equation:

FP=1-(FI sample-FI buffer)/(FI zero enzyme-FI buffer),

[0423] wherein FP is fraction product, and FI=fluorescence units.

[0424] 7.1.11 Cultivation of Fusarium verticillioides and Purification of Major Hemicellulase Activities Detected in the Extracellular Protein

[0425] Wild type Fusarium verticillioides source was as described in Table 1 of Fuchs et al., Fungal Genetics and Biology 2004, 41:852-863. The fungus was grown using destarched corn grain fiber using a modification of the method described in Li et al., Applied Biochemistry and Biotechnology 2005, (121-124):321-334.

[0426] Starch was removed from the dried corn pericarp fraction from a corn dry mill fractionation by slurrying 200 g of corn pericarp in 3 L of tap water and heating to 80.degree. C. with 5 mL (500 mg) of high temperature amylase Liquozyme SC DS (Novozymes, Denmark). The mixture was stirred occasionally with a spatula and held at 80.degree. C. for 30 min then allowed to cool to room temperature for about 2 h. The resulting slurry was decanted onto a 20 mesh screen to partially de-water. The solids on the screen were further washed with 4 L of tap water then air dried overnight before a final drying in an oven at 60.degree. C. The dried material was ground to pass a 1 mm screen in a knife mill before use.

[0427] The F. verticillioides culture was maintained on potato dextrose agar (Sigma P6685) and a rich growth media of 24 g/L potato dextrose was inoculated with mycelia from the plate. The growth culture was incubated for 3 days at 30.degree. C. with agitation at 130 rpm. After 3 days growth the 150 mL of the resulting cell mass and spent media were used to inoculate a corn pericarp induction media. The induction media was 80 g de-starched corn pericarp in 1 L base media (15 g KH.sub.2PO.sub.4, 5 g ammonium sulfate, 20 g yeast extract, 0.5 g magnesium sulfate and 1 g Tween 80 at pH4.8). The culture was maintained at 30.degree. C. with agitation at 130 rpm for 7 days.

[0428] Extracellular protein was separated from the spent grain and fungal cell mass by centrifugation at 2,000.times.G for 20 min. The partially clarified supernatant was passed first through a 0.45 .mu.m filter and then through a 0.22 .mu.m filter. The clarified filtrate was stored at 4.degree. C. until used. The filtrate was concentrated approximately 14-fold with Amicon ultra-4 centrifugal filters with a 30 kD cutoff (Millipore).

[0429] L-.alpha.-arabinofuranosidase (LARF) and .beta.-xylosidase (BXL) activities were purified from the fungal supernatant by first exchanging the proteins into 50 mM pH 6.3 (N-Morpholino) ethanesulphonic acid-NaOH (MES) buffer, then applying protein onto a GE Health Sciences Q-Sepharose Fast Flow quaternary amine anion exchange column (20 mL bed volume equilibrated with MES buffer). The protein purification system used was a GH Health Sciences Akta system using Unicorn software. Proteins were eluted with a 0-0.5 M NaCl gradient in MES buffer.

[0430] Activity in column fractions was monitored using either 4-nitrophenyl xylopyranoside (for BXL activity) or 4-nitrophenyl .alpha.-L-arabinofuranoside (for LARF activity). In both cases, 2 .mu.L of the column fraction was added to 95 .mu.L of 50 mM pH 5.0 Sodium Acetate buffer and 5 .mu.L of the p-nitrophenyl substrate, mixed and incubated 2 min at 23.degree. C. 100 .mu.L of 1 N sodium carbonate stop solution was added and absorbance at 405 nm was read.

[0431] Active enzyme fractions from the anion exchange system were collected, concentrated on Amicon Centriprep filtration, and applied to a GE Health Sciences SP-Sepharose Fast Flow sulfopropyl cation exchange column (20 mL bed equilibrated with Sodium Acetate buffer) using the same protein purification system described above. Proteins were eluted with a 0-1 M NaCl gradient in Sodium Acetate buffer, and activity in the fractions was monitored as described above. Active fractions were then collected, pooled and concentrated if necessary. Proteins in active fractions were identified by MS-MS.

[0432] 7.1.12 Protein Identification by MS-MS

[0433] The identification of a representative set of Fusarium verticillioides family GH54 L-.alpha.-arabinofuranosidase (LARF), family GH51 LARF, two family GH3 .beta.-xylosidases and a family GH30 .beta.-xylosidase (BXL) is described below.

[0434] For the identification of GH54 LARF, 200 .mu.L of 1 N HCl (EM Industries, Hawthorne, N.Y.) was added to 250 .mu.L of "Fraction B3" (0.35 mg protein) from the purification procedure described above and the sample iced for 10 min. Next, 200 .mu.L of 50% TCA (Sigma-Aldrich, St. Louis, Mo.) was added and samples were iced for another 10 min to precipitate protein. Samples were centrifuged for 2 min at 16,000 rcf and the supernatants were discarded. The pelleted protein was washed with 90% cold acetone (J. T. Baker, Phillipsburg, N.J.), then centrifuged for 1 min at 16,000 rcf. The supernatant was discarded and the pellet was left to air dry. First, 30 .mu.L of 8 M urea (MP Biomedicals, Solon, Ohio) was added to the pellet, then 4 .mu.L of 0.2 M DTT (Sigma-Aldrich, St. Louis, Mo.). Sample was incubated at 52.degree. C. for 30 min and then 4 .mu.L of 0.44 M iodoacetamide (Sigma-Aldrich, St. Louis, Mo.) was added and the solution incubated at room temperature in the dark for 30 min. Next, 120 .mu.L of 0.1% n-octyl B-D-glucopyranoside water (Sigma-Aldrich, St. Louis, Mo.) was added slowly, and then sample was divided into two equal parts. To half the sample, 7.5 .mu.g Trypsin (Promega Corp., Madison, Wis.) was added, and to the other half, 4 .mu.g of AspN (Roche Applied Science, Indianapolis, Ind.) was added. Both samples were incubated at 37.degree. C. for 1 h. Samples were quenched with 10% Trifluoroacetic Acid (Thermoscientific, Waltham, Mass.) for a final volume of 0.1% TFA. Both samples were run on a Thermofinnigan (San Jose, Calif.) LCQ-Deca electrospray ionization (ESI) ion-trap mass spectrometer. A Vydac C18 column (5 u, 300A, 0.2.times.150 mm, Michrom Bioresources, Auburn, Calif.) was run at a flow rate of 200 .mu.L/min. The injection volumes were 50 .mu.L, and were filtered through an on-line trapping cartridge (Peptide CapTrap, Michrom Bioresources, Auburn, Calif.) before loading onto the column. Separation of the digests was performed with a gradient as follows (Solvent A: 0.1% trifluoroacetic acid in H.sub.2O (J. T. Baker, Phillipsburg, N.J.), Solvent B: 0.08% trifluoroacetic acid in acetonitrile (J. T. Baker, Phillipsburg, N.J.)). For the identification of GH51 LARF, the two GH3 .beta.-xylosidases, and GH30 .beta.-xylosidase, the method described above was used, except samples were digested with Chymotrypsin (7.5 .mu.g, Sigma-Aldrich, St. Louis, Mo.) in addition to Trypsin and AspN. The TurboSequest search engine within Bioworks 3.31 (Thermo, San Jose) was used to identify proteins from the Fusarium verticillioides protein database. The protein database was downloaded from the BROAD Institute at http://www.broadinstitute.org/annotation/genome/fusarium_verticillioides/- MultiDownloads.html.

[0435] 7.1.13 Induction of Fusarium verticillioides Enzymes by Dilute Aqueous Ammonia Pretreated Switchgrass

[0436] For induction of Fusarium verticillioides enzymes in response to dilute aqueous ammonia pretreated switchgrass, switchgrass was ground to <1 mm diameter, then pretreated with 6% ammonia based on dry weight at 50% initial dry matter at 160.degree. C. maximum temperature for 90 min. The resulting material was 43.3% solids and contained 0.336% residual ammonia.

[0437] Wild type F. verticillioides culture was maintained on potato dextrose agar (Sigma P6685) and a rich growth media of 24 g/L potato dextrose was inoculated with mycelia from the plate. The growth culture was incubated for 3 days at 24.degree. C. with agitation at 140 rpm, during which time it became turbid with Fusarium cells. After 3 days growth, 4 flasks each containing 100 mL of base Christakapoulos media (0.1% KH.sub.2PO.sub.4, 0.03% CaCl.sub.2, 0.03% MgSO.sub.4.times.7H.sub.2O, 2.61% Na.sub.2HPO.sub.4.times.7H.sub.2O, 0.134% NaH.sub.2PO.sub.4.times.1H.sub.2O, 1.0% anhydrous ammonium phosphate) were inoculated with 4 mL of the resulting cell suspension. To the suspension, two grams dry matter of dilute aqueous ammonia-pretreated switchgrass was added as the sole carbon source.

[0438] After addition of the pretreated switchgrass, the pH was adjusted to 6.5 and the flask was swirled at 180 rpm at 23.degree. C. for 168 h. Prior to size exclusion chromatography, samples were analyzed at different time points by Bradford assay for protein, p-nitrophenyl arabinofuranosidase, p-nitrophenyl xylosidase and p-nitrophenyl glucosidase activities, and by SDS-PAGE gels for induction of enzymes. Intact non-pretreated switchgrass, 2% glucose and no carbon source were included as parallel controls and were found to lead to much lower levels of enzyme induction than was the case for dilute aqueous ammonia-pretreated switchgrass carbon source.

[0439] 7.1.14 Size Exclusion Chromatography Fractionation of the Fusarium verticillioides Culture Broth

[0440] The Fusarium verticillioides culture media containing the expressed enzymes from the 168-h induction described above was centrifuged at 3,500.times.g to remove debris and cells. The supernatant was filtered through a 0.4 .mu.m filter yielding a clear deep yellow liquid. The liquid was concentrated (.about.4.times.) two times in a large Amicon ultra 10K MW cutoff concentrator (UFC901024). A 1.7 mL concentrated sample was set aside for size exclusion chromatography (SEC) and assayed for protein content by BCA assay as described earlier. The concentrated protein sample was loaded onto a Superdex 16/60 SEC column equilibrated with 50 mM Sodium Acetate buffer pH 5.0. The column was eluted at 1 mL/min at 4.degree. C. Absorbance of the sample at 214 nm and 280 nm were monitored. After elution of the void volume, 1 mL fractions were collected in a deep well polypropylene microtiter plate. The protein-containing fractions were aggregated into a separate deep well plate and all fractions were measured for total protein concentration by BCA assay, and p-nitrophenyl arabinosidase, p-nitrophenyl xylosidase, and p-nitrophenyl glucosidase activities as described in Example 1 (Synthetic substrate activity assays).

[0441] 7.1.15 Purification of a NUMBER OF GH43, GH51 and GH3 Homologs (Section 8.1 Below) from T. Reesei Fermentation by Cation Exchange Chromatography

[0442] Shake flask-scale enzyme production from wild type Trichoderma reesei cultures was performed as described in WO 2005/001036. The extracellular enzyme preparations were concentrated by Amicon Centriprep filtration if necessary, and applied to a GE Health Sciences SP-Sepharose Fast Flow sulfopropyl cation exchange column (20 mL bed equilibrated with Sodium Acetate buffer) using a GE Health Sciences Akta system protein purification system using Unicorn software. Proteins were eluted with a 0-1 M NaCl gradient in Sodium Acetate buffer and activity in the fractions were monitored as described above. Active fractions were then collected, pooled and concentrated if necessary. Activity in column fractions was monitored using either 4-nitrophenyl .beta.-D-xylopyranoside (for BXL activity) or 4-nitrophenyl .alpha.-L-arabinofuranoside (for LARF activity). In both cases, 2 .mu.L of the column fraction was added to 95 .mu.L of 50 mM pH 5.0 Sodium Acetate buffer and 5 .mu.L of the p-nitrophenyl substrate, mixed and incubated 2 min at 23.degree. C. One hundred microliters of 1 N sodium carbonate stop solution was added and the absorbance was measured at 405 nm. In some cases an anion exchange protein purification step was useful before the cation exchange. In this case, the enzyme fractions were exchanged into 50 mM pH 6.3 (N-Morpholino) ethanesulphonic acid-NaOH (MES) buffer, and then the protein sample was applied onto a GE Health Sciences Q-Sepharose Fast Flow quaternary amine anion exchange column (20 mL bed volume equilibrated with MES buffer). The Akta protein purification system as described for the cation exchange step was used. Proteins were eluted with a 0-0.5 M NaCl gradient in MES buffer. The active enzymes were then exchanged into 50 mM, pH 5.0 Sodium Acetate buffer before cation exchange chromatography.

[0443] 7.1.16 Purification of Xylanases

[0444] Xylanase purification was conducted in two stages:

[0445] Stage 1:

[0446] Hydrophobic interaction chromatography (HIC). (NH.sub.4).sub.2SO.sub.4 was added to fermentation supernatant to achieve a final concentration of 1 M. The separation column used was a Hiprep Phenyl (highsub), 16/10, 20 mL. Buffer A: 20 mM sodium phosphate, pH 6.0. Buffer B: Buffer A+1 M (NH.sub.4).sub.2SO.sub.4. Stepwise Elution: 45% B; 0% B; water+10% glycerol.

[0447] Stage 2:

[0448] Gel filtration (GF). Eluate of 0% B from the HIC column was collected and loaded on a HiLoad 26/60 Superdex 75 prep grade (320 mL, GE Healthcare) column. The mobile phase was 20 mM sodium phosphate, pH 6.8, containing 0.15 M NaCl. The elution profile is shown in FIG. 26. FIG. 27 shows the SDS-PAGE detection of the two step separation of AfuXyn 5.

[0449] The Applied Biosystems BIOCAD.RTM. Vision and Amersham Pharmacia Biotech AKTA Explorer were used for protein purification of T. reesei XYN3. Approximately 150 mL of Xylanase 3, from an ultrafiltration concentrate, was loaded onto a Sephadex G-25M desalting column (total volume .about.525 mL) equilibrated in 10 mM TES buffer, pH 6.8. 300-400 mL of this desalted sample was then loaded onto an anion-exchange column (high density quaternary amine resin; Applied Biosystems Inc.). The bound protein was then eluted using a salt gradient between 0-1 M sodium chloride using 8-column volumes of 25 mM TES buffer. Elution of the protein occurred between 0-250 mM sodium chloride, and was detected using 10% Bis-Tris NUPAGE.RTM. SDS-PAGE (Novex).

[0450] The eluted Xylanase 3-containing samples were concentrated to 10 mL with Vivaspin 5,000 MWCO (molecular weight cut-off) membrane concentrators (Vivascience; GE Healthcare). The concentrate was then loaded onto a High Load 26/60 Superdex 200 (ID no 17-1071-01; GE Healthcare). The column was equilibrated with 25 mM TES buffer, pH 6.8, containing 100 mM sodium chloride, and the bound protein was eluted over 8-column volumes of the TES buffer with sodium chloride.

[0451] The purity of the eluted protein was assessed using 10% Bis-Tris NUPAGE.RTM. SDS-PAGE (Novex), and determined to be greater than 95% pure.

[0452] 7.1.17 Purification of Fv43D

[0453] The ultrafiltration concentrate (UFC) of Fusarium verticillioides 43D was buffer exchanged and dialyzed against 50 mM Sodium Acetate buffer, pH 4.0, overnight. The dialyzed material was passed through a HiTrap 1 mL column prepacked with sulfopropyl sepharose fast flow resin (GE Healthcare) designed for use with a syringe. The purified Fv43D was eluted with 50 mM Sodium Acetate buffer, pH 4.0, with 250 mM sodium chloride. The UFC and Sodium Acetate buffer were pushed through the column using a 5 mL syringe fitted with a GE Healthcare connector. The purified Fv43D was dialyzed overnight against 50 mM Sodium Acetate buffer, pH 4.0. The purified protein was assayed by SDS-PAGE, HPLC, and mass spectroscopy to demonstrate homogeneity.

[0454] 7.1.18 Purification of Fv51A

[0455] The ultrafiltration concentrate (UFC) of Fusarium verticillioides 51A was buffer exchanged and dialyzed against 50 mM Sodium Acetate buffer, pH 5.0, overnight. The dialyzed material was passed through a RESOURCE 15 6 mL column prepacked with methyl sulfonate media (GE Healthcare). The UFC was loaded at 1 mL/min against 50 mM Sodium Acetate buffer, pH 5.0, and eluted at 5 mL/min against 50 mM Sodium Acetate buffer, pH 5.0, using a 0 to 250 mM sodium chloride gradient. The eluted fractions were collected and assayed by SDS-PAGE. Fractions with purified Fv51A were concentrated using a 10,000 MWCO Vivaspin concentrator from Sartorius Stemdim Biotech. The purified Fv51A was dialyzed against 50 mM Sodium Acetate buffer, pH 5.0, overnight. The purified protein was assayed by SDS-PAGE, HPLC, and mass spectroscopy to demonstrate homogeneity. The AKTA Explorer 100 system from GE Healthcare was used for the purification of Fv51A.

8. EXAMPLE 2

Expression of Individual Hemicellulase Genes from Various Species in Trichoderma reesei

[0456] 8.1 Fusarium verticillioides Genes

[0457] The sequence for Fv51A was obtained by searching the Fusarium verticillioides genome in the Broad Institute database (http://www.broadinstitute.org/) for GH51 arabinofuranosidase homologs.

[0458] The following genes from Fusarium verticillioides were expressed in Trichoderma reesei: Fv3A, Fv43A, Fv43B, Fv43D, Fv51A, Fv3B, Fv43C, Fv39A, Fv43E, Fv30A, Fv30B, and Fv43F. Fv3A sequence was obtained by searching for GH3 .beta.-xylosidase homologs in Fusarium verticillioides genome. The annotated sequence lacked a signal sequence and the gene prediction program Augustus (http://augustus.gobics.de/) was used to identify upstream sequence which contained a signal sequence. Sequences for Fv39A, Fv43A, Fv43B, Fv43D, Fv43E, and Fv30A were obtained by searching the Fusarium verticillioides genome for GH39, GH30, and GH43 .beta.-xylosidase homologs.

[0459] Open reading frames of the hemicellulase genes of interest were amplified by PCR using purified/extracted genomic DNA from Fusarium verticillioides as the template. The PCR thermocycler used was DNA Engine Tetrad 2 Peltier Thermal Cycler (BioRad Laboratories). The DNA polymerase used was PfuUltra II Fusion HS DNA Polymerase (Stratagene). The primers used to amplify the open reading frames were as follows:

TABLE-US-00001 Fv3A: Forward primer MH124 (SEQ ID NO: 52) (5'-CACCCATGCTGCTCAATCTTCAG-3') Reverse primer MH125 (SEQ ID NO: 53) (5'-TTACGCAGACTTGGGGTCTTGAG-3') Fv43A: Forward primer MH075 (SEQ ID NO: 54) (5'-CACCATGTGGCTGACCTCCCCATT-3') Reverse primer MH076 (SEQ ID NO: 55) (5'-TTAGCTAAACTGCCACCAGTTGAAGTTG-3') Fv43B: Forward primer MH077 (SEQ ID NO: 56) (5'-CACCATGCGCTTCTCTTGGCTATTGT-3') Reverse primer MH078 (SEQ ID NO: 57) (5'-CTACAATTCTGATTTCACAAAAACACC-3') Fv43D: Forward primer MH081 (SEQ ID NO: 58) (5'-CACCATGCAGCTCAAGTTTCTG-3') Reverse primer MH082 (SEQ ID NO: 59) (5'-CTAAATCTTAGGACGAGTAAGC-3') Fv51A: Forward primer SK1159 (SEQ ID NO: 60) (5'-CACCATGGTTCGCTTCAGTTCAATCCTAG-3') Reverse primer SK1160 (SEQ ID NO: 61) (5'-CTAGCTAGAGTAAGGCTTTCC-3') Fv39A: Forward: MH116 (SEQ ID NO: 62) (5'-CACCATGCACTACGCTACCCTCACCAC-3') Reverse: MH117 (SEQ ID NO: 63) (5'-TCAAGTAGAGGGGCTGCTCACC-3') Fv3B: Forward primer MH126 (SEQ ID NO: 64) (5'-CAC CAT GAA ACT CTC TAG CTA CCT CTG-3') Reverse primer MH127 (SEQ ID NO: 65) (5'-CTA CGA AAC TGT GAC AGT CAC GTT G-3') Fv30A: Forward primer MH112 (SEQ ID NO: 66) (5'-CAC CAT GCT CTT CTC GCT CGT TCT TCC TAC-3') Reverse primer MH113 (SEQ ID NO: 67) (5'-TTA GTT GGT GCA GTG GCC ACG-3') Fv30B: Forward primer MH114 (SEQ ID NO: 68) (5'-CAC CAT GAA TCC TTT ATC TCT CGG CCT TG-3') Reverse primer MH115 (SEQ ID NO: 69) (5'-CAG CCC TCA TAG TCG TCT TCT TC-3') Fv43C: Forward primer MH079 (SEQ ID NO: 70) (5'-CAC CAT GCG TCT TCT ATC GTT TCC-3') Reverse primer MH080 (SEQ ID NO: 71) (5'-CTA CAA AGG CCT AGG ATC AA-3') Fv39A: Forward primer MH116 (SEQ ID NO: 72) (5'-CAC CAT GCA CTA CGC TAC CCT CAC CAC-3') Reverse primer MH117 (SEQ ID NO: 73) (5'-TCA AGT AGA GGG GCT GCT CAC C-3') Fv43E: Forward primer MH147 (SEQ ID NO: 74) (5'-CAC CAT GAA GGT ATA CTG GCT CGT GG-3') Reverse primer MH148 (SEQ ID NO: 75) (5'-CTA TGC AGC TGT GAA AGA CTC AAC C-3') Fv43F: Forward primer MH149 (SEQ ID NO: 76) (5'-CACCATGTGGAAACTCCTCGTCAGC-3') Reverse primer MH150 (SEQ ID NO: 77) (5'-CTA ATA AGC AAC AGG CCA GCC ATT G-3')

[0460] The forward primers included four additional nucleotides (sequences--CACC) at the 5'-end to facilitate directional cloning into pENTR/D-TOPO (Invitrogen, Carlsbad, Calif.) (FIG. 28). The PCR conditions for amplifying the open reading frames were as follows (except for Fv51A): Step 1: 94.degree. C. for 2 min. Step 2: 94.degree. C. for 30 sec. Step 3: 57.degree. C. for 30 sec. Step 4: 72.degree. C. for 30-45 sec. Steps 2, 3 and 4 were repeated for an additional 29 cycles. Step 5: 72.degree. C. for 2 min. For Fv51A, the following conditions were used: Step 1: 94.degree. C. for 2 min. Step 2: 94.degree. C. for 30 sec. Step 3: 56.degree. C. for 30 sec. Step 4: 72.degree. C. for 45 sec. Steps 2, 3, 4 were repeated for an additional 25 cycles. Step 5: 4.degree. C. hold.

[0461] The PCR products of the corresponding hemicellulase open reading frames were purified using a Qiaquick PCR Purification Kit (Qiagen, Valencia, Calif.). The purified PCR products were cloned into the pENTR/D-TOPO vector, transformed into TOP10 chemically competent E. coli cells (Invitrogen, Carlsbad, Calif.) and plated on LA plates with 50 ppm kanamycin. Plasmid DNA was obtained from the E. coli transformants using a QIAspin plasmid preparation kit (Qiagen). Sequence data for the DNA inserted in the pENTR/D-TOPO vector was obtained using M13 forward and reverse primers (Sequetech, Mountain View, Calif.). A pENTR/D-TOPO vector with the correct DNA sequence of the corresponding hemicellulase open reading frame was recombined with the pTrex3gM destination vector (WO 05/001036, FIG. 29) using LR clonase reaction mixture (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. The product of the LR clonase reaction was subsequently transformed into TOP10 chemically competent E. coli cells which were then plated on LA containing 50 ppm carbenicillin. The resulting expression vectors were pTrex3gM plasmids containing the corresponding hemicellulase open reading frames that resulted from the recombination event between the attR1 and attR2 sites of pTrex3gM and the attL1 and attL2 sites of pENTR/D-TOPO; and the Aspergillus nidulans acetamidase selection marker (amdS). DNA of the expression vectors containing the corresponding hemicellulase open reading frames were isolated using a Qiagen miniprep kit and used for biolistic transformation of Trichoderma reesei spores.

[0462] Biolistic transformation of Trichoderma reesei with the pTrex3gM expression vector containing the corresponding hemicellulase open reading frame was performed using the following protocol. Transformation of the Trichoderma reesei cellulase quad delete (.DELTA.cbh1, .DELTA.cbh2, .DELTA.eg1, .DELTA.eg2) strain by helium-bombardment was accomplished using a Biolistic.RTM. PDS-1000/He Particle Delivery System from Bio-Rad (Hercules, Calif.) following the manufacturer's instructions (see patent publications WO 05/001036 and US 2006/0003408). Transformants were transferred to fresh acetamide selection plates (see patent publication WO 2009114380). Stable transformants were inoculated into filter microtiter plates (Corning), containing 200 .mu.L/well of Glycine Minimal media (6.0 g/L glycine; 4.7 g/L (NH.sub.4).sub.2SO.sub.4; 5.0 g/L KH.sub.2PO.sub.4; 1.0 g/L MgSO.sub.4.7H.sub.2O; 33.0 g/L PIPPS; pH 5.5) with post sterile addition of .about.2% glucose/sophorose mixture (U.S. Pat. No. 7,713,725) as the carbon source, 10 mL/L of 100 g/L of CaCl.sub.2, 2.5 mL/L of T. reesei trace elements (400.times.): 175 g/L Citric acid anhydrous; 200 g/L FeSO.sub.4.7H.sub.2O; 16 g/L ZnSO.sub.4.7H.sub.2O; 3.2 g/L CuSO.sub.4.5H.sub.2O; 1.4 g/L MnSO.sub.4.H.sub.2O; 0.8 g/L H.sub.3Bo.sub.3). Transformants were grown in liquid culture for 5 days in an O.sub.2-rich chamber housed in a 28.degree. C. incubator. The supernatant samples from the filter microtiter plate were collected on a vacuum manifold. Supernatant samples were run on 4-12% NuPAGE gels (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions to check for expression. The gel was stained with Simply Blue stain (Invitrogen, Carlsbad, Calif.). Purification of GH43, GH51 and GH3 enzymes from T. reesei fermentations was performed by cation exchange chromatography as described in Example 1.

[0463] 8.2 Genes from Other Species

[0464] Pf43A and Pf51A sequences were obtained by sequencing of the Penicillium funiculosum genome. The queries used for the searches of the P. funiculosum genome were other fungal GH43 and GH51 homologs available in the public genomes. A gene prediction program named Augustus (http://augustus.gobics.de/) was used to verify the intron sequences, along with the start codon. Fv43D sequence was used to query the Gibberella zeae (Fusarium graminearum) and Fusarium oxysporum genomes available in the Broad Institute database and retrieve sequences for Gz43A and Fo43A respectively. The genes for Gz43A and Fo43A were synthesized by GeneArt (Geneart GmbH, Regensburg, Germany) with the CBH1 signal sequence in place of the native signal sequence. Neither gene contained introns. The Pf51A gene was codon-optimized and synthesized by GeneArt with the CBH1 signal sequence in place of the native signal sequence.

[0465] Pf43A and Pf51A were cloned and expressed in Trichoderma reesei using the following primers:

TABLE-US-00002 Pf43A: MH151 (SEQ ID NO: 78) (5'-CACCATGCTTCAGCGATTTGCTTATATTTTACC-3') Pf43A: MH152 (SEQ ID NO: 79) (5'-TTATGCGAACTGCCAATAATCAAAGTTG-3') Pf51A: SK1168 (SEQ ID NO: 80) (5'-CACCATGTACCGGAAGCTCGCCGTG-3') Pf51A: SK1169: (SEQ ID NO: 81) (5'-CTACTCCGTCTTCAGCACAGCCAC-3')

[0466] Three genes were cloned from Aspergillus fumigatus for expression in Trichoderma reesei: GH11 xylanase 2 (AfuXyn2), GH11 xylanase 5 (AfuXyn5), and GH43 Af43A. The primers for AfuXyn2, AfuXyn5, and Af43A cloning primers are shown below:

TABLE-US-00003 AfuXyn2: A.fumi-Q4WG11-F: (SEQ ID NO: 82) (5'-CCGCGGCCGCACCATGGTTTCTTTCTCCTACCTGCTGCTG-3') A.fumi-Q4WG11-R: (SEQ ID NO: 83) (5'-CCGGCGCGCCCTTACTAGTAGACAGTGATGGAAGCAGATCCG-3') AfuXyn5: A.fumi-Q4WFZ8-F: (SEQ ID NO: 84) (5'-CCGCGGCCGCACCATGATCTCCATTTCCTCGCTCAGCT-3') A.fumi-Q4WFZ8-R: (SEQ ID NO: 85) (5'-CCGGCGCGCCCTTATCACTTGGATATAACCCTGCAAGAAGGTA-3') Af43A: SK1203: (SEQ ID NO: 86) (5'-CACCATGGCAGCTCCAAGTTTATCC-3') SK1204- (SEQ ID NO: 87) (5' TCAGTAGCTCGGGACCACTC-3')

[0467] The methods used for cloning and expression of all these genes were similar to the procedure described for cloning of the Fusarium genes. Additional genes including those listed in Table 1B, were cloned in a similar manner.

9. EXAMPLE 3

Testing for Activity of Novel Hemicellulases on Synthetic Substrates

[0468] The activities of Fv3A, Fv43A, Fv43B, Fv43D and a number of other proteins in, for example, Table 2, were tested on synthetic substrates pNPX and pNPA as described in Synthetic substrate activity assay in Example 1. T. reesei Bxl1 was used in at 0.7 g/L. The other enzyme samples, and the Quad-delete host control, were added by volume from growth cultures (microtiter plate or shake flask scale). Therefore, the absolute activity cannot be compared across samples but is an indication of an active expressed protein with the relative pNPX and pNPA activity shown in Table 2. Activity on para-nitrophenyl substrates is not used as a predictor of performance in biomass saccharification.

10. EXAMPLE 4

Hydrolysis of Pretreated Corncob by Cellulase and Hemicellulase Preparations

[0469] 10.1 Saccharification Performance of Expressed Proteins

[0470] The saccharification performance of expressed proteins as additions to an enzyme mixture with an L-.alpha.-arabinofuranosidase deficiency was evaluated. L-.alpha.-arabinofuranosidase candidates were evaluated in a 4-day cob saccharification assay by addition to an enzyme mixture of Accellerase.RTM. 1500/T. reesei Xyn3/Fv3A. The screen was conducted as described in the corncob Saccharification Assay (Example 1) with the following enzymes and amounts/concentrations: [0471] Accellerase.RTM. 1500, TP (Total Nitrogen) 54.2 mg/mL [0472] Trichoderma reesei Xyn3, 2.9 mg/mL TP (purified) [0473] Fv3A, 3.2 mg/mL TP (purified) [0474] Fv51A, 7.8 mg/mL TP (purified) [0475] Mg51A, 6.8 mg/mL TP (TCA/BCA) [0476] At51A, 6.7 mg/mL TP (TCA/BCA) [0477] Pt51A, 3.3 mg/mL TP (TCA/BCA) [0478] Ss51A, 3.0 mg/mL TP (TCA/BCA) [0479] Vd51A, 6.8 mg/mL TP (TCA/BCA) [0480] Cg51B, 3.6 mg/mL TP (TCA/BCA) [0481] Af43A, 2.6 mg/mL TP (TCA/BCA) [0482] Pf43A, 2 mg/mL TP (TCA/BCA) [0483] Fv43E, 1.37 mg/mL TP (TCA/BCA)

[0484] The total protein (TP) of purified samples was determined by A280 unless otherwise noted. The total protein of unpurified samples was determined by BCA according to manufacturer instructions, unless otherwise noted. Accellerase.RTM. 1500 was added at 20 mg protein/g cellulose; Trichoderma reesei Xyn3 was added at 5 mg protein/g cellulose; Fv3A was added at 5 mg protein/g cellulose. Fv51A, Mg51A, At51A, Pt51A, Ss51A, Vd51A, Cg51B, Af43A, Pf43A, or Fv43E were added at 1, 3, and 5 mg protein/g cellulose. Following 4 days incubation, 50.degree. C., 200 rpm, the assay plate was quenched and analyzed by HPLC for soluble sugars.

[0485] Enzymes were found that enhanced glucose or xylose or arabinose yield, or reduced cellobiose or xylobiose concentration in this enzyme mixture. Results are shown in FIGS. 30A and 30B.

[0486] The saccharification performance of expressed proteins was also evaluated as additions to the enzyme mixture with an L-.alpha.-arabinofuranosidase and .beta.-xylosidase deficiency. .beta.-xylosidase candidates were evaluated in a 3 day cob saccharification assay by addition to an enzyme mixture of Accellerase.RTM. 1500/T. reesei Xyn3. The screen was conducted as described in the corncoob saccharification assay (Example 1) with the following enzymes and amounts/concentrations: [0487] Accellerase.RTM. 1500, TP (total nitrogen) 54.2 mg/mL [0488] Trichoderma reesei Xyn3, 2.9 mg/mL (purified) [0489] Fv3A, 3.2 mg/mL (purified) [0490] Fv43D, 6.8 mg/mL (purified) [0491] Pf43A, 2 mg/mL TP (BCA) [0492] Pf43B, 2.7 mg/mL TP (BCA) [0493] Fv43E, 1.37 mg/mL TP (BCA) [0494] Fv43F, 2.8 mg/mL TP (BCA) [0495] Fv30A, 2.7 mg/mL TP (BCA)

[0496] Accellerase.RTM. 1500 was added at 17.9 mg protein/g cellulose; T. reesei Xyn3 was added at 5 mg protein/g cellulose. Fv3A, Fv43D, Pf43A, Pf43B, Fv43E, Fv43F, or Fv30A were added at 1, 3, and 5 mg protein/g cellulose (Fv43E was only added at 1 and 3 mg/g). Following 3 days incubation, 50.degree. C., 200 rpm, the assay plate was quenched and analyzed by HPLC for soluble sugars. Results are shown in FIG. 31. Enzymes were found that enhanced glucose, or xylose or arabinose yield, or reduced cellobiose or xylobiose concentration in this enzyme mixture.

[0497] The saccharification performance of expressed proteins was also evaluated as additions to the enzyme mix with a xylanase deficiency. During the construction of the Trichoderma reesei integrated expression strains (described in Example 9 below), one T. reesei strain (strain #44) was isolated that over-expressed Bgl1, Fv3A, Fv51A, Fv43D proteins but did not over-express endo-xylanase. This strain was used as the background to which candidate xylanases were added for performance screening. Endo-xylanase candidates were evaluated in a 3 day cob saccharification assay by addition to the enzyme products from strain #44. The screen was conducted as described in the corncob saccharification assay (Example 1) with the following enzymes and amounts/concentrations: [0498] Strain #44 enzyme product 78.6 mg/mL TP (modified Biuret) [0499] Trichoderma reesei Xyn3 2.9 mg/mL TP (purified) [0500] AfuXyn2 3.3 mg/mL TP (purified) [0501] AfuXyn3 5.8 mg/mL TP (purified) [0502] AfuXyn5 14.8 mg/mL TP (purified) [0503] PfuXyn1 1.9 mg/mL TP (purified) [0504] SspXyn1 1.2 mg/mL TP (purified)

[0505] The enzyme composition produced by Strain #44 was added at 20 mg protein/g cellulose; candidate xylanase enzymes were added at 3 and 7 mg protein/g cellulose. Following 3 days incubation, at 50.degree. C., 200 rpm, the assay plate was quenched and analyzed by HPLC for soluble sugars. Enzymes were found that enhanced xylose, or glucose, or arabinose yield, or reduced cellobiose or xylobiose concentration in this enzyme mixture. Results are shown in FIG. 32.

[0506] The saccharification performance of expressed Fv51A and Pa51A proteins in enzyme mixtures with L-.alpha.-arabinofuranosidase deficiency was evaluated and compared. Fv51A and Pa51A were evaluated in a 3 day cob saccharification assay by addition to an enzyme mixture of Accellerase.RTM. 1000/T. reesei Xyn2/Bxl1 or Fv3A. The screen was conducted as described in the corncob saccharification assay (Example 1) with the following enzymes and amounts/concentrations: [0507] Accellerase.RTM. 1000, 60.6 mg/mL TP (total nitrogen) [0508] Trichoderma reesei Xyn2 4.1 mg/mL TP (purified) [0509] Trichoderma reesei Bxl1 69 mg/mL TP (TCA/total nitrogen) [0510] Fv3A 65 mg/mL TP (TCA/total nitrogen) [0511] Fv51A 43 mg/mL TP (TCA/BCA) [0512] Pa51A 85.4 mg/mL TP (TCA/total nitrogen)

[0513] Accellerase.RTM. 1000 was added at 20 mg protein/g cellulose; Trichoderma reesei Xyn2 was added at 5 mg protein/g cellulose; Trichoderma reesei Bxl1 or Fv3A was added at 5 mg protein/g cellulose. Fv51A was added at 5 mg protein/g cellulose. Pa51A was added at 1, 2, or 5 mg protein/g cellulose. Following 3 days incubation, at 50.degree. C., 200 rpm, the assay plate was quenched and analyzed by HPLC for soluble sugars. Enzyme combinations were found that enhanced xylose, or glucose, or arabinose yield, or reduced cellobiose or xylobiose concentration in this enzyme mixture. Results are shown in FIG. 33.

[0514] Fv51A and Pa51A also were evaluated in a 3 day cob saccharification assay by addition to an enzyme mixture of Accellerase.RTM. 1000/Trichoderma reesei Xyn2. Purified Fv51A (29 mg/mL TP) and Pa51A (29 mg/mL) were used in this part of the study. Accellerase.RTM. 1000 was added at 17.5 mg protein/g cellulose; Trichoderma reesei Xyn2 was added at 4.4 mg protein/g cellulose. Fv51A was added at 4.4 mg protein/g cellulose. Pa51A was added at 0.9, 1.8, and 4.4 mg protein/g cellulose. Following 3 days incubation, at 50.degree. C., 200 rpm, the assay plate was quenched and analyzed by HPLC for soluble sugars. Enzyme combinations were found that enhanced xylose, or glucose, or arabinose yield in this enzyme mixture. Results are shown in FIG. 34.

[0515] The saccharification performance of expressed proteins was also evaluated as additions to the enzyme mix with .beta.-xylosidase deficiency. .beta.-xylosidase candidates were evaluated in a 3 day cob saccharification assay by addition to an enzyme mixture of Accellerase.RTM. 1000/Trichoderma reesei Xyn2/Fv51A. The screen was conducted as described in the corncob saccharification assay (Example 1) with the following enzymes and amounts/concentrations: [0516] Accellerase.RTM. 1000, TP (TCA/Total nitrogen) 60.6 mg/mL [0517] Trichoderma reesei Xyn2, 4.1 mg/mL TP (purified) [0518] Fv3A, 65 mg/mL TP (TCA/Total nitrogen) [0519] Fv3B, 62.9 mg/mL TP (TCA/Total nitrogen) [0520] Fv39A, 47.5 mg/mL TP (TCA/Total nitrogen) [0521] Fv30B, 62.9 mg/mL TP (TCA/Total nitrogen) [0522] Fv51A, 43 mg/mL TP (TCA/BCA)

[0523] Accellerase.RTM. 1000 was added at 20 mg protein/g cellulose; Trichoderma reesei Xyn2 was added at 5 mg protein/g cellulose; Fv51A was added at 5 mg protein/g cellulose. Fv39A, Fv30B, Fv3A, or Fv3B were added at 1, 2, or 5 mg protein/g cellulose. Following 3 days incubation, at 50.degree. C., 200 rpm, the assay plate was quenched and analyzed by HPLC for soluble sugars. Enzymes and combinations were found that enhanced glucose or xylose or arabinose yield, or reduced cellobiose or xylobiose concentration in this enzyme mixture, with or without another .beta.-xylosidase (T. reesei Bxl1). Results are shown in FIGS. 35A-35C.

[0524] 10.2 Activity of Candidate Endo-Xylanases with Birchwood Xylan

[0525] The activity of candidate endo-xylanases was evaluated using birchwood xylan as a substrate using the following assay. Ninety microliters of a 1% (wt/vol) birchwood xylan (Sigma X0502) stock solution was added to wells in a 96-well microtiter plate and pre-incubated at 50.degree. C. for 10 min. Enzyme dilutions and xylose standards were added (10 .mu.L) to the microtiter plate and the plates incubated at 50.degree. C. for 10 min. Meanwhile, 100 .mu.L of DNS solution was added to PCR tubes. Following the 10-min incubation, 60 .mu.L of the enzyme reaction was transferred to the PCR tubes containing the DNS solution. The tubes were incubated in a thermocycler at 95.degree. C. for 5 min, and then cooled to 4.degree. C. One hundred microliters of the reaction mixture was transferred to a 96-well plate, and absorbance at 540 nm was measured. A xylose standard curve was generated and used to calculate the activity. One xylanase unit is defined as the amount of enzyme required to generate 1 .mu.mole of xylose reducing sugar equivalents per minute under the conditions of the assay. Results are shown in Table 3.

[0526] 10.3 Enzyme Hydrolysis of Arabinoxylan Oligomers from Saccharified Corncob

[0527] In this study, enzyme hydrolysis of the arabinoxylan oligomers remaining after digestion of dilute ammonia pretreated corncob with cellulase and hemicellulase preparations was monitored. Preparation of crude oligomers is described in Example 1. Total oligomer sugars were determined by HPLC (see Example 1) after acid hydrolysis of the crude oligomers with 2% (v/v) sulfuric acid in a sealed vial at 121.degree. C. for 30 min. The sugar concentrations were corrected for a small amount of sugar degradation as determined by control samples of known sugar mixtures treated by the same procedure. The concentration of total sugars in the crude oligomer preparation determined by this method was 45 g/L glucose, 168 g/L xylose, and 46 g/L arabinose. When accounting for monomer sugar present in crude oligomers before acid hydrolysis, 86% of the glucose, 90% of the xylose, and 43% of the arabinose was present in oligomeric form. Various .beta.-xylosidases, arabinofuranosidases, and mixtures thereof were tested for increased conversion of arabinose monomer from the crude oligomers preparation. The crude oligomer preparation was diluted 20-fold to 12 g/L oligomers in 50 mM Sodium Acetate buffer, pH 5.0, and maintained at 50.degree. C. in a heating block in capped 1.5 mL Eppendorf tubes. Enzymes were added at final concentrations of 0.06-0.09 g/L and incubated for 24 h to reach completion. Samples were then removed for HPLC analysis of momomer sugars as described in Example 1. The results are listed in Table 4 as % conversion to monomer sugar based on total sugar as determined by acid hydrolysis.

[0528] To obtain the highest yields (44-71%) of arabinose from the remaining arabinoxylan oligomers in the crude oligomer mix, the data in Table 4 show that binary combinations of Fv3A+Fv51A, Fv3A+Fv43B, and Fv43A+Fv43B provide the best results. From the sequence families and activity on artificial substrates, it is deduced that Fv3A is a .beta.-xylosidase and Fv51A is an L-.alpha.-arabinofuranosidase.

[0529] It is known that arabinose sugars in arabinoxylan from corncob are frequently linked to xylose at both the 2 and 3 carbon positions of the arabinose sugar. Thus the activity of Fv3A is likely to hydrolyze the xyl(1-2)ara linkage which then makes available the ara(1-3) xyl linkage to hydrolysis by the L-.alpha.-arabinofuranosidase. Of the .beta.-xylosidases tested only Fv3A and Fv43A appeared to have this activity. Also in this screening only Fv43B appeared to have L-.alpha.-arabinofuranosidase activity among the Family 43 members from Fusarium verticillioides. Results are shown in Table 4.

11. EXAMPLE 5

Substrate Range of B-Xylosidases for Effective Corncob Hydrolysis

[0530] In this example, the substrate range of 3 .beta.-xylosidases and their relation to effective conversion of corncob xylooligomers to monomer sugars were determined. Preparation of corncob hydrolysate containing oligomeric sugars and assay of monomer sugars was performed as described in Example 1. The proton NMR spectra of oligomeric sugars with degree of polymerization (DP) greater than 2 as separated by size exclusion chromatography on Bio-Gel P2 were determined (FIG. 36 and FIG. 37). The spectra of oligomers before enzyme treatment is labeled "MD07 oligomers" in the bottom panel of FIG. 36 and spectra of the same oligo containing fractions after enzyme treatments are in the remaining panels of FIG. 36 and FIG. 37 labeled with the treatment enzyme.

[0531] The Bio-gel P2 fractions containing oligomers of greater than DP2 (5-10 mg) were lyophilized then dissolved in 0.7 mL of a D.sub.2O solution containing 0.5 mM 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as internal standard. Aliquots of 0.55 mL were used for the NMR samples in order to optimize suppression of the residual water peak. Standard versions of the Varian 2D correlation pulse sequences were used, with optimization of the .sup.13C spectral width for the heteronuclear experiments. Spectra were acquired on a Varian Unity Inova using a high sensitivity cryoprobe operating at 500 MHz. Structure elucidation was done by identifying the correlations that characterize the spin-system for the individual sugar residues and then identifying the inter-glycosidic correlations.

[0532] The arabinose containing oligomers as determined by NMR, are dominated by one or more branched structures in which arabinose is linked .beta.-1.fwdarw.3 to a xylose residue in a polymer fragment. The arabinose residue in the resulting branch is further substituted by a xylose residue linked .beta.-1.fwdarw.2 to the arabinose. Little arabinose without this second substitution is present in the remaining xylo-oligomers. T. reesei Bxl1 is not very effective at cleaving the furthest out xylose 1.fwdarw.2 to arabinose bond as evidenced by the remaining signals at 5.5 to 5.55 ppm in the spectra. The combination of the Fv43A that is effective on longer chain xylose oligomers and the Fv43B L-.alpha.-arabinofuranosidase removes most but not all of the branched species with signals in the 5.5 ppm range and unlike treatment with the T. reesei Bxl1, leaves none of the signal at 5.35 that is attributable to remaining arabinose branched to xylose oligomer.

[0533] The anomeric proton region of the spectra of the remaining xylo-oligomers after treatment with the Fv51A alone or in combination with the Fv3A show different results. The L-.alpha.-arabinofuranosidase alone is not effective at reducing the complexity of signals in the region. The Fv3A .beta.-xylosidase removes essentially all of the xylose subtending the arabinose branch leaving mostly simply linked arabinose 1.fwdarw.3 xylose oligomer structures. Addition of the L-.alpha.-arabinofuranosidase to the .beta.-xylosidase results in a fairly complete conversion to monomer sugars as evidenced by the increase in signal coming from the .alpha. and .beta. anomeric protons of reducing sugars.

[0534] It can thus be concluded that the increased effectiveness of one .beta.-xylosidase over another is likely to be due to the substrate range in terms of the structural complexity of the aglycon allowable as substrate.

12. EXAMPLE 6

Sequence Comparison and Critical Residues in Determining Substrate Range in GH3 B-Xylosidases

[0535] The T. reesei .beta.-xylosidase (Bxl1) is 61% similar to the F. verticillioides ortholog (Fv3A) and shares 42% sequence identity as shown in the alignment of FIG. 38. Fv3A has much broader substrate range than Bxl1, which is likely to be attributable to the non-conserved amino acids among the two proteins.

13. EXAMPLE 7

Determination of Defined Hemicellulase Activity for Corncob Hydrolysis to Monomer Sugars

[0536] Pretreated corncob as described in Example 1 was used as water slurry adjusted to about pH 5 with H.sub.2SO.sub.4 in water at 18.6 g dry corncob solids/100 g of total slurry. 0.78 g of the slurry was added to 4 mL glass vials and sufficient pH 5.0, 50 mM Sodium Acetate buffer was added to give a total reaction weight of 1.06 g after the desired enzyme additions. The hemicellulases were added as the purified preparations described in Example 1. Supernatant from the quad deleted T. reesei strain (Quad in Tables 5 and 6) is the concentrate of background proteins expressed by the T. reesei strain deleted in 4 major cellulase activities (described in WO 05/001036). Accellerase.RTM. 1000 is a whole cellulase mixture with high .beta.-glucosidase activity. The vials were incubated at 230 rpm in an orbital shaker at 48.degree. C. for 72 h then 2 mL of water was added. A sub-sample was taken and further diluted, centrifuged and filtered for HPLC analysis for monomer sugars as described in Example 1. Experimental results defining useful amounts of defined hemicellulase activity for hydrolyzing pretreated corncob to monomer sugars is shown in Table 5.

[0537] The results from the design-of-experiments (DoE) were fit to a surface model and used to determine best ratios of the 7 enzyme components for best yield of glucose, xylose and arabinose at the two total protein concentrations tested. Results of the ratios for the seven enzyme components are shown in Table 6.

[0538] Another exploration of the ratios (Table 7) was conducted including Fv3A, and again including Fv43D and holding that activity constant at a low level. The reaction set up and reaction conditions were identical to those described for the full DoE experiment.

[0539] Reactions (run numbers) 20, 21, and 22 contain only the whole cellulase enzyme mix and at the 21 mg/g of glucan loading monomerized about 48% of the glucose present in the cob and 24% of the xylose. Addition of the endoxylanase, Trichoderma reesei Xyn3 allowed decrease of the whole cellulase protein load while retaining about the same glucose monomer yield and increased the xylose monomer yield to about 40% (run#1). All the combinations that gave arabinose yields of above 40% required the combination of Fv43D, Fv43A and Fv43B or Fv51A or of Fv3A and Fv51A or Fv43B. Those combinations also tended to have highest release of xylose to monomer sugar.

[0540] Another set of reactions aimed at refining the required mix of hemicellulases was run holding both the loading of whole cellulase constant and the loading of the endoxylanase constant. Accellerase.RTM. 1000 whole cellulase preparation was held constant at 12 mg/g glucan and purified T. reesei Xyn3 endoxylanase was held at 6 mg/g xylan. Fv51A was the only L-.alpha.-arabinofuranosidase in the mixture, at different doses. Other reaction conditions remained the same but the hydrolysate was analyzed by size exclusion chromatography as described in Example 1. The quantitation of individual sugars was performed by peak area only and the results are shown in Table 8.

[0541] All combinations released similar amounts of glucose and about the same amount of total soluble xylose. The degree of reduction to monomer xylose varied by treatment. Without added activity to convert oligomer to monomer about 50% of the solubilized xylose remained oligomeric unless at least a .beta.-xylosidase was added. Fv3A at 2 mg Fv3A protein/g xylan reduced >DP2 oligomers to 3.6 mg/mL. Addition of 2 mg Fv51A protein/g xylan to the 2 mg Fv3A protein/g xylan further decreased the >DP2 oligomers to 1.5 mg/mL. About 2 mg of Fv51A/g xylan appeared to be sufficient to reduce the >DP2 oligomers to a minimum when the required 2 mg/g Fv3A was present (Table 8).

[0542] A mix of 6 mg/g xylan T. reesei Xyn3, 2 mg/g Fv3A and either 1 or 2 mg/g Fv51A is a suitable loading to reduce total cob arabinoxylan to monomer sugars. The addition of Fv43D to the mix aids in taking the xylobiose or other DP2 oligomers to monomer. Arabinose hydrolysis to monomer was not measured in this experiment.

14. EXAMPLE 8

Effectiveness of Hemicellulases at Producing Monomer Sugars from Corncob

[0543] In this example, the effectiveness of a set of purified hemicellulase activities at producing monomer xylose and arabinose sugars when acting alone on diluted ammonia pretreated corncob is demonstrated. Three mixtures (Mixes A, B, & C) of purified hemicellulases were prepared and used to hydrolyze hemicellulose in pretreated cob in 1 g total, 14% solids reactions prepared as in Example 1 and run under the conditions described in Example 1. Monomer sugars were analyzed by HPLC as described in Example 1 after 72 h of reaction and the amounts obtained are shown in Table 9. [0544] Mix A: 6 mg Trichoderma reesei Xyn3; 4 mg Fv3A; 1 mg Fv51A/g xylan [0545] Mix B: 6 mg Trichoderma reesei Xyn3; 1 mg Fv43D; 3 mg Fv43A; 3 mg Fv43B/g xylan [0546] Mix C: 6 mg Trichoderma reesei Xyn3; 3 mg Fv3A; 1 mg Fv43D; 1 mg Fv51A/g xylan

[0547] In this experiment the defined hemicellulase sets yielded slightly less monomer sugar than seen in earlier experiments which included activities to solubilize cellulose. The yields were still greater than those seen with endoxylanase-only addition to whole cellulase preparations. The hemicellulase activities are effective in taking xylan to monomer.

[0548] The same set of mixtures was used on hemicellulose preparations made from corncob, total stover from grain sorghum, switchgrass and sugar cane bagasse using the procedures in the general methods in accordance with Example 1. Stock suspensions of each hemicellulose preparation at 100 mg/mL in 50 mM pH 5.0 Sodium Acetate buffer were made and the pH was checked. Each of them was diluted to 10 mg preparation per mL with more 50 mM acetate buffer. Aliquots of each enzyme mixture were added to 100 .mu.L of the 10 mg/mL suspension and the reactions were run in duplicate, incubated at 48.degree. C. for 6 h with agitation. Reactions were diluted with 100 .mu.L of water, centrifuged and filtered before HPLC analysis for monomer sugars as described in Example 1. 200 .mu.L of each hemicellulose suspension was diluted with 200 .mu.L of 0.8 NH.sub.2SO.sub.4, autoclaved at 121.degree. C. for 30 min on liquid cycle then filtered and sugars analyzed by HPLC as described in Example 1. Results shown in Table 10 are reported as the average monomer sugar released by the enzyme mixture as a percentage of the acid hydrolysable sugar present in the reaction.

[0549] Mixes A and C performed well on hemicellulose from cob and as seen in other experiments on whole pretreated cob. Mixtures containing Fv3A increase conversion of arabinose to monomer and give a slight advantage in conversion of xylose to monomer. All 3 mixtures work well on hemicellulose purified from other monocots. The mixing of one endoxylanase, either one or two .beta.-xylosidases, one of which has substrate specificity beyond two or three xylose units linked .beta. 1.fwdarw.4 and an L .alpha.-arabinofuranosidase results in an effective hemicellulase blend against monocot hemicellulose.

15. EXAMPLE 9

Construction of the Integrated Expression Strain of Trichoderma reesei

[0550] An integrated expression strain of Trichoderma reesei was constructed that co-expressed five genes: T. reesei .beta.-glucosidase gene bgl1, T. reesei endoxylanase gene xyn3, F. verticillioides .beta.-xylosidase gene fv3A, F. verticillioides .beta.-xylosidase gene fv43D, and F. verticillioides .alpha.-arabinofuranosidase gene fv51A.

[0551] The construction of the expression cassettes for these different genes and the transformation of T. reesei strain are described below.

[0552] 15.1 Construction of the .beta.-Glucosidase Expression Cassette

[0553] The N-terminal portion of the native T. reesei .beta.-glucosidase gene bgl1 was codon optimized by DNA 2.0 (Menlo Park, USA). This synthesized portion comprised of the first 447 bases of the coding region. This fragment was PCR amplified using primers SK943 and SK941. The remaining region of the native bgl1 gene was PCR amplified from a genomic DNA sample extracted from T. reesei strain RL-P37, using primer SK940 and SK942. These two PCR fragments of the bgl1 gene were fused together in a fusion PCR reaction, using primers SK943 and SK942:

TABLE-US-00004 Forward Primer SK943: (SEQ ID NO: 88) (5'-CACCATGAGATATAGAACAGCTGCCGCT-3') Reverse Primer SK941: (SEQ ID NO: 89)) (5'-CGACCGCCCTGCGGAGTCTTGCCCAGTGGTCCCGCGACAG-3') Forward Primer SK940: (SEQ ID NO: 90) (5'-CTGTCGCGGGACCACTGGGCAAGACTCCGCAGGGCGGTCG-3') Reverse Primer SK942: (SEQ ID NO: 91) (5'-CCTACGCTACCGACAGAGTG-3')

[0554] The resulting fusion PCR fragments were cloned into the Gateway.RTM. Entry vector pENTRT.TM./D-TOPO.RTM., and transformed into E. coli One Shot.RTM. TOP10 Chemically Competent cells (Invitrogen) resulting in the intermediate vector, pENTRY-943/942 (FIG. 39). The nucleotide sequence of the inserted DNA was determined. The pENTRY-943/942 vector with the correct bgl1 sequence was recombined with pTrex3g using a LR Clonase.RTM. reaction protocol outlined by Invitrogen. The LR clonase reaction mixture was transformed into E. coli One Shot.RTM. TOP10 Chemically Competent cells (Invitrogen), resulting in the final expression vector, pTrex3g 943/942 (FIG. 40). The vector also contains the Aspergillus nidulans amdS gene encoding acetamidase as a selectable marker for transformation of T. reesei. The expression cassette was PCR amplified with primers SK745 and SK771 to generate product for transformation of the strain, using the electroporation method described in WO 08153712.

TABLE-US-00005 Forward Primer SK771: (SEQ ID NO: 94) (5'-GTCTAGACTGGAAACGCAAC-3') Reverse Primer SK745: (SEQ ID NO: 95) (5'-GAGTTGTGAAGTCGGTAATCC-3')

[0555] 15.2 Construction of the Endoxylanase Expression Cassette

[0556] The native T. reesei endoxylanase gene xyn3 was PCR amplified from a genomic DNA sample extracted from T. reesei, using primers xyn3F-2 and xyn3R-2.

TABLE-US-00006 Forward Primer xyn3F-2: (SEQ ID NO: 94) (5'-CACCATGAAAGCAAACGTCATCTTGTGCCTCCTGG-3') Reverse Primer (xyn3R-2): (SEQ ID NO: 95) (5'-CTATTGTAAGATGCCAACAATGCTGTTATATGCCGGCTTGGGG-3')

[0557] The resulting PCR fragments were cloned into the Gateway.RTM. Entry vector pENTR.TM./D-TOPO.RTM., and transformed into E. coli One Shot.RTM. TOP10 Chemically Competent cells (Invitrogen) resulting in the intermediate vector, pENTR/Xyn3 (FIG. 41). The nucleotide sequence of the inserted DNA was determined. The pENTR/Xyn3 vector with the correct xyn3 sequence was recombined with pTrex3g using a LR Clonase.RTM. reaction protocol outlined by Invitrogen. The LR clonase reaction mixture was transformed into E. coli One Shot.RTM. TOP10 Chemically Competent cells (Invitrogen), resulting in the final expression vector, pTrex3g/Xyn3 (FIG. 42). The vector also contains the Aspergillus nidulans amdS gene encoding acetamidase as a selectable marker for transformation of T. reesei. The expression cassette was PCR amplified with primers SK745 and SK822 to generate product for transformation of the strain, using the electroporation method.

TABLE-US-00007 Forward Primer SK745: (SEQ ID NO: 96) (5'-GAGTTGTGAAGTCGGTAATCC-3') Reverse Primer SK822: (SEQ ID NO: 97) (5'-CACGAAGAGCGGCGATTC-3')

[0558] 15.3 Construction of the .beta.-Xylosidase Fv3A Expression Cassette

[0559] The F. verticilloides .beta.-xylosidase fv3A gene was amplified from a F. verticilloides genomic DNA sample using the primers MH124 and MH125.

TABLE-US-00008 Forward Primer MH124: (SEQ ID NO: 98) (5'-CAC CCA TGC TGC TCA ATC TTC AG-3') Reverse Primer MH125: (SEQ ID NO: 99) (5'-TTA CGC AGA CTT GGG GTC TTG AG-3')

[0560] The PCR fragments were cloned into the Gateway.RTM. Entry vector pENTR.TM./D-TOPO.RTM., and transformed into E. coli One Shot.RTM. TOP10 Chemically Competent cells (Invitrogen) resulting in the intermediate vector, pENTR-Fv3A (FIG. 43). The nucleotide sequence of the inserted DNA was determined. The pENTRY-Fv3A vector with the correct fv3A sequence was recombined with pTrex6g using a LR Clonase.RTM. reaction protocol outlined by Invitrogen. The LR clonase reaction mixture was transformed into E. coli One Shot.RTM. TOP10 Chemically Competent cells (Invitrogen), resulting in the final expression vector, pTrex6g/Fv3A (FIG. 44). The vector also contains a chlorimuron ethyl resistant mutant of the native T. reesei acetolactate synthase (als) gene, designated alsR, which is used together with its native promoter and terminator as a selectable marker for transformation of T. reesei (WO2008/039370 A1). The expression cassette was PCR amplified with primers SK1334, SK1335 and SK1299 to generate product for transformation of T. reesei, using the electroporation method (see, e.g., WO2008153712 A2).

TABLE-US-00009 Forward Primer SK1334: (SEQ ID NO: 100) (5'-GCTTGAGTGTATCGTGTAAG-3') Forward Primer SK1335: (SEQ ID NO: 101) (5'-GCAACGGCAAAGCCCCACTTC-3') Reverse Primer SK1299: (SEQ ID NO: 102) (5'-GTAGCGGCCGCCTCATCTCATCTCATCCATCC-3')

[0561] 15.4 Construction of the .beta.-Xylosidase Fv43D Expression Cassette

[0562] For the construction of the F. verticilloides .beta.-xylosidase Fv43D expression cassette, the fv43D gene product was amplified from F. verticilloides genomic DNA using the primers SK1322 and SK1297. A region of the promoter of the endoglucanase gene egl1 was PCR amplified from T. reesei genomic DNA extracted from strain RL-P37, using the primers SK1236 and SK1321. These two PCR amplified DNA fragments were subsequently fused together in a fusion PCR reaction using the primers SK1236 and SK1297. The resulting fusion PCR fragment was cloned into pCR-Blunt II-TOPO vector (Invitrogen) to give the plasmid TOPO Blunt/Pegl1-Fv43D (FIG. 45) and E. coli One Shot.RTM. TOP10 Chemically Competent cells (Invitrogen) were transformed using this plasmid. Plasmid DNA was extracted from several E. coli clones and confirmed by restriction digest.

TABLE-US-00010 Forward Primer SK1322: (SEQ ID NO: 103) (5'-CACCATGCAGCTCAAGTTTCTGTC-3') Reverse Primer SK1297: (SEQ ID NO: 104) (5'-GGTTACTAGTCAACTGCCCGTTCTGTAGCGAG-3') Forward Primer SK1236: (SEQ ID NO: 105) (5'-CATGCGATCGCGACGTTTTGGTCAGGTCG-3') Reverse Primer SK1321: (SEQ ID NO: 106) (5'-GACAGAAACTTGAGCTGCATGGTGTGGGACAACAAGAAGG-3')

[0563] The expression cassette was PCR amplified from TOPO Blunt/Pegl1-Fv43D with primers SK1236 and SK1297 to generate product for transformation of T. reesei, using the electroporation method as described in WO2008153712A2.

[0564] 15.5 Construction of the .alpha.-Arabinofuranosidase Expression Cassette

[0565] For the construction of the F. verticilloides .alpha.-arabinofuranosidase gene fv51A expression cassette, the fv51A gene product was amplified from F. verticilloides genomic DNA using the primers SK1159 and SK1289. A region of the promoter of the endoglucanase gene egl1 was PCR amplified from T. reesei genomic DNA sample extracted from strain RL-P37, using the primers SK1236 and SK1262. These two PCR amplified DNA fragments were subsequently fused together in a fusion PCR reaction using the primers SK1236 and SK1289. The resulting fusion PCR fragment was cloned into pCR-Blunt II-TOPO vector (Invitrogen) to give the plasmid TOPO Blunt/Pegl1-Fv51A (FIG. 46) and E. coli One Shot.RTM. TOP10 Chemically Competent cells (Invitrogen) were transformed using this plasmid.

TABLE-US-00011 Forward Primer SK1159: (SEQ ID NO: 107) (5'-CACCATGGTTCGCTTCAGTTCAATCCTAG-3') Reverse Primer SK1289: (SEQ ID NO: 108) (5'-GTGGCTAGAAGATATCCAACAC-3') Forward Primer SK1236: (SEQ ID NO: 109) (5'-CATGCGATCGCGACGTTTTGGTCAGGTCG-3') Reverse Primer SK1262: (SEQ ID NO: 110) (5'-GAACTGAAGCGAACCATGGTGTGGGACAACAAGAAGGAC-3')

[0566] The expression cassette was PCR amplified with primers SK1298 and SK1289 to generate product for transformation of T. reesei using the electroporation method.

TABLE-US-00012 Forward Primer SK1298: (SEQ ID NO: 111) (5'-GTAGTTATGCGCATGCTAGAC-3') Reverse Primer SK1289: (SEQ ID NO: 112) (5'-GTGGCTAGAAGATATCCAACAC-3')

[0567] 15.6 Co-Transformation of T. reesei with the .beta.-Glucosidase and Endoxylanase Expression Cassettes

[0568] A Trichoderma reesei mutant strain, derived from RL-P37 (Sheir-Neiss, G et al. Appl. Microbiol. Biotechnol. 1984, 20:46-53) and selected for high cellulase production was co-transformed with the .beta.-glucosidase expression cassette (cbh1 promoter, T. reesei.beta.-glucosidase) gene, cbh1 terminator, and amdS marker), and the endoxylanase expression cassette (cbh1 promoter, T. reesei xyn3, and cbh1 terminator) using PEG-mediated transformation (Penttila, M et al. Gene 1987, 61(2):155-64). Numerous transformants were isolated and examined for .beta.-glucosidase and endoxylanase production. One transformant called T. reesei strain #229 was used for transformation with the other expression cassettes.

[0569] 15.7 Co-Transformation of T. reesei Strain #229 with two .beta.-Xylosidase and .alpha.-Arabinofuranosidase Expression Cassettes

[0570] T. reesei strain #229 was co-transformed with the 3-xylosidase fv3A expression cassette (cbh1 promoter, fv3A gene, cbh1 terminator, and alsR marker), the 3-xylosidase fv43D expression cassette (egl1 promoter, fv43D gene, native fv43D terminator), and the fv51A .alpha.-arabinofuranosidase expression cassette (egl1 promoter, fv51A gene, fv51A native terminator) using electroporation. Transformants were selected on Vogels agar plates containing chlorimuron ethyl (80 ppm). Vogels agar was prepared as follows, per liter.

TABLE-US-00013 50 x Vogels Stock Solution (below) 20 mL BBL Agar 20 g With deionized H.sub.2O bring to post-sterile addition: 980 mL 50% Glucose 20 mL

50.times. Vogels Stock Solution (WO 2005/001036), per liter: In 750 mL deionized H2O, dissolve successively:

TABLE-US-00014 Na.sub.3Citrate*2H.sub.2O 125.00 g KH.sub.2PO.sub.4 (Anhydrous) 250.00 g NH.sub.4NO.sub.3 (Anhydrous) 100 g MgSO.sub.4*7H.sub.2O 10.00 g CaCl.sub.2*2H.sub.2O 5.00 g Vogels Trace Element Solution 5.0 mL Vogels Biotin Solution 2.5 mL With deionized H.sub.2O, bring to 1 L

[0571] Numerous transformants were isolated and examined for .beta.-xylosidase and L-.alpha.-arabinofuranosidase production. Transformants were also screened for biomass conversion performance according to the cob saccharification assay described in Example 1. Examples of T. reesei integrated expression strains described herein are H3A, 39A, A10A, 11A, and G9A, which express all of the genes for T. reesei beta-glucosidase 1, T. reesei Xyn3, Fv3A, Fv51A, and Fv43D, at different ratios (Table 11). Examples of T. reesei integrated expression strains described herein also include 44A, 69A, G6A and 102, and each includes most of the genes for T. reesei beta-glucosidase 1, T. reesei XYN3, Fv3A, Fv51A, and Fv43D, expressed at different ratios. Strain 44A lacked overexpressed T. reesei XYN3; strain 69A lacked Fv51A (confirmed by Western Blot, not shown); strains G6A and 102 lacked Fv3A (Table 11), as determined by HPLC protein analysis (Example 1).

16. EXAMPLE 10

Saccharification Performance of T. reesei Integrated Expression Strains on Ammonia Pretreated Corncob

[0572] The saccharification performance of enzyme compositions produced by T. reesei integrated expression strains on dilute ammonia pretreated corncob was evaluated. T. reesei enzyme samples were generated as either ultrafiltration concentrates (UFC) or centrate. For the generation of UFC, T. reesei fermentation broths (14 L-scale) were obtained after cell separation by centrifugation, concentrated using membrane-ultrafiltration through a Millipore 10 kD molecular weight cut off membrane. Then pH was adjusted to 4.8. The cell-separated broth was then polished by filtration, using FW6 Buchner filtration. Each enzyme sample was assayed for total protein concentration using the modified Biuret method.

[0573] The saccharification performance was evaluated in vials or in shake flasks. Each enzyme preparation was assayed for saccharification performance on 20% dry solids (DS) loading of dilute ammonia pretreated corncob (see, WO2006/110901). All saccharification reactions were then titrated with sulfuric acid to pH 5.0 and sodium azide was added to a final concentration of 0.01% (w/v), for microbial contamination control. Each saccharification reaction was then dosed with 20 mg of total protein (TP) enzyme preparation per g of substrate glucan or xylan, as appropriate. Accellerase.RTM. 1500 (Ac1500) and the integrated strain UFC's were dosed at 20 mg total protein/g glucan. An enzyme blend was prepared in a ratio of 25:9:4:3:1 Accellerase.RTM. 1500:Xyn3:Fv3A:Fv51A: FV43D. In the blend, Accellerase.RTM. 1500 was dosed at 20 mg total protein/g glucan and the hemicellulases were dosed per g xylan (4.3 mg Xyn3/g of xylan, 1.7 mg Fv3A/g of xylan, 1.4 mg Fv51A/g of xylan, 0.5 mg Fv43D/g of xylan). Each saccharification reaction was incubated at 50.degree. C. in a rotary shaker set to 200 rpm, then sampled and diluted 10.times.(v/v) before monomeric sugar concentration was determined using HPLC analysis (detailed in Section 16.1 below under the "monomeric HPLC analysis" section) after 1, 2, 3 and 7 days of saccharification (FIGS. 47A, 47B). On day 3 of saccharification, each reaction was also sampled by weight (w/w) for oligomeric sugar concentration by HPLC analysis (Section 16.1, FIG. 47C). These results show that the enzyme compositions produced by integrated strains H3A and G9A provide better glucose and xylose yields than Accellerase.RTM. 1500 or enzyme blends created from individually expressed enzymes.

[0574] A chromatographic comparison of the enzyme composition produced by three different integrated strains is shown in FIG. 47D.

[0575] 16.1 HPLC Analysis

[0576] Monomeric Sugar HPLC Analysis:

[0577] Each sample was analyzed by HPLC using a BioRad Aminex HPX-87H ion exclusion column (300 mm.times.7.8 mm). All day 1, 2, 3, and 7 samples were diluted 10.times. volumetrically with 5 mM sulfuric acid, filtered through a 0.2 .mu.m filter before injection into the HPLC and run under manufacture specifications.

[0578] Oligomeric Sugar HPLC Analysis (Acid Hydrolysis):

[0579] Day 3 saccharification samples were diluted 10.times. by weight with Milli-Q water, then sulfuric acid was added to the final concentration of 4% (w/w). A xylose and glucose standard (sugar recovery standard--"SRS") of known concentration was also prepared and measured for monomeric sugar HPLC analysis as stated above, and oligomer sugar HPLC analysis. Oligomer HPLC samples and were then autoclaved at 121.degree. C. for 15 min, and filtered through a 0.2 .mu.m filter before injection into the HPLC BioRad Aminex HPX-87H ion exclusion column (300 mm.times.7.8 mm), and run under manufacturer specifications. Oligomer sugar concentration was determined by multiplying the percent retained xylose and glucose concentration of the sugar recovery standard (SRS) after acid hydrolysis, by the post acid hydrolysis HPLC sugar concentrations of each sample, then subtracting the monomeric sugar concentration determined in the "monomeric sugar HPLC Analysis" section above.

17. EXAMPLE 11

Identification of Phylogenetically Broad Enzyme Classes which can Improve the Saccharification Performance of T. reesei Integrated Expression Strain

[0580] In this example, enzyme activities which limit the efficacy of enzyme compositions produced by a T. reesei integrated expression strain were identified and enzymes from different species which can compensate for those limiting activities or which could substitute for integrated strain components are exemplified.

[0581] 0.95 g of pretreated corncob as described in Example 1 was added to 20 mL glass vials. Sufficient pH 5.0, 50 mM Sodium Acetate buffer and 1 NH.sub.2SO.sub.4 were added to give a total reaction weight of 3.00 g at 22 g dry corncob solids/100 g of total slurry, pH 5.0 post enzyme additions. The T. reesei enzyme composition produced by integrated strain H3A (FIGS. 48A, 49A, and 50A) was added as an ultrafiltered (i.e., cell-free) concentrate to 7 mg total protein per g of glucan and xylan combined in the feedstock to all vials. In addition, candidate hemicellulases were added as the ultrafiltered preparations from 14 L fermentation cultures expressed by T. reesei Quad delete strain (described in WO 05/001036). The candidate hemicellulases were added at 0, 0.5, 1.0 or 3.0 mg enzyme protein per g of glucan and xylan combined. The hemicellulases included constituents of the enzyme composition produced by the integrated strain itself including: [0582] Fusarium verticillioides Fv3A [0583] Fusarium verticillioides Fv51A [0584] Fusarium verticillioides Fv43D As well as enzymes from different fungi [0585] Fusarium oxysporum Fo43A [0586] Gibberella zeae Gz43A [0587] Penicillium funiculosum Pf43A [0588] Aspergillus fumigatus Af43A [0589] Podospora anserina Pa51A [0590] Penicillium funiculosum Pf51A

[0591] The vials were incubated at 180 rpm in an orbital shaker at 48.degree. C. for 72 h. Then 12 mL of water was added. A sub-sample was taken and further diluted, centrifuged and filtered for HPLC analysis for monomer sugars as described in Example 1.

[0592] The results shown in FIG. 48A and FIG. 48B demonstrate that addition of .beta.-xylosidase activities such as Fusarium verticillioides Fv43D and Fusarium oxysporum Fo43A markedly improved monomer xylose release relative to 7 mg T. reesei integrated strain total protein per g of Glucan and Xylan combined. Significant improvements were even observed at the low additions of 0.5 and 1.0 mg/g.

[0593] The results shown in FIG. 49A and FIG. 49B demonstrate that addition of all of the hemicellulases lead to a significant increase in monomer glucose, even >10% at hemicellulase loadings of 1-3 mg per g of Glucan and Xylan combined.

[0594] The results shown in FIG. 50A and FIG. 50B demonstrate that addition of GH51 enzymes such as Fusarium verticillioides Fv51A, and especially Podospora anserine Pa51A and Penicillium funiculosum Pfu51A, led to an increase in monomer arabinose level.

18. EXAMPLE 12

Saccarification of Variously Pretreated Switchgrass by Cellulase and Hemicellulase Preparations

[0595] The saccharification performance of expressed cellulases and hemicellulases on pretreated raw switchgrass was evaluated. A range of conditions for dilute ammonia pretreatment of switchgrass were evaluated for saccharification performance with an enzyme cocktail composed of enzymes described herein. Pretreatment conditions vary and pretreatment efficacy affects enzymatic hydrolysis performance.

[0596] Pretreatment of raw switchgrass was performed in sealed, 6''.times.1/2'', stainless steel tubes that were immersed in a heated sand bath. A slurry of raw switchgrass, water, and ammonium hydroxide (.about.28% solution) was mixed to the desired percent solids and percent ammonia and then loaded into a pretreatment tube. Tubes were then held at the desired temperature (+/-2.degree. C.) for the desired time and then quenched in ice water for approximately 1 min before being brought to room temperature. The pretreated slurry was removed from the tubes and allowed to dry overnight in the hood (>90% solids attainable).

[0597] Dried pretreated solids were then saccharified at 10% solids, pH 5, 50.degree. C., 200 rpm using Accellerase.RTM. 1500, Xyn 3, Fv3A, Fv51A, and Fv43D (25, 9, 7, 3, 1 mg total protein/g glucan or xylan respectively). A 5 mL total hydrolysate volume in 20 mL scintillation vials was used.

[0598] Glucan and xylan yields were based on monomeric glucose and xylose released compared to the glucan or xylan available from the raw biomass. Monomeric sugar concentrations were measured by HPLC (BioRad Aminex HPX 87-H column).

[0599] Pretreatment time, temperature, percent solids, and percent NH.sub.3, were varied over a wide range in order to optimize saccharification results. Each pretreatment condition that had both glucan and xylan yields better than .about.50% is considered a strong performer. The pretreatment parameters that performed strongly are listed in Table 12 along with their respective glucan, xylan, and total percent yields. Glucan and Xylan conversions are based on monomeric sugars released during saccharification as compared to glucan or xylan theoretically available in the raw switchgrass. Total conversion is glucose and xylose only (FIG. 51).

19. EXAMPLE 13

Saccharification of Pretreated Switchgrass by Cellulase and Hemicellulase Preparations

[0600] In addition to the above Examples, the saccharification performance of enzyme mixes and enzyme compositions produced by an integrated strain was tested on several substrates, pretreatments and conditions. These experiments show the range of performance using the enzyme mix or an integrated strain product. They demonstrate good performance across a range of substrates and pretreatments, pH, and temperatures.

[0601] Dilute ammonia pretreated switchgrass was prepared according to the methods and process ranges in WO06110901A: Switchgrass (38.7% glucan, 22.2% xylan, 2.5% arabinan, 23.2% lignin) was hammer-milled to pass through a 1 mm screen, then pretreated at 160.degree. C. for 90 min with 6% NH.sub.3 (weight/weight DM, added as NH.sub.4OH). This pretreated substrate was treated with enzyme mixes containing Accellerase.RTM. 1500, Multifect.RTM. Xylanase (both commercial products of Danisco A/S, Genencor Division, Palo Alto, Calif.), Fv3A, Fv51A, and Fv43D in a total reaction mass of 50 g at 15% solids. The total protein (TP) of the commercial products was determined by Biuret assay. The other enzymes were ultra-filtration concentrates (UFCs) following expression in cellulase quad-deleted strains of T. reesei, with TP determined by Total Nitrogen analysis of TCA-precipitable protein. All reactions were dosed with Accellerase.RTM. 1500 at 25 mg TP/g Glucan and Multifect.RTM. Xylanase (MF Xyl) at 9 mg TP/g Xylan, and Fv3A, Fv51A, and Fv43D were added as indicated in FIG. 56A-56B at 3.6 mg TP/g Xylan, 3.0 mg TP/g Xylan and 1.0 mg TP/g Xylan, respectively. All enzymes were dosed relative to the starting carbohydrate contents of the switchgrass before pretreatment. The saccharification reactions were carried out at 47.degree. C. and 33.degree. C. at pH 5.3 for three days.

[0602] The results at 33.degree. C. showed that addition of Fv3A, Fv51A, and Fv43D increased glucan conversion (FIG. 56A) and more than doubled the xylan conversion (FIG. 56B). The results at 47.degree. C. showed that addition of Fv3A, Fv51A, and Fv43D gave some increased glucan conversion (FIG. 56A) and more than doubled the xylan conversion (FIG. 56B). Additions of Fv51A or Fv43D, alone, gave large increases in xylan conversion, especially to xylo-oligomers or xylose monomers, respectively. Addition of Fv3A alone increased xylose yields, but in combination with Fv51A gave a large increase in xylan conversion to monomer.

20. EXAMPLE 14

Saccharification of Pretreated Switchgrass by an Integrated T. Reesei Strain

[0603] The saccharification performance of an enzyme composition produced by an integrated T. reesei strain (H3A) was evaluated on dilute ammonia pretreated switchgrass prepared according to the methods and process ranges in WO06110901A: Switchgrass (37% glucan, 21% xylan, 5% arabinan, 18% lignin) was hammer-milled to pass through a 1 mm screen, then pretreated at 160.degree. C. for 90 min with 10.0% NH.sub.3 (weight/weight DM, added as NH.sub.4OH). In duplicate 500 mL glass Erlenmeyer flasks, 50 g of pretreated slurry at 25% solids was saccharified at 48.degree. C., pH 5.3 for 7 days, with supernatant from the integrated strain was dosed at 14 mg TP/g of carbohydrate (glucan plus xylan). TP of H3A was determined by Total Nitrogen analysis of TCA-precipitable protein. Enzymes were dosed relative to the starting carbohydrate contents of the switchgrass before pretreatment.

[0604] At the end of 7 days, high levels of glucan conversion (52-55%, FIG. 57A) and xylan conversion (51%-53%, (FIG. 57B) were measured by HPLC. These results show that the enzyme composition produced by the integrated strain can saccharify dilute ammonia pretreated switchgrass at high solids (25% dry matter).

21. EXAMPLE 15

Saccharification of Hardwood Pulp by an Integrated T. reesei Strain

[0605] The saccharification performance of an enzyme composition produced by integrated T reesei strain H3A was evaluated on industrial hardwood unbleached pulp (derived from Kraft process and oxygen delignification, Smurfit Kappa Cellulose Du Pin, Biganos, France) with the following composition: Glucan 75.1%, Xylan 19.1%, Acid soluble lignin 2.2%. The enzymatic saccharification studies were carried out using NREL standard assay method LAP-009 "Enzymatic Saccharification of Lignocellulosic Biomass" (http://www.nrel.gov/biomass/pdfs/42629.pdf), except that the cellulose loading was different (varying from 9.3-20%) and a total mass of 100 g was used. The experimental condition was 200 rpm and pH 5.0, 50.degree. C. Enzyme was dosed at 20 mg TP/g glucan (based on final dry matter) at the start of the experiment. Samples were taken at timed intervals if they were liquefied, and then analyzed by HPLC for sugar concentration. Glucose, xylose, and cellobiose concentration were determined using a Waters HPLC system (Alliance system, Waters Corp., Milford, Mass.). The HPLC column used for sugar analysis was from BioRad (Aminex HPX-87H ion exclusion column (300 mm.times.7.8 mm), BioRad Inc., Hercules, Calif.). All samples were diluted 10.times. with 5 mM sulfuric acid, filtered through a 0.2 .mu.m filter before injection into the HPLC. As indicated (TABLE 13) two experiments were carried out in "fed-batch" mode: Pretreated hardwood pulp to an initial dry matter content of 7.0% at Time 0 and the rest of the substrate was added at discrete times in four equal portions during the first 24 h to bring the final dry solids loading to 20%.

[0606] Results showed high levels of glucan and xylan conversion to monomers (up to 89% and 90%, respectively) with the enzyme composition produced by integrated strain H3A. Conversions increased with higher enzyme loadings and longer saccharification times. Conversion was lower when the solids were at 20% than at 15%, but this lower conversion at 20% could be partially mitigated by using a fed-batch process.

22. EXAMPLE 16

Saccharification of Hardwood Pulp by an Integrated T. reesei Strain Over a Range of Temperature and pH

[0607] The saccharification performance of an enzyme composition produced by an integrated T. reesei strain (H3A) on industrial hardwood unbleached pulp (as used in the preceding example) at 7% cellulose loading and 20 mg TP/g glucan of integrated strain supernatant (based on final dry matter and composition of the pretreated substrate) was tested at temperatures from 45.degree. C.-60.degree. C. and pH's from 4.65-5.4 (buffered in 0.1 M sodium citrate). Results after 2 days saccharification are shown in TABLE 14 and show good glucan and xylan conversions over the whole range of conditions tested. These results suggested optimum conditions for saccharification of this substrate as pH 4.9 and 50.degree. C. but good conversions are seen even at pH 5.0, 60.degree. C. In a follow-up experiment at 50.degree. C., including lower pH's (and otherwise unchanged experimental conditions) good saccharifications were seen at pH 3.8, pH 4.0 and pH 4.25, with glucose & xylose titers of 45.1 & 9.7 g/L; 50.0 & 11.4 g/L; and 57.2 & 13.2 g/L, respectively.

23. EXAMPLE 17

Saccharification of Steam-Expanded Sugarcane Bagasse with an Enzyme Composition Produced by an Integrated Strain at Different Enzyme Doses

[0608] The saccharification performance of an enzyme composition produced by an integrated T reesei strain (H3A) on steam-expanded sugarcane bagasse was evaluated at 7% cellulose loading. The bagasse was pretreated by steam injection in a StakeTech reactor at 210 psig, 200.degree. C. with a 4 min residence time. The pretreated material had the following composition: Glucan 40.9%, Xylan 20.8%, Lignin 27%. The integrated strain supernatant was dosed at 10, 20, 30, 50 or 80 mg TP/g glucan (based on final dry matter and composition of the pretreated substrate). Saccharification was carried out in a 5 mL reaction volume at 50.degree. C., pH 5 for 3 days. The results (FIG. 58A-C) showed that the integrated strain product out-performed Accellerase.RTM. 1500 (20 mg protein/g glucan) in glucan conversion to glucose (FIG. 58A and FIG. 58C) and, especially, xylan conversion to xylose (FIG. 58B and FIG. 58C) even at half the Accellerase.RTM. 1500 dose. Glucan conversion, especially at Day 1, increased significantly as the dose of H3A total protein increased (FIG. 58A and FIG. 58C), whereas xylan conversion was more rapid, with little increase from Day 1 to Day 3, and very high, even at the lowest dose of H3A total protein (FIG. 58B and FIG. 58C).

24. EXAMPLE 18

Saccharification of Dilute-Acid Pretreated Corn Fiber with Various Enzymes

[0609] The saccharification performance of enzyme mixtures on dilute sulfuric acid pretreated corn fiber was evaluated in a 250 mL shake flask. In a typical experiment, corn fiber (initial composition 38% C6 sugars and 27% C5 sugars) was adjusted to 15% DS (dry solids) and 0.36% (w/w %) sulfuric acid was added. Corn fiber slurry was then autoclaved at 121.degree. C. for 60 min. The slurry was then adjusted to pH 5.0 using 6 N NaOH. The sugar content of the pretreated sample was 21 g/L glucose and 12 g/L xylose. Enzymes were added to the pretreated substrate as follows (as indicated in FIG. 59A, 59B, 59C); Accellerase.RTM. 1500 (AC 1500): 20 mg TP/g glucan and 45 mg TP/g glucan; Accellerase.RTM. 1500+Multifect.RTM. Xylanase (MF): (25 mg/g glucan+9 mg TP/g xylan) and (25 mg TP/g glucan+20 mg TP/g xylan); Accellerase.RTM. 1500+Xyn 3+Fv3A+Fv51A+Fv43D: (25 mg TP/g glucan+9 mg TP/g xylan+7 mg TP/g xylan+3 mg TP/g xylan+1 mg TP/g xylan) (the full enzyme "blend"). The total protein (TP) of the commercial products (Accellerase.RTM. 1500 and Multifect.RTM. Xylanase: Danisco US Inc., Genencor) were determined by Biuret assay. The other enzymes were ultra-filtration concentrates (UFCs) following expression in cellulase quad-deleted strains of T. reesei (as described earlier)--their TP were determined by Total Nitrogen analysis of TCA-precipitable protein. All enzymes were dosed relative to the starting carbohydrate contents of the corn fiber before pretreatment. Enzymatic saccharification was carried out at 200 rpm and 50.degree. C. for 24 h, 48 h and 120 h. Samples were withdrawn at different time intervals and analyzed for the formation of glucose and xylose sugars by HPLC. The adjusted sugar (glucose or xylose) reflected the sugar being produced from the enzymatic step, with the starting sugars subtracted.

[0610] The results show that the full enzyme blend out-performed the (Accellerase.RTM. 1500+Multifect.RTM. Xylanase) ("AC 1500+ME") in glucan conversion and in xylan conversion, even when both were dosed at the same total protein. The full enzyme blend gave almost complete glucan conversion of this substrate after 5 days saccharification.

25. SPECIFIC EMBODIMENTS AND INCORPORATION BY REFERENCE

[0611] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0612] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure(s).

Sequence CWU 1

1

11712358DNAFusarium verticillioides 1atgctgctca atcttcaggt cgctgccagc gctttgtcgc tttctctttt aggtggattg 60gctgaggctg ctacgccata tacccttccg gactgtacca aaggaccttt gagcaagaat 120ggaatctgcg atacttcgtt atctccagct aaaagagcgg ctgctctagt tgctgctctg 180acgcccgaag agaaggtggg caatctggtc aggtaaaata tacccccccc cataatcact 240attcggagat tggagctgac ttaacgcagc aatgcaactg gtgcaccaag aatcggactt 300ccaaggtaca actggtggaa cgaagccctt catggcctcg ctggatctcc aggtggtcgc 360tttgccgaca ctcctcccta cgacgcggcc acatcatttc ccatgcctct tctcatggcc 420gctgctttcg acgatgatct gatccacgat atcggcaacg tcgtcggcac cgaagcgcgt 480gcgttcacta acggcggttg gcgcggagtc gacttctgga cacccaacgt caaccctttt 540aaagatcctc gctggggtcg tggctccgaa actccaggtg aagatgccct tcatgtcagc 600cggtatgctc gctatatcgt caggggtctc gaaggcgata aggagcaacg acgtattgtt 660gctacctgca agcactatgc tggaaacgac tttgaggact ggggaggctt cacgcgtcac 720gactttgatg ccaagattac tcctcaggac ttggctgagt actacgtcag gcctttccag 780gagtgcaccc gtgatgcaaa ggttggttcc atcatgtgcg cctacaatgc cgtgaacggc 840attcccgcat gcgcaaactc gtatctgcag gagacgatcc tcagagggca ctggaactgg 900acgcgcgata acaactggat cactagtgat tgtggcgcca tgcaggatat ctggcagaat 960cacaagtatg tcaagaccaa cgctgaaggt gcccaggtag cttttgagaa cggcatggat 1020tctagctgcg agtatactac taccagcgat gtctccgatt cgtacaagca aggcctcttg 1080actgagaagc tcatggatcg ttcgttgaag cgccttttcg aagggcttgt tcatactggt 1140ttctttgacg gtgccaaagc gcaatggaac tcgctcagtt ttgcggatgt caacaccaag 1200gaagctcagg atcttgcact cagatctgct gtggagggtg ctgttcttct taagaatgac 1260ggcactttgc ctctgaagct caagaagaag gatagtgttg caatgatcgg attctgggcc 1320aacgatactt ccaagctgca gggtggttac agtggacgtg ctccgttcct ccacagcccg 1380ctttatgcag ctgagaagct tggtcttgac accaacgtgg cttggggtcc gacactgcag 1440aacagctcat ctcatgataa ctggaccacc aatgctgttg ctgcggcgaa gaagtctgat 1500tacattctct actttggtgg tcttgacgcc tctgctgctg gcgaggacag agatcgtgag 1560aaccttgact ggcctgagag ccagctgacc cttcttcaga agctctctag tctcggcaag 1620ccactggttg ttatccagct tggtgatcaa gtcgatgaca ccgctctttt gaagaacaag 1680aagattaaca gtattctttg ggtcaattac cctggtcagg atggcggcac tgcagtcatg 1740gacctgctca ctggacgaaa gagtcctgct ggccgactac ccgtcacgca atatcccagt 1800aaatacactg agcagattgg catgactgac atggacctca gacctaccaa gtcgttgcca 1860gggagaactt atcgctggta ctcaactcca gttcttccct acggctttgg cctccactac 1920accaagttcc aagccaagtt caagtccaac aagttgacgt ttgacatcca gaagcttctc 1980aagggctgca gtgctcaata ctccgatact tgcgcgctgc cccccatcca agttagtgtc 2040aagaacaccg gccgcattac ctccgacttt gtctctctgg tctttatcaa gagtgaagtt 2100ggacctaagc cttaccctct caagaccctt gcggcttatg gtcgcttgca tgatgtcgcg 2160ccttcatcga cgaaggatat ctcactggag tggacgttgg ataacattgc gcgacgggga 2220gagaatggtg atttggttgt ttatcctggg acttacactc tgttgctgga tgagcctacg 2280caagccaaga tccaggttac gctgactgga aagaaggcta ttttggataa gtggcctcaa 2340gaccccaagt ctgcgtaa 23582766PRTFusarium verticillioides 2Met Leu Leu Asn Leu Gln Val Ala Ala Ser Ala Leu Ser Leu Ser Leu1 5 10 15Leu Gly Gly Leu Ala Glu Ala Ala Thr Pro Tyr Thr Leu Pro Asp Cys 20 25 30Thr Lys Gly Pro Leu Ser Lys Asn Gly Ile Cys Asp Thr Ser Leu Ser 35 40 45Pro Ala Lys Arg Ala Ala Ala Leu Val Ala Ala Leu Thr Pro Glu Glu 50 55 60Lys Val Gly Asn Leu Val Ser Asn Ala Thr Gly Ala Pro Arg Ile Gly65 70 75 80Leu Pro Arg Tyr Asn Trp Trp Asn Glu Ala Leu His Gly Leu Ala Gly 85 90 95Ser Pro Gly Gly Arg Phe Ala Asp Thr Pro Pro Tyr Asp Ala Ala Thr 100 105 110Ser Phe Pro Met Pro Leu Leu Met Ala Ala Ala Phe Asp Asp Asp Leu 115 120 125Ile His Asp Ile Gly Asn Val Val Gly Thr Glu Ala Arg Ala Phe Thr 130 135 140Asn Gly Gly Trp Arg Gly Val Asp Phe Trp Thr Pro Asn Val Asn Pro145 150 155 160Phe Lys Asp Pro Arg Trp Gly Arg Gly Ser Glu Thr Pro Gly Glu Asp 165 170 175Ala Leu His Val Ser Arg Tyr Ala Arg Tyr Ile Val Arg Gly Leu Glu 180 185 190Gly Asp Lys Glu Gln Arg Arg Ile Val Ala Thr Cys Lys His Tyr Ala 195 200 205Gly Asn Asp Phe Glu Asp Trp Gly Gly Phe Thr Arg His Asp Phe Asp 210 215 220Ala Lys Ile Thr Pro Gln Asp Leu Ala Glu Tyr Tyr Val Arg Pro Phe225 230 235 240Gln Glu Cys Thr Arg Asp Ala Lys Val Gly Ser Ile Met Cys Ala Tyr 245 250 255Asn Ala Val Asn Gly Ile Pro Ala Cys Ala Asn Ser Tyr Leu Gln Glu 260 265 270Thr Ile Leu Arg Gly His Trp Asn Trp Thr Arg Asp Asn Asn Trp Ile 275 280 285Thr Ser Asp Cys Gly Ala Met Gln Asp Ile Trp Gln Asn His Lys Tyr 290 295 300Val Lys Thr Asn Ala Glu Gly Ala Gln Val Ala Phe Glu Asn Gly Met305 310 315 320Asp Ser Ser Cys Glu Tyr Thr Thr Thr Ser Asp Val Ser Asp Ser Tyr 325 330 335Lys Gln Gly Leu Leu Thr Glu Lys Leu Met Asp Arg Ser Leu Lys Arg 340 345 350Leu Phe Glu Gly Leu Val His Thr Gly Phe Phe Asp Gly Ala Lys Ala 355 360 365Gln Trp Asn Ser Leu Ser Phe Ala Asp Val Asn Thr Lys Glu Ala Gln 370 375 380Asp Leu Ala Leu Arg Ser Ala Val Glu Gly Ala Val Leu Leu Lys Asn385 390 395 400Asp Gly Thr Leu Pro Leu Lys Leu Lys Lys Lys Asp Ser Val Ala Met 405 410 415Ile Gly Phe Trp Ala Asn Asp Thr Ser Lys Leu Gln Gly Gly Tyr Ser 420 425 430Gly Arg Ala Pro Phe Leu His Ser Pro Leu Tyr Ala Ala Glu Lys Leu 435 440 445Gly Leu Asp Thr Asn Val Ala Trp Gly Pro Thr Leu Gln Asn Ser Ser 450 455 460Ser His Asp Asn Trp Thr Thr Asn Ala Val Ala Ala Ala Lys Lys Ser465 470 475 480Asp Tyr Ile Leu Tyr Phe Gly Gly Leu Asp Ala Ser Ala Ala Gly Glu 485 490 495Asp Arg Asp Arg Glu Asn Leu Asp Trp Pro Glu Ser Gln Leu Thr Leu 500 505 510Leu Gln Lys Leu Ser Ser Leu Gly Lys Pro Leu Val Val Ile Gln Leu 515 520 525Gly Asp Gln Val Asp Asp Thr Ala Leu Leu Lys Asn Lys Lys Ile Asn 530 535 540Ser Ile Leu Trp Val Asn Tyr Pro Gly Gln Asp Gly Gly Thr Ala Val545 550 555 560Met Asp Leu Leu Thr Gly Arg Lys Ser Pro Ala Gly Arg Leu Pro Val 565 570 575Thr Gln Tyr Pro Ser Lys Tyr Thr Glu Gln Ile Gly Met Thr Asp Met 580 585 590Asp Leu Arg Pro Thr Lys Ser Leu Pro Gly Arg Thr Tyr Arg Trp Tyr 595 600 605Ser Thr Pro Val Leu Pro Tyr Gly Phe Gly Leu His Tyr Thr Lys Phe 610 615 620Gln Ala Lys Phe Lys Ser Asn Lys Leu Thr Phe Asp Ile Gln Lys Leu625 630 635 640Leu Lys Gly Cys Ser Ala Gln Tyr Ser Asp Thr Cys Ala Leu Pro Pro 645 650 655Ile Gln Val Ser Val Lys Asn Thr Gly Arg Ile Thr Ser Asp Phe Val 660 665 670Ser Leu Val Phe Ile Lys Ser Glu Val Gly Pro Lys Pro Tyr Pro Leu 675 680 685Lys Thr Leu Ala Ala Tyr Gly Arg Leu His Asp Val Ala Pro Ser Ser 690 695 700Thr Lys Asp Ile Ser Leu Glu Trp Thr Leu Asp Asn Ile Ala Arg Arg705 710 715 720Gly Glu Asn Gly Asp Leu Val Val Tyr Pro Gly Thr Tyr Thr Leu Leu 725 730 735Leu Asp Glu Pro Thr Gln Ala Lys Ile Gln Val Thr Leu Thr Gly Lys 740 745 750Lys Ala Ile Leu Asp Lys Trp Pro Gln Asp Pro Lys Ser Ala 755 760 76531338DNAPenicillium funiculosum 3atgcttcagc gatttgctta tattttacca ctggctctat tgagtgttgg agtgaaagcc 60gacaacccct ttgtgcagag catctacacc gctgatccgg caccgatggt atacaatgac 120cgcgtttatg tcttcatgga ccatgacaac accggagcta cctactacaa catgacagac 180tggcatctgt tctcgtcagc agatatggcg aattggcaag atcatggcat tccaatgagc 240ctggccaatt tcacctgggc caacgcgaat gcgtgggccc cgcaagtcat ccctcgcaac 300ggccaattct acttttatgc tcctgtccga cacaacgatg gttctatggc tatcggtgtg 360ggagtgagca gcaccatcac aggtccatac catgatgcta tcggcaaacc gctagtagag 420aacaacgaga ttgatcccac cgtgttcatc gacgatgacg gtcaggcata cctgtactgg 480ggaaatccag acctgtggta cgtcaaattg aaccaagata tgatatcgta cagcgggagc 540cctactcaga ttccactcac cacggctgga tttggtactc gaacgggcaa tgctcaacgg 600ccgaccactt ttgaagaagc tccatgggta tacaaacgca acggcatcta ctatatcgcc 660tatgcagccg attgttgttc tgaggatatt cgctactcca cgggaaccag tgccactggt 720ccgtggactt atcgaggcgt catcatgccg acccaaggta gcagcttcac caatcacgag 780ggtattatcg acttccagaa caactcctac tttttctatc acaacggcgc tcttcccggc 840ggaggcggct accaacgatc tgtatgtgtg gagcaattca aatacaatgc agatggaacc 900attccgacga tcgaaatgac caccgccggt ccagctcaaa ttgggactct caacccttac 960gtgcgacagg aagccgaaac ggcggcatgg tcttcaggca tcactacgga ggtttgtagc 1020gaaggcggaa ttgacgtcgg gtttatcaac aatggcgatt acatcaaagt taaaggcgta 1080gctttcggtt caggagccca ttctttctca gcgcgggttg cttctgcaaa tagcggcggc 1140actattgcaa tacacctcgg aagcacaact ggtacgctcg tgggcacttg tactgtcccc 1200agcactggcg gttggcagac ttggactacc gttacctgtt ctgtcagtgg cgcatctggg 1260acccaggatg tgtattttgt tttcggtggt agcggaacag gatacctgtt caactttgat 1320tattggcagt tcgcataa 13384445PRTPenicillium funiculosum 4Met Leu Gln Arg Phe Ala Tyr Ile Leu Pro Leu Ala Leu Leu Ser Val1 5 10 15Gly Val Lys Ala Asp Asn Pro Phe Val Gln Ser Ile Tyr Thr Ala Asp 20 25 30Pro Ala Pro Met Val Tyr Asn Asp Arg Val Tyr Val Phe Met Asp His 35 40 45Asp Asn Thr Gly Ala Thr Tyr Tyr Asn Met Thr Asp Trp His Leu Phe 50 55 60Ser Ser Ala Asp Met Ala Asn Trp Gln Asp His Gly Ile Pro Met Ser65 70 75 80Leu Ala Asn Phe Thr Trp Ala Asn Ala Asn Ala Trp Ala Pro Gln Val 85 90 95Ile Pro Arg Asn Gly Gln Phe Tyr Phe Tyr Ala Pro Val Arg His Asn 100 105 110Asp Gly Ser Met Ala Ile Gly Val Gly Val Ser Ser Thr Ile Thr Gly 115 120 125Pro Tyr His Asp Ala Ile Gly Lys Pro Leu Val Glu Asn Asn Glu Ile 130 135 140Asp Pro Thr Val Phe Ile Asp Asp Asp Gly Gln Ala Tyr Leu Tyr Trp145 150 155 160Gly Asn Pro Asp Leu Trp Tyr Val Lys Leu Asn Gln Asp Met Ile Ser 165 170 175Tyr Ser Gly Ser Pro Thr Gln Ile Pro Leu Thr Thr Ala Gly Phe Gly 180 185 190Thr Arg Thr Gly Asn Ala Gln Arg Pro Thr Thr Phe Glu Glu Ala Pro 195 200 205Trp Val Tyr Lys Arg Asn Gly Ile Tyr Tyr Ile Ala Tyr Ala Ala Asp 210 215 220Cys Cys Ser Glu Asp Ile Arg Tyr Ser Thr Gly Thr Ser Ala Thr Gly225 230 235 240Pro Trp Thr Tyr Arg Gly Val Ile Met Pro Thr Gln Gly Ser Ser Phe 245 250 255Thr Asn His Glu Gly Ile Ile Asp Phe Gln Asn Asn Ser Tyr Phe Phe 260 265 270Tyr His Asn Gly Ala Leu Pro Gly Gly Gly Gly Tyr Gln Arg Ser Val 275 280 285Cys Val Glu Gln Phe Lys Tyr Asn Ala Asp Gly Thr Ile Pro Thr Ile 290 295 300Glu Met Thr Thr Ala Gly Pro Ala Gln Ile Gly Thr Leu Asn Pro Tyr305 310 315 320Val Arg Gln Glu Ala Glu Thr Ala Ala Trp Ser Ser Gly Ile Thr Thr 325 330 335Glu Val Cys Ser Glu Gly Gly Ile Asp Val Gly Phe Ile Asn Asn Gly 340 345 350Asp Tyr Ile Lys Val Lys Gly Val Ala Phe Gly Ser Gly Ala His Ser 355 360 365Phe Ser Ala Arg Val Ala Ser Ala Asn Ser Gly Gly Thr Ile Ala Ile 370 375 380His Leu Gly Ser Thr Thr Gly Thr Leu Val Gly Thr Cys Thr Val Pro385 390 395 400Ser Thr Gly Gly Trp Gln Thr Trp Thr Thr Val Thr Cys Ser Val Ser 405 410 415Gly Ala Ser Gly Thr Gln Asp Val Tyr Phe Val Phe Gly Gly Ser Gly 420 425 430Thr Gly Tyr Leu Phe Asn Phe Asp Tyr Trp Gln Phe Ala 435 440 44551593DNAFusarium verticillioides 5atgaaggtat actggctcgt ggcgtgggcc acttctttga cgccggcact ggctggcttg 60attggacacc gtcgcgccac caccttcaac aatcctatca tctactcaga ctttccagat 120aacgatgtat tcctcggtcc agataactac tactacttct ctgcttccaa cttccacttc 180agcccaggag cacccgtttt gaagtctaaa gatctgctaa actgggatct catcggccat 240tcaattcccc gcctgaactt tggcgacggc tatgatcttc ctcctggctc acgttattac 300cgtggaggta cttgggcatc atccctcaga tacagaaaga gcaatggaca gtggtactgg 360atcggctgca tcaacttctg gcagacctgg gtatacactg cctcatcgcc ggaaggtcca 420tggtacaaca agggaaactt cggtgataac aattgctact acgacaatgg catactgatc 480gatgacgatg ataccatgta tgtcgtatac ggttccggtg aggtcaaagt atctcaacta 540tctcaggacg gattcagcca ggtcaaatct caggtagttt tcaagaacac tgatattggg 600gtccaagact tggagggtaa ccgcatgtac aagatcaacg ggctctacta tatcctaaac 660gatagcccaa gtggcagtca gacctggatt tggaagtcga aatcaccctg gggcccttat 720gagtctaagg tcctcgccga caaagtcacc ccgcctatct ctggtggtaa ctcgccgcat 780cagggtagtc tcataaagac tcccaatggt ggctggtact tcatgtcatt cacttgggcc 840tatcctgccg gccgtcttcc ggttcttgca ccgattacgt ggggtagcga tggtttcccc 900attcttgtca agggtgctaa tggcggatgg ggatcatctt acccaacact tcctggcacg 960gatggtgtga caaagaattg gacaaggact gataccttcc gcggaacctc acttgctccg 1020tcctgggagt ggaaccataa tccggacgtc aactccttca ctgtcaacaa cggcctgact 1080ctccgcactg ctagcattac gaaggatatt taccaggcga ggaacacgct atctcaccga 1140actcatggtg atcatccaac aggaatagtg aagattgatt tctctccgat gaaggacggc 1200gaccgggccg ggctttcagc gtttcgagac caaagtgcat acatcggtat tcatcgagat 1260aacggaaagt tcacaatcgc tacgaagcat gggatgaata tggatgagtg gaacggaaca 1320acaacagacc tgggacaaat aaaagccaca gctaatgtgc cttctggaag gaccaagatc 1380tggctgagac ttcaacttga taccaaccca gcaggaactg gcaacactat cttttcttac 1440agttgggatg gagtcaagta tgaaacactg ggtcccaact tcaaactgta caatggttgg 1500gcattcttta ttgcttaccg attcggcatc ttcaacttcg ccgagacggc tttaggaggc 1560tcgatcaagg ttgagtcttt cacagctgca tag 15936530PRTFusarium verticillioides 6Met Lys Val Tyr Trp Leu Val Ala Trp Ala Thr Ser Leu Thr Pro Ala1 5 10 15Leu Ala Gly Leu Ile Gly His Arg Arg Ala Thr Thr Phe Asn Asn Pro 20 25 30Ile Ile Tyr Ser Asp Phe Pro Asp Asn Asp Val Phe Leu Gly Pro Asp 35 40 45Asn Tyr Tyr Tyr Phe Ser Ala Ser Asn Phe His Phe Ser Pro Gly Ala 50 55 60Pro Val Leu Lys Ser Lys Asp Leu Leu Asn Trp Asp Leu Ile Gly His65 70 75 80Ser Ile Pro Arg Leu Asn Phe Gly Asp Gly Tyr Asp Leu Pro Pro Gly 85 90 95Ser Arg Tyr Tyr Arg Gly Gly Thr Trp Ala Ser Ser Leu Arg Tyr Arg 100 105 110Lys Ser Asn Gly Gln Trp Tyr Trp Ile Gly Cys Ile Asn Phe Trp Gln 115 120 125Thr Trp Val Tyr Thr Ala Ser Ser Pro Glu Gly Pro Trp Tyr Asn Lys 130 135 140Gly Asn Phe Gly Asp Asn Asn Cys Tyr Tyr Asp Asn Gly Ile Leu Ile145 150 155 160Asp Asp Asp Asp Thr Met Tyr Val Val Tyr Gly Ser Gly Glu Val Lys 165 170 175Val Ser Gln Leu Ser Gln Asp Gly Phe Ser Gln Val Lys Ser Gln Val 180 185 190Val Phe Lys Asn Thr Asp Ile Gly Val Gln Asp Leu Glu Gly Asn Arg 195 200 205Met Tyr Lys Ile Asn Gly Leu Tyr Tyr Ile Leu Asn Asp Ser Pro Ser 210 215 220Gly Ser Gln Thr Trp Ile Trp Lys Ser Lys Ser Pro Trp Gly Pro Tyr225 230 235 240Glu Ser Lys Val Leu Ala Asp Lys Val Thr Pro Pro Ile Ser Gly Gly 245 250 255Asn Ser Pro His Gln Gly Ser Leu Ile Lys Thr Pro Asn Gly Gly Trp 260 265 270Tyr Phe Met Ser Phe Thr Trp Ala Tyr Pro Ala Gly Arg Leu Pro Val 275 280 285Leu Ala Pro Ile Thr Trp Gly Ser Asp Gly Phe Pro Ile Leu Val Lys 290 295 300Gly Ala Asn Gly Gly Trp Gly Ser Ser Tyr Pro Thr Leu Pro Gly Thr305 310 315 320Asp Gly Val Thr Lys Asn Trp Thr Arg Thr Asp Thr Phe Arg Gly Thr 325 330 335Ser Leu Ala Pro Ser Trp Glu Trp Asn His Asn Pro

Asp Val Asn Ser 340 345 350Phe Thr Val Asn Asn Gly Leu Thr Leu Arg Thr Ala Ser Ile Thr Lys 355 360 365Asp Ile Tyr Gln Ala Arg Asn Thr Leu Ser His Arg Thr His Gly Asp 370 375 380His Pro Thr Gly Ile Val Lys Ile Asp Phe Ser Pro Met Lys Asp Gly385 390 395 400Asp Arg Ala Gly Leu Ser Ala Phe Arg Asp Gln Ser Ala Tyr Ile Gly 405 410 415Ile His Arg Asp Asn Gly Lys Phe Thr Ile Ala Thr Lys His Gly Met 420 425 430Asn Met Asp Glu Trp Asn Gly Thr Thr Thr Asp Leu Gly Gln Ile Lys 435 440 445Ala Thr Ala Asn Val Pro Ser Gly Arg Thr Lys Ile Trp Leu Arg Leu 450 455 460Gln Leu Asp Thr Asn Pro Ala Gly Thr Gly Asn Thr Ile Phe Ser Tyr465 470 475 480Ser Trp Asp Gly Val Lys Tyr Glu Thr Leu Gly Pro Asn Phe Lys Leu 485 490 495Tyr Asn Gly Trp Ala Phe Phe Ile Ala Tyr Arg Phe Gly Ile Phe Asn 500 505 510Phe Ala Glu Thr Ala Leu Gly Gly Ser Ile Lys Val Glu Ser Phe Thr 515 520 525Ala Ala 53071374DNAFusarium verticillioides 7atgcactacg ctaccctcac cactttggtg ctggctctga ccaccaacgt cgctgcacag 60caaggcacag caactgtcga cctctccaaa aatcatggac cggcgaaggc ccttggttca 120ggcttcatat acggctggcc tgacaacgga acaagcgtcg acacctccat accagatttc 180ttggtaactg acatcaaatt caactcaaac cgcggcggtg gcgcccaaat cccatcactg 240ggttgggcca gaggtggcta tgaaggatac ctcggccgct tcaactcaac cttatccaac 300tatcgcacca cgcgcaagta taacgctgac tttatcttgt tgcctcatga cctctggggt 360gcggatggcg ggcagggttc aaactccccg tttcctggcg acaatggcaa ttggactgag 420atggagttat tctggaatca gcttgtgtct gacttgaagg ctcataatat gctggaaggt 480cttgtgattg atgtttggaa tgagcctgat attgatatct tttgggatcg cccgtggtcg 540cagtttcttg agtattacaa tcgcgcgacc aaactacttc ggtgagtcta ctactgatcc 600atacgtattt acagtgagct gactggtcga attagaaaaa cacttcccaa aactcttctc 660agtggcccag ccatggcaca ttctcccatt ctgtccgatg ataaatggca tacctggctt 720caatcagtag cgggtaacaa gacagtccct gatatttact cctggcatca gattggcgct 780tgggaacgtg agccggacag cactatcccc gactttacca ccttgcgggc gcaatatggc 840gttcccgaga agccaattga cgtcaatgag tacgctgcac gcgatgagca aaatccagcc 900aactccgtct actacctctc tcaactagag cgtcataacc ttagaggtct tcgcgcaaac 960tggggtagcg gatctgacct ccacaactgg atgggcaact tgatttacag cactaccggt 1020acctcggagg ggacttacta ccctaatggt gaatggcagg cttacaagta ctatgcggcc 1080atggcagggc agagacttgt gaccaaagca tcgtcggact tgaagtttga tgtctttgcc 1140actaagcaag gccgtaagat taagattata gccggcacga ggaccgttca agcaaagtat 1200aacatcaaaa tcagcggttt ggaagtagca ggacttccta agatgggtac ggtaaaggtc 1260cggacttatc ggttcgactg ggctgggccg aatggaaagg ttgacgggcc tgttgatttg 1320ggggagaaga agtatactta ttcggccaat acggtgagca gcccctctac ttga 13748439PRTFusarium verticillioides 8Met His Tyr Ala Thr Leu Thr Thr Leu Val Leu Ala Leu Thr Thr Asn1 5 10 15Val Ala Ala Gln Gln Gly Thr Ala Thr Val Asp Leu Ser Lys Asn His 20 25 30Gly Pro Ala Lys Ala Leu Gly Ser Gly Phe Ile Tyr Gly Trp Pro Asp 35 40 45Asn Gly Thr Ser Val Asp Thr Ser Ile Pro Asp Phe Leu Val Thr Asp 50 55 60Ile Lys Phe Asn Ser Asn Arg Gly Gly Gly Ala Gln Ile Pro Ser Leu65 70 75 80Gly Trp Ala Arg Gly Gly Tyr Glu Gly Tyr Leu Gly Arg Phe Asn Ser 85 90 95Thr Leu Ser Asn Tyr Arg Thr Thr Arg Lys Tyr Asn Ala Asp Phe Ile 100 105 110Leu Leu Pro His Asp Leu Trp Gly Ala Asp Gly Gly Gln Gly Ser Asn 115 120 125Ser Pro Phe Pro Gly Asp Asn Gly Asn Trp Thr Glu Met Glu Leu Phe 130 135 140Trp Asn Gln Leu Val Ser Asp Leu Lys Ala His Asn Met Leu Glu Gly145 150 155 160Leu Val Ile Asp Val Trp Asn Glu Pro Asp Ile Asp Ile Phe Trp Asp 165 170 175Arg Pro Trp Ser Gln Phe Leu Glu Tyr Tyr Asn Arg Ala Thr Lys Leu 180 185 190Leu Arg Lys Thr Leu Pro Lys Thr Leu Leu Ser Gly Pro Ala Met Ala 195 200 205His Ser Pro Ile Leu Ser Asp Asp Lys Trp His Thr Trp Leu Gln Ser 210 215 220Val Ala Gly Asn Lys Thr Val Pro Asp Ile Tyr Ser Trp His Gln Ile225 230 235 240Gly Ala Trp Glu Arg Glu Pro Asp Ser Thr Ile Pro Asp Phe Thr Thr 245 250 255Leu Arg Ala Gln Tyr Gly Val Pro Glu Lys Pro Ile Asp Val Asn Glu 260 265 270Tyr Ala Ala Arg Asp Glu Gln Asn Pro Ala Asn Ser Val Tyr Tyr Leu 275 280 285Ser Gln Leu Glu Arg His Asn Leu Arg Gly Leu Arg Ala Asn Trp Gly 290 295 300Ser Gly Ser Asp Leu His Asn Trp Met Gly Asn Leu Ile Tyr Ser Thr305 310 315 320Thr Gly Thr Ser Glu Gly Thr Tyr Tyr Pro Asn Gly Glu Trp Gln Ala 325 330 335Tyr Lys Tyr Tyr Ala Ala Met Ala Gly Gln Arg Leu Val Thr Lys Ala 340 345 350Ser Ser Asp Leu Lys Phe Asp Val Phe Ala Thr Lys Gln Gly Arg Lys 355 360 365Ile Lys Ile Ile Ala Gly Thr Arg Thr Val Gln Ala Lys Tyr Asn Ile 370 375 380Lys Ile Ser Gly Leu Glu Val Ala Gly Leu Pro Lys Met Gly Thr Val385 390 395 400Lys Val Arg Thr Tyr Arg Phe Asp Trp Ala Gly Pro Asn Gly Lys Val 405 410 415Asp Gly Pro Val Asp Leu Gly Glu Lys Lys Tyr Thr Tyr Ser Ala Asn 420 425 430Thr Val Ser Ser Pro Ser Thr 43591350DNAFusarium verticillioides 9atgtggctga cctccccatt gctgttcgcc agcaccctcc tgggcctcac tggcgttgct 60ctagcagaca accccatcgt ccaagacatc tacaccgcag acccagcacc aatggtctac 120aatggccgcg tctacctctt cacaggccat gacaacgacg gctctaccga cttcaacatg 180acagactggc gtctcttctc gtcagcagac atggtcaact ggcagcacca tggtgtcccc 240atgagcttaa agaccttcag ctgggccaac agcagagcct gggctggtca agtcgttgcc 300cgaaacggaa agttttactt ctatgttcct gtccgtaatg ccaagacggg tggaatggct 360attggtgtcg gtgttagtac caacatcctt gggccctaca ctgatgccct tggaaagcca 420ttggtcgaga acaatgagat cgacccaact gtctacatcg acactgatgg ccaggcctat 480ctctactggg gcaaccctgg attgtactac gtcaagctca accaagacat gctctcctac 540agtggtagca tcaacaaagt atcgctcaca acagctggat tcggcagccg cccgaacaac 600gcgcagcgtc ctactacttt cgaggaagga ccgtggctgt acaagcgtgg aaatctctac 660tacatgatct acgcagccaa ctgctgttcc gaggacattc gctactcaac tggacccagc 720gccactggac cttggactta ccgcggtgtc gtgatgaaca aggcgggtcg aagcttcacc 780aaccatcctg gcatcatcga ctttgagaac aactcgtact tcttttacca caatggcgct 840cttgatggag gtagcggtta tactcggtct gtggctgtcg agagcttcaa gtatggttcg 900gacggtctga tccccgagat caagatgact acgcaaggcc cagcgcagct caagtctctg 960aacccatatg tcaagcagga ggccgagact atcgcctggt ctgagggtat cgagactgag 1020gtctgcagcg aaggtggtct caacgttgct ttcatcgaca atggtgacta catcaaggtc 1080aagggagtcg actttggcag caccggtgca aagacgttca gcgcccgtgt tgcttccaac 1140agcagcggag gcaagattga gcttcgactt ggtagcaaga ccggtaagtt ggttggtacc 1200tgcacggtaa cgactacggg aaactggcag acttataaga ctgtggattg ccccgtcagt 1260ggtgctactg gtacgagcga tctattcttt gtcttcacgg gctctgggtc tggctctctg 1320ttcaacttca actggtggca gtttagctaa 135010449PRTFusarium verticillioides 10Met Trp Leu Thr Ser Pro Leu Leu Phe Ala Ser Thr Leu Leu Gly Leu1 5 10 15Thr Gly Val Ala Leu Ala Asp Asn Pro Ile Val Gln Asp Ile Tyr Thr 20 25 30Ala Asp Pro Ala Pro Met Val Tyr Asn Gly Arg Val Tyr Leu Phe Thr 35 40 45Gly His Asp Asn Asp Gly Ser Thr Asp Phe Asn Met Thr Asp Trp Arg 50 55 60Leu Phe Ser Ser Ala Asp Met Val Asn Trp Gln His His Gly Val Pro65 70 75 80Met Ser Leu Lys Thr Phe Ser Trp Ala Asn Ser Arg Ala Trp Ala Gly 85 90 95Gln Val Val Ala Arg Asn Gly Lys Phe Tyr Phe Tyr Val Pro Val Arg 100 105 110Asn Ala Lys Thr Gly Gly Met Ala Ile Gly Val Gly Val Ser Thr Asn 115 120 125Ile Leu Gly Pro Tyr Thr Asp Ala Leu Gly Lys Pro Leu Val Glu Asn 130 135 140Asn Glu Ile Asp Pro Thr Val Tyr Ile Asp Thr Asp Gly Gln Ala Tyr145 150 155 160Leu Tyr Trp Gly Asn Pro Gly Leu Tyr Tyr Val Lys Leu Asn Gln Asp 165 170 175Met Leu Ser Tyr Ser Gly Ser Ile Asn Lys Val Ser Leu Thr Thr Ala 180 185 190Gly Phe Gly Ser Arg Pro Asn Asn Ala Gln Arg Pro Thr Thr Phe Glu 195 200 205Glu Gly Pro Trp Leu Tyr Lys Arg Gly Asn Leu Tyr Tyr Met Ile Tyr 210 215 220Ala Ala Asn Cys Cys Ser Glu Asp Ile Arg Tyr Ser Thr Gly Pro Ser225 230 235 240Ala Thr Gly Pro Trp Thr Tyr Arg Gly Val Val Met Asn Lys Ala Gly 245 250 255Arg Ser Phe Thr Asn His Pro Gly Ile Ile Asp Phe Glu Asn Asn Ser 260 265 270Tyr Phe Phe Tyr His Asn Gly Ala Leu Asp Gly Gly Ser Gly Tyr Thr 275 280 285Arg Ser Val Ala Val Glu Ser Phe Lys Tyr Gly Ser Asp Gly Leu Ile 290 295 300Pro Glu Ile Lys Met Thr Thr Gln Gly Pro Ala Gln Leu Lys Ser Leu305 310 315 320Asn Pro Tyr Val Lys Gln Glu Ala Glu Thr Ile Ala Trp Ser Glu Gly 325 330 335Ile Glu Thr Glu Val Cys Ser Glu Gly Gly Leu Asn Val Ala Phe Ile 340 345 350Asp Asn Gly Asp Tyr Ile Lys Val Lys Gly Val Asp Phe Gly Ser Thr 355 360 365Gly Ala Lys Thr Phe Ser Ala Arg Val Ala Ser Asn Ser Ser Gly Gly 370 375 380Lys Ile Glu Leu Arg Leu Gly Ser Lys Thr Gly Lys Leu Val Gly Thr385 390 395 400Cys Thr Val Thr Thr Thr Gly Asn Trp Gln Thr Tyr Lys Thr Val Asp 405 410 415Cys Pro Val Ser Gly Ala Thr Gly Thr Ser Asp Leu Phe Phe Val Phe 420 425 430Thr Gly Ser Gly Ser Gly Ser Leu Phe Asn Phe Asn Trp Trp Gln Phe 435 440 445Ser 111725DNAFusarium verticillioides 11atgcgcttct cttggctatt gtgccccctt ctagcgatgg gaagtgctct tcctgaaacg 60aagacggatg tttcgacata caccaaccct gtccttccag gatggcactc ggatccatcg 120tgtatccaga aagatggcct ctttctctgc gtcacttcaa cattcatctc cttcccaggt 180cttcccgtct atgcctcaag ggatctagtc aactggcgtc tcatcagcca tgtctggaac 240cgcgagaaac agttgcctgg cattagctgg aagacggcag gacagcaaca gggaatgtat 300gcaccaacca ttcgatacca caagggaaca tactacgtca tctgcgaata cctgggcgtt 360ggagatatta ttggtgtcat cttcaagacc accaatccgt gggacgagag tagctggagt 420gaccctgtta ccttcaagcc aaatcacatc gaccccgatc tgttctggga tgatgacgga 480aaggtttatt gtgctaccca tggcatcact ctgcaggaga ttgatttgga aactggagag 540cttagcccgg agcttaatat ctggaacggc acaggaggtg tatggcctga gggtccccat 600atctacaagc gcgacggtta ctactatctc atgattgccg agggtggaac tgccgaagac 660cacgctatca caatcgctcg ggcccgcaag atcaccggcc cctatgaagc ctacaataac 720aacccaatct tgaccaaccg cgggacatct gagtacttcc agactgtcgg tcacggtgat 780ctgttccaag ataccaaggg caactggtgg ggtctttgtc ttgctactcg catcacagca 840cagggagttt cacccatggg ccgtgaagct gttttgttca atggcacatg gaacaagggc 900gaatggccca agttgcaacc agtacgaggt cgcatgcctg gaaacctcct cccaaagccg 960acgcgaaacg ttcccggaga tgggcccttc aacgctgacc cagacaacta caacttgaag 1020aagactaaga agatccctcc tcactttgtg caccatagag tcccaagaga cggtgccttc 1080tctttgtctt ccaagggtct gcacatcgtg cctagtcgaa acaacgttac cggtagtgtg 1140ttgccaggag atgagattga gctatcagga cagcgaggtc tagctttcat cggacgccgc 1200caaactcaca ctctgttcaa atatagtgtt gatatcgact tcaagcccaa gtccgatgat 1260caggaagctg gaatcaccgt tttccgcacg cagttcgacc atatcgatct tggcattgtt 1320cgtcttccta caaaccaagg cagcaacaag aaatctaagc ttgccttccg attccgggcc 1380acaggagctc agaatgttcc tgcaccgaag gtagtaccgg tccccgatgg ctgggagaag 1440ggcgtaatca gtctacatat cgaggcagcc aacgcgacgc actacaacct tggagcttcg 1500agccacagag gcaagactct cgacatcgcg acagcatcag caagtcttgt gagtggaggc 1560acgggttcat ttgttggtag tttgcttgga ccttatgcta cctgcaacgg caaaggatct 1620ggagtggaat gtcccaaggg aggtgatgtc tatgtgaccc aatggactta taagcccgtg 1680gcacaagaga ttgatcatgg tgtttttgtg aaatcagaat tgtag 172512574PRTFusarium verticillioides 12Met Arg Phe Ser Trp Leu Leu Cys Pro Leu Leu Ala Met Gly Ser Ala1 5 10 15Leu Pro Glu Thr Lys Thr Asp Val Ser Thr Tyr Thr Asn Pro Val Leu 20 25 30Pro Gly Trp His Ser Asp Pro Ser Cys Ile Gln Lys Asp Gly Leu Phe 35 40 45Leu Cys Val Thr Ser Thr Phe Ile Ser Phe Pro Gly Leu Pro Val Tyr 50 55 60Ala Ser Arg Asp Leu Val Asn Trp Arg Leu Ile Ser His Val Trp Asn65 70 75 80Arg Glu Lys Gln Leu Pro Gly Ile Ser Trp Lys Thr Ala Gly Gln Gln 85 90 95Gln Gly Met Tyr Ala Pro Thr Ile Arg Tyr His Lys Gly Thr Tyr Tyr 100 105 110Val Ile Cys Glu Tyr Leu Gly Val Gly Asp Ile Ile Gly Val Ile Phe 115 120 125Lys Thr Thr Asn Pro Trp Asp Glu Ser Ser Trp Ser Asp Pro Val Thr 130 135 140Phe Lys Pro Asn His Ile Asp Pro Asp Leu Phe Trp Asp Asp Asp Gly145 150 155 160Lys Val Tyr Cys Ala Thr His Gly Ile Thr Leu Gln Glu Ile Asp Leu 165 170 175Glu Thr Gly Glu Leu Ser Pro Glu Leu Asn Ile Trp Asn Gly Thr Gly 180 185 190Gly Val Trp Pro Glu Gly Pro His Ile Tyr Lys Arg Asp Gly Tyr Tyr 195 200 205Tyr Leu Met Ile Ala Glu Gly Gly Thr Ala Glu Asp His Ala Ile Thr 210 215 220Ile Ala Arg Ala Arg Lys Ile Thr Gly Pro Tyr Glu Ala Tyr Asn Asn225 230 235 240Asn Pro Ile Leu Thr Asn Arg Gly Thr Ser Glu Tyr Phe Gln Thr Val 245 250 255Gly His Gly Asp Leu Phe Gln Asp Thr Lys Gly Asn Trp Trp Gly Leu 260 265 270Cys Leu Ala Thr Arg Ile Thr Ala Gln Gly Val Ser Pro Met Gly Arg 275 280 285Glu Ala Val Leu Phe Asn Gly Thr Trp Asn Lys Gly Glu Trp Pro Lys 290 295 300Leu Gln Pro Val Arg Gly Arg Met Pro Gly Asn Leu Leu Pro Lys Pro305 310 315 320Thr Arg Asn Val Pro Gly Asp Gly Pro Phe Asn Ala Asp Pro Asp Asn 325 330 335Tyr Asn Leu Lys Lys Thr Lys Lys Ile Pro Pro His Phe Val His His 340 345 350Arg Val Pro Arg Asp Gly Ala Phe Ser Leu Ser Ser Lys Gly Leu His 355 360 365Ile Val Pro Ser Arg Asn Asn Val Thr Gly Ser Val Leu Pro Gly Asp 370 375 380Glu Ile Glu Leu Ser Gly Gln Arg Gly Leu Ala Phe Ile Gly Arg Arg385 390 395 400Gln Thr His Thr Leu Phe Lys Tyr Ser Val Asp Ile Asp Phe Lys Pro 405 410 415Lys Ser Asp Asp Gln Glu Ala Gly Ile Thr Val Phe Arg Thr Gln Phe 420 425 430Asp His Ile Asp Leu Gly Ile Val Arg Leu Pro Thr Asn Gln Gly Ser 435 440 445Asn Lys Lys Ser Lys Leu Ala Phe Arg Phe Arg Ala Thr Gly Ala Gln 450 455 460Asn Val Pro Ala Pro Lys Val Val Pro Val Pro Asp Gly Trp Glu Lys465 470 475 480Gly Val Ile Ser Leu His Ile Glu Ala Ala Asn Ala Thr His Tyr Asn 485 490 495Leu Gly Ala Ser Ser His Arg Gly Lys Thr Leu Asp Ile Ala Thr Ala 500 505 510Ser Ala Ser Leu Val Ser Gly Gly Thr Gly Ser Phe Val Gly Ser Leu 515 520 525Leu Gly Pro Tyr Ala Thr Cys Asn Gly Lys Gly Ser Gly Val Glu Cys 530 535 540Pro Lys Gly Gly Asp Val Tyr Val Thr Gln Trp Thr Tyr Lys Pro Val545 550 555 560Ala Gln Glu Ile Asp His Gly Val Phe Val Lys Ser Glu Leu 565 570132251DNAPodospora anserina 13atgatccacc tcaagccagc cctcgcggcg ttgttggcgc tgtcgacgca atgtgtggct 60attgatttgt ttgtcaagtc ttcggggggg aataagacga ctgatatcat gtatggtctt 120atgcacgagg tatgtgtttt gcgagatctc ccttttgttt ttgcgcactg ctgacatgga 180gactgcaaac aggatatcaa caactccggc gacggcggca tctacgccga gctaatctcc 240aaccgcgcgt tccaagggag tgagaagttc ccctccaacc tcgacaactg gagccccgtc 300ggtggcgcta cccttaccct tcagaagctt gccaagcccc tttcctctgc

gttgccttac 360tccgtcaatg ttgccaaccc caaggagggc aagggcaagg gcaaggacac caaggggaag 420aaggttggct tggccaatgc tgggttttgg ggtatggatg tcaagaggca gaagtacact 480ggtagcttcc acgttactgg tgagtacaag ggtgactttg aggttagctt gcgcagcgcg 540attaccgggg agacctttgg caagaaggtg gtgaagggtg ggagtaagaa ggggaagtgg 600accgagaagg agtttgagtt ggtgcctttc aaggatgcgc ccaacagcaa caacaccttt 660gttgtgcagt gggatgccga ggtatgtgct tctttgatat tggctgagat agaagttggg 720ttgacatgat gtggtgcagg gcgcaaagga cggatctttg gatctcaact tgatcagctt 780gttccctccg acattcaagg gaaggaagaa tgggctgaga attgatcttg cgcagacgat 840ggttgagctc aagccggtaa gtcctctcta gtcagaaaag tagagccttt gttaacgctt 900gacagacctt cttgcgcttc cccggtggca acatgctcga gggtaacacc ttggacactt 960ggtggaagtg gtacgagacc attggccctc tgaaggatcg cccgggcatg gctggtgtct 1020gggagtacca gcaaaccctt ggcttgggtc tggtcgagta catggagtgg gccgatgaca 1080tgaacttgga gcccagtatg tgatcccatt ttctggagtg acttctcttg ctaacgtatc 1140cacagttgtc ggtgtcttcg ctggtcttgc cctcgatggc tcgttcgttc ccgaatccga 1200gatgggatgg gtcatccaac aggctctcga cgaaatcgag ttcctcactg gcgatgctaa 1260gaccaccaaa tggggtgccg tccgcgcgaa gcttggtcac cccaagcctt ggaaggtcaa 1320gtgggttgag atcggtaacg aggattggct tgccggacgc cctgctggct tcgagtcgta 1380catcaactac cgcttcccca tgatgatgaa ggccttcaac gaaaagtacc ccgacatcaa 1440gatcatcgcc tcgccctcca tcttcgacaa catgacaatc cccgcgggtg ctgccggtga 1500tcaccacccg tacctgactc ccgatgagtt cgttgagcga ttcgccaagt tcgataactt 1560gagcaaggat aacgtgacgc tcatcggcga ggctgcgtcg acgcatccta acggtggtat 1620cgcttgggag ggagatctca tgcccttgcc ttggtggggc ggcagtgttg ctgaggctat 1680cttcttgatc agcactgaga gaaacggtga caagatcatc ggtgctactt acgcgcctgg 1740tcttcgcagc ttggaccgct ggcaatggag catgacctgg gtgcagcatg ccgccgaccc 1800ggccctcacc actcgctcga ccagttggta tgtctggaga atcctcgccc accacatcat 1860ccgtgagacg ctcccggtcg atgccccggc cggcaagccc aactttgacc ctctgttcta 1920cgttgccgga aagagcgaga gtggcaccgg tatcttcaag gctgccgtct acaactcgac 1980tgaatcgatc ccggtgtcgt tgaagtttga tggtctcaac gagggagcgg ttgccaactt 2040gacggtgctt actgggccgg aggatccgta tggatacaac gaccccttca ctggtatcaa 2100tgttgtcaag gagaagacca ccttcatcaa ggccggaaag ggcggcaagt tcaccttcac 2160cctgccgggc ttgagtgttg ctgtgttgga gacggccgac gcggtcaagg gtggcaaggg 2220aaagggcaag ggcaagggaa agggtaactg a 225114676PRTPodospora anserina 14Met Ile His Leu Lys Pro Ala Leu Ala Ala Leu Leu Ala Leu Ser Thr1 5 10 15Gln Cys Val Ala Ile Asp Leu Phe Val Lys Ser Ser Gly Gly Asn Lys 20 25 30Thr Thr Asp Ile Met Tyr Gly Leu Met His Glu Asp Ile Asn Asn Ser 35 40 45Gly Asp Gly Gly Ile Tyr Ala Glu Leu Ile Ser Asn Arg Ala Phe Gln 50 55 60Gly Ser Glu Lys Phe Pro Ser Asn Leu Asp Asn Trp Ser Pro Val Gly65 70 75 80Gly Ala Thr Leu Thr Leu Gln Lys Leu Ala Lys Pro Leu Ser Ser Ala 85 90 95Leu Pro Tyr Ser Val Asn Val Ala Asn Pro Lys Glu Gly Lys Gly Lys 100 105 110Gly Lys Asp Thr Lys Gly Lys Lys Val Gly Leu Ala Asn Ala Gly Phe 115 120 125Trp Gly Met Asp Val Lys Arg Gln Lys Tyr Thr Gly Ser Phe His Val 130 135 140Thr Gly Glu Tyr Lys Gly Asp Phe Glu Val Ser Leu Arg Ser Ala Ile145 150 155 160Thr Gly Glu Thr Phe Gly Lys Lys Val Val Lys Gly Gly Ser Lys Lys 165 170 175Gly Lys Trp Thr Glu Lys Glu Phe Glu Leu Val Pro Phe Lys Asp Ala 180 185 190Pro Asn Ser Asn Asn Thr Phe Val Val Gln Trp Asp Ala Glu Gly Ala 195 200 205Lys Asp Gly Ser Leu Asp Leu Asn Leu Ile Ser Leu Phe Pro Pro Thr 210 215 220Phe Lys Gly Arg Lys Asn Gly Leu Arg Ile Asp Leu Ala Gln Thr Met225 230 235 240Val Glu Leu Lys Pro Thr Phe Leu Arg Phe Pro Gly Gly Asn Met Leu 245 250 255Glu Gly Asn Thr Leu Asp Thr Trp Trp Lys Trp Tyr Glu Thr Ile Gly 260 265 270Pro Leu Lys Asp Arg Pro Gly Met Ala Gly Val Trp Glu Tyr Gln Gln 275 280 285Thr Leu Gly Leu Gly Leu Val Glu Tyr Met Glu Trp Ala Asp Asp Met 290 295 300Asn Leu Glu Pro Ile Val Gly Val Phe Ala Gly Leu Ala Leu Asp Gly305 310 315 320Ser Phe Val Pro Glu Ser Glu Met Gly Trp Val Ile Gln Gln Ala Leu 325 330 335Asp Glu Ile Glu Phe Leu Thr Gly Asp Ala Lys Thr Thr Lys Trp Gly 340 345 350Ala Val Arg Ala Lys Leu Gly His Pro Lys Pro Trp Lys Val Lys Trp 355 360 365Val Glu Ile Gly Asn Glu Asp Trp Leu Ala Gly Arg Pro Ala Gly Phe 370 375 380Glu Ser Tyr Ile Asn Tyr Arg Phe Pro Met Met Met Lys Ala Phe Asn385 390 395 400Glu Lys Tyr Pro Asp Ile Lys Ile Ile Ala Ser Pro Ser Ile Phe Asp 405 410 415Asn Met Thr Ile Pro Ala Gly Ala Ala Gly Asp His His Pro Tyr Leu 420 425 430Thr Pro Asp Glu Phe Val Glu Arg Phe Ala Lys Phe Asp Asn Leu Ser 435 440 445Lys Asp Asn Val Thr Leu Ile Gly Glu Ala Ala Ser Thr His Pro Asn 450 455 460Gly Gly Ile Ala Trp Glu Gly Asp Leu Met Pro Leu Pro Trp Trp Gly465 470 475 480Gly Ser Val Ala Glu Ala Ile Phe Leu Ile Ser Thr Glu Arg Asn Gly 485 490 495Asp Lys Ile Ile Gly Ala Thr Tyr Ala Pro Gly Leu Arg Ser Leu Asp 500 505 510Arg Trp Gln Trp Ser Met Thr Trp Val Gln His Ala Ala Asp Pro Ala 515 520 525Leu Thr Thr Arg Ser Thr Ser Trp Tyr Val Trp Arg Ile Leu Ala His 530 535 540His Ile Ile Arg Glu Thr Leu Pro Val Asp Ala Pro Ala Gly Lys Pro545 550 555 560Asn Phe Asp Pro Leu Phe Tyr Val Ala Gly Lys Ser Glu Ser Gly Thr 565 570 575Gly Ile Phe Lys Ala Ala Val Tyr Asn Ser Thr Glu Ser Ile Pro Val 580 585 590Ser Leu Lys Phe Asp Gly Leu Asn Glu Gly Ala Val Ala Asn Leu Thr 595 600 605Val Leu Thr Gly Pro Glu Asp Pro Tyr Gly Tyr Asn Asp Pro Phe Thr 610 615 620Gly Ile Asn Val Val Lys Glu Lys Thr Thr Phe Ile Lys Ala Gly Lys625 630 635 640Gly Gly Lys Phe Thr Phe Thr Leu Pro Gly Leu Ser Val Ala Val Leu 645 650 655Glu Thr Ala Asp Ala Val Lys Gly Gly Lys Gly Lys Gly Lys Gly Lys 660 665 670Gly Lys Gly Asn 675151023DNAGibberella zeae 15atgaagtcca agttgttatt cccactcctc tctttcgttg gtcaaagtct tgccaccaac 60gacgactgtc ctctcatcac tagtagatgg actgcggatc cttcggctca tgtctttaac 120gacaccttgt ggctctaccc gtctcatgac atcgatgctg gatttgagaa tgatcctgat 180ggaggccagt acgccatgag agattaccat gtctactcta tcgacaagat ctacggttcc 240ctgccggtcg atcacggtac ggccctgtca gtggaggatg tcccctgggc ctctcgacag 300atgtgggctc ctgacgctgc ccacaagaac ggcaaatact acctatactt ccctgccaaa 360gacaaggatg atatcttcag aatcggcgtt gctgtctcac caacccccgg cggaccattc 420gtccccgaca agagttggat ccctcacact ttcagcatcg accccgccag tttcgtcgat 480gatgatgaca gagcctactt ggcatggggt ggtatcatgg gtggccagct tcaacgatgg 540caggataaga acaagtacaa cgaatctggc actgagccag gaaacggcac cgctgccttg 600agccctcaga ttgccaagct gagcaaggac atgcacactc tggcagagaa gcctcgcgac 660atgctcattc ttgaccccaa gactggcaag ccgctccttt ctgaggatga agaccgacgc 720ttcttcgaag gaccctggat tcacaagcgc aacaagattt actacctcac ctactctact 780ggcacaaccc actatcttgt ctatgcgact tcaaagaccc cctatggtcc ttacacctac 840cagggcagaa ttctggagcc agttgatggc tggactactc actctagtat cgtcaagtac 900cagggtcagt ggtggctatt ttatcacgat gccaagacat ctggcaagga ctatcttcgc 960caggtaaagg ctaagaagat ttggtacgat agcaaaggaa agatcttgac aaagaagcct 1020tga 102316340PRTGibberella zeae 16Met Lys Ser Lys Leu Leu Phe Pro Leu Leu Ser Phe Val Gly Gln Ser1 5 10 15Leu Ala Thr Asn Asp Asp Cys Pro Leu Ile Thr Ser Arg Trp Thr Ala 20 25 30Asp Pro Ser Ala His Val Phe Asn Asp Thr Leu Trp Leu Tyr Pro Ser 35 40 45His Asp Ile Asp Ala Gly Phe Glu Asn Asp Pro Asp Gly Gly Gln Tyr 50 55 60Ala Met Arg Asp Tyr His Val Tyr Ser Ile Asp Lys Ile Tyr Gly Ser65 70 75 80Leu Pro Val Asp His Gly Thr Ala Leu Ser Val Glu Asp Val Pro Trp 85 90 95Ala Ser Arg Gln Met Trp Ala Pro Asp Ala Ala His Lys Asn Gly Lys 100 105 110Tyr Tyr Leu Tyr Phe Pro Ala Lys Asp Lys Asp Asp Ile Phe Arg Ile 115 120 125Gly Val Ala Val Ser Pro Thr Pro Gly Gly Pro Phe Val Pro Asp Lys 130 135 140Ser Trp Ile Pro His Thr Phe Ser Ile Asp Pro Ala Ser Phe Val Asp145 150 155 160Asp Asp Asp Arg Ala Tyr Leu Ala Trp Gly Gly Ile Met Gly Gly Gln 165 170 175Leu Gln Arg Trp Gln Asp Lys Asn Lys Tyr Asn Glu Ser Gly Thr Glu 180 185 190Pro Gly Asn Gly Thr Ala Ala Leu Ser Pro Gln Ile Ala Lys Leu Ser 195 200 205Lys Asp Met His Thr Leu Ala Glu Lys Pro Arg Asp Met Leu Ile Leu 210 215 220Asp Pro Lys Thr Gly Lys Pro Leu Leu Ser Glu Asp Glu Asp Arg Arg225 230 235 240Phe Phe Glu Gly Pro Trp Ile His Lys Arg Asn Lys Ile Tyr Tyr Leu 245 250 255Thr Tyr Ser Thr Gly Thr Thr His Tyr Leu Val Tyr Ala Thr Ser Lys 260 265 270Thr Pro Tyr Gly Pro Tyr Thr Tyr Gln Gly Arg Ile Leu Glu Pro Val 275 280 285Asp Gly Trp Thr Thr His Ser Ser Ile Val Lys Tyr Gln Gly Gln Trp 290 295 300Trp Leu Phe Tyr His Asp Ala Lys Thr Ser Gly Lys Asp Tyr Leu Arg305 310 315 320Gln Val Lys Ala Lys Lys Ile Trp Tyr Asp Ser Lys Gly Lys Ile Leu 325 330 335Thr Lys Lys Pro 340171047DNAFusarium oxysporum 17atgcagctca agtttctgtc ttcagcattg ctgttctctc tgaccagcaa atgcgctgcg 60caagacacta atgacattcc tcccctgatc accgacctct ggtccgcaga tccctcggct 120catgttttcg aaggcaagct ctgggtttac ccatctcacg acatcgaagc caatgttgtc 180aacggcacag gaggcgctca atacgccatg agggattacc atacctactc catgaagagc 240atctatggta aagatcccgt tgtcgaccac ggcgtcgctc tctcagtcga tgacgttccc 300tgggcgaagc agcaaatgtg ggctcctgac gcagctcata agaacggcaa atattatctg 360tacttccccg ccaaggacaa ggatgagatc ttcagaattg gagttgctgt ctccaacaag 420cccagcggtc ctttcaaggc cgacaagagc tggatccctg gcacgtacag tatcgatcct 480gctagctacg tcgacactga taacgaggcc tacctcatct ggggcggtat ctggggcggc 540cagctccaag cctggcagga taaaaagaac tttaacgagt cgtggattgg agacaaggct 600gctcctaacg gcaccaatgc cctatctcct cagatcgcca agctaagcaa ggacatgcac 660aagatcaccg aaacaccccg cgatctcgtc attctcgccc ccgagacagg caagcctctt 720caggctgagg acaacaagcg acgattcttc gagggccctt ggatccacaa gcgcggcaag 780ctttactacc tcatgtactc caccggtgat acccacttcc ttgtctacgc tacttccaag 840aacatctacg gtccttatac ctaccggggc aagattcttg atcctgttga tgggtggact 900actcatggaa gtattgttga gtataaggga cagtggtggc ttttctttgc tgatgcgcat 960acgtctggta aggattacct tcgacaggtg aaggcgagga agatctggta tgacaagaac 1020ggcaagatct tgcttcaccg tccttag 104718348PRTFusarium oxysporum 18Met Gln Leu Lys Phe Leu Ser Ser Ala Leu Leu Phe Ser Leu Thr Ser1 5 10 15Lys Cys Ala Ala Gln Asp Thr Asn Asp Ile Pro Pro Leu Ile Thr Asp 20 25 30Leu Trp Ser Ala Asp Pro Ser Ala His Val Phe Glu Gly Lys Leu Trp 35 40 45Val Tyr Pro Ser His Asp Ile Glu Ala Asn Val Val Asn Gly Thr Gly 50 55 60Gly Ala Gln Tyr Ala Met Arg Asp Tyr His Thr Tyr Ser Met Lys Ser65 70 75 80Ile Tyr Gly Lys Asp Pro Val Val Asp His Gly Val Ala Leu Ser Val 85 90 95Asp Asp Val Pro Trp Ala Lys Gln Gln Met Trp Ala Pro Asp Ala Ala 100 105 110His Lys Asn Gly Lys Tyr Tyr Leu Tyr Phe Pro Ala Lys Asp Lys Asp 115 120 125Glu Ile Phe Arg Ile Gly Val Ala Val Ser Asn Lys Pro Ser Gly Pro 130 135 140Phe Lys Ala Asp Lys Ser Trp Ile Pro Gly Thr Tyr Ser Ile Asp Pro145 150 155 160Ala Ser Tyr Val Asp Thr Asp Asn Glu Ala Tyr Leu Ile Trp Gly Gly 165 170 175Ile Trp Gly Gly Gln Leu Gln Ala Trp Gln Asp Lys Lys Asn Phe Asn 180 185 190Glu Ser Trp Ile Gly Asp Lys Ala Ala Pro Asn Gly Thr Asn Ala Leu 195 200 205Ser Pro Gln Ile Ala Lys Leu Ser Lys Asp Met His Lys Ile Thr Glu 210 215 220Thr Pro Arg Asp Leu Val Ile Leu Ala Pro Glu Thr Gly Lys Pro Leu225 230 235 240Gln Ala Glu Asp Asn Lys Arg Arg Phe Phe Glu Gly Pro Trp Ile His 245 250 255Lys Arg Gly Lys Leu Tyr Tyr Leu Met Tyr Ser Thr Gly Asp Thr His 260 265 270Phe Leu Val Tyr Ala Thr Ser Lys Asn Ile Tyr Gly Pro Tyr Thr Tyr 275 280 285Arg Gly Lys Ile Leu Asp Pro Val Asp Gly Trp Thr Thr His Gly Ser 290 295 300Ile Val Glu Tyr Lys Gly Gln Trp Trp Leu Phe Phe Ala Asp Ala His305 310 315 320Thr Ser Gly Lys Asp Tyr Leu Arg Gln Val Lys Ala Arg Lys Ile Trp 325 330 335Tyr Asp Lys Asn Gly Lys Ile Leu Leu His Arg Pro 340 345191677DNAAspergillus fumigatus 19atggcagctc caagtttatc ctaccccaca ggtatccaat cgtataccaa tcctctcttc 60cctggttggc actccgatcc cagctgtgcc tacgtagcgg agcaagacac ctttttctgc 120gtgacgtcca ctttcattgc cttccccggt cttcctcttt atgcaagccg agatctgcag 180aactggaaac tggcaagcaa tattttcaat cggcccagcc agatccctga tcttcgcgtc 240acggatggac agcagtcggg tatctatgcg cccactctgc gctatcatga gggccagttc 300tacttgatcg tttcgtacct gggcccgcag actaagggct tgctgttcac ctcgtctgat 360ccgtacgacg atgccgcgtg gagcgatccg ctcgaattcg cggtacatgg catcgacccg 420gatatcttct gggatcacga cgggacggtc tatgtcacgt ccgccgagga ccagatgatt 480aagcagtaca cactcgatct gaagacgggg gcgattggcc cggttgacta cctctggaac 540ggcaccggag gagtctggcc cgagggcccg cacatttaca agagagacgg atactactac 600ctcatgatcg cagagggagg taccgagctc ggccactcgg agaccatggc gcgatctaga 660acccggacag gtccctggga gccatacccg cacaatccgc tcttgtcgaa caagggcacc 720tcggagtact tccagactgt gggccatgcg gacttgttcc aggatgggaa cggcaactgg 780tgggccgtgg cgttgagcac ccgatcaggg cctgcatgga agaactatcc catgggtcgg 840gagacggtgc tcgcccccgc cgcttgggag aagggtgagt ggcctgtcat tcagcctgtg 900agaggccaaa tgcaggggcc gtttccacca ccaaataagc gagttcctcg cggcgagggc 960ggatggatca agcaacccga caaagtggat ttcaggcccg gatcgaagat accggcgcac 1020ttccagtact ggcgatatcc caagacagag gattttaccg tctcccctcg gggccacccg 1080aatactcttc ggctcacacc ctccttttac aacctcaccg gaactgcgga cttcaagccg 1140gatgatggcc tgtcgcttgt tatgcgcaaa cagaccgaca ccttgttcac gtacactgtg 1200gacgtgtctt ttgaccccaa ggttgccgat gaagaggcgg gtgtgactgt tttccttacc 1260cagcagcagc acatcgatct tggtattgtc cttctccaga caaccgaggg gctgtcgttg 1320tccttccggt tccgcgtgga aggccgcggt aactacgaag gtcctcttcc agaagccacc 1380gtgcctgttc ccaaggaatg gtgtggacag accatccggc ttgagattca ggccgtgagt 1440gacaccgagt atgtctttgc ggctgccccg gctcggcacc ctgcacagag gcaaatcatc 1500agccgcgcca actcgttgat tgtcagtggt gatacgggac ggtttactgg ctcgcttgtt 1560ggcgtgtatg ccacgtcgaa cgggggtgcc ggatccacgc ccgcatatat cagcagatgg 1620agatacgaag gacggggcca gatgattgat tttggtcgag tggtcccgag ctactga 167720558PRTAspergillus fumigatus 20Met Ala Ala Pro Ser Leu Ser Tyr Pro Thr Gly Ile Gln Ser Tyr Thr1 5 10 15Asn Pro Leu Phe Pro Gly Trp His Ser Asp Pro Ser Cys Ala Tyr Val 20 25 30Ala Glu Gln Asp Thr Phe Phe Cys Val Thr Ser Thr Phe Ile Ala Phe 35 40 45Pro Gly Leu Pro Leu Tyr Ala Ser Arg Asp Leu Gln Asn Trp Lys Leu 50 55 60Ala Ser Asn Ile Phe Asn Arg Pro Ser Gln Ile Pro Asp Leu Arg Val65 70 75 80Thr Asp Gly Gln Gln Ser Gly Ile Tyr Ala Pro Thr Leu Arg Tyr His 85 90 95Glu Gly Gln Phe Tyr Leu Ile Val Ser Tyr Leu Gly Pro Gln Thr Lys 100 105 110Gly Leu Leu Phe Thr Ser Ser Asp Pro Tyr Asp Asp Ala Ala Trp Ser 115 120 125Asp Pro Leu Glu Phe Ala Val His Gly Ile Asp

Pro Asp Ile Phe Trp 130 135 140Asp His Asp Gly Thr Val Tyr Val Thr Ser Ala Glu Asp Gln Met Ile145 150 155 160Lys Gln Tyr Thr Leu Asp Leu Lys Thr Gly Ala Ile Gly Pro Val Asp 165 170 175Tyr Leu Trp Asn Gly Thr Gly Gly Val Trp Pro Glu Gly Pro His Ile 180 185 190Tyr Lys Arg Asp Gly Tyr Tyr Tyr Leu Met Ile Ala Glu Gly Gly Thr 195 200 205Glu Leu Gly His Ser Glu Thr Met Ala Arg Ser Arg Thr Arg Thr Gly 210 215 220Pro Trp Glu Pro Tyr Pro His Asn Pro Leu Leu Ser Asn Lys Gly Thr225 230 235 240Ser Glu Tyr Phe Gln Thr Val Gly His Ala Asp Leu Phe Gln Asp Gly 245 250 255Asn Gly Asn Trp Trp Ala Val Ala Leu Ser Thr Arg Ser Gly Pro Ala 260 265 270Trp Lys Asn Tyr Pro Met Gly Arg Glu Thr Val Leu Ala Pro Ala Ala 275 280 285Trp Glu Lys Gly Glu Trp Pro Val Ile Gln Pro Val Arg Gly Gln Met 290 295 300Gln Gly Pro Phe Pro Pro Pro Asn Lys Arg Val Pro Arg Gly Glu Gly305 310 315 320Gly Trp Ile Lys Gln Pro Asp Lys Val Asp Phe Arg Pro Gly Ser Lys 325 330 335Ile Pro Ala His Phe Gln Tyr Trp Arg Tyr Pro Lys Thr Glu Asp Phe 340 345 350Thr Val Ser Pro Arg Gly His Pro Asn Thr Leu Arg Leu Thr Pro Ser 355 360 365Phe Tyr Asn Leu Thr Gly Thr Ala Asp Phe Lys Pro Asp Asp Gly Leu 370 375 380Ser Leu Val Met Arg Lys Gln Thr Asp Thr Leu Phe Thr Tyr Thr Val385 390 395 400Asp Val Ser Phe Asp Pro Lys Val Ala Asp Glu Glu Ala Gly Val Thr 405 410 415Val Phe Leu Thr Gln Gln Gln His Ile Asp Leu Gly Ile Val Leu Leu 420 425 430Gln Thr Thr Glu Gly Leu Ser Leu Ser Phe Arg Phe Arg Val Glu Gly 435 440 445Arg Gly Asn Tyr Glu Gly Pro Leu Pro Glu Ala Thr Val Pro Val Pro 450 455 460Lys Glu Trp Cys Gly Gln Thr Ile Arg Leu Glu Ile Gln Ala Val Ser465 470 475 480Asp Thr Glu Tyr Val Phe Ala Ala Ala Pro Ala Arg His Pro Ala Gln 485 490 495Arg Gln Ile Ile Ser Arg Ala Asn Ser Leu Ile Val Ser Gly Asp Thr 500 505 510Gly Arg Phe Thr Gly Ser Leu Val Gly Val Tyr Ala Thr Ser Asn Gly 515 520 525Gly Ala Gly Ser Thr Pro Ala Tyr Ile Ser Arg Trp Arg Tyr Glu Gly 530 535 540Arg Gly Gln Met Ile Asp Phe Gly Arg Val Val Pro Ser Tyr545 550 555212320DNAPenicillium funiculosum 21atgggaaaga tgtggcattc gatcttggtt gtgttgggct tattgtctgt cgggcatgcc 60atcactatca acgtgtccca aagtggcggc aataagacca gtcctttgca atatggtctg 120atgttcgagg taatccttct cttataccac atataaaagt tgcgtcattt ctaagacaag 180tcaaggacat aaatcacggc ggtgatggcg gtctgtatgc agagcttgtt cgaaaccgag 240cattccaagg tagcaccgtc tatccagcaa acctcgatgg atacgactcg gtcaatggag 300caatcctagc gcttcagaat ttgacaaacc ctctatcacc ctccatgcct agctctctca 360acgtcgccaa ggggtccaac aatggaagca tcggtttcgc aaatgaaggc tggtggggga 420tagaagtcaa gccgcaaaga tacgcgggct cattctacgt ccagggggac tatcaaggag 480atttcgacat ctctcttcag tcgaaattga cacaagaagt cttcgcaacg gcaaaagtca 540ggtcctcggg caaacacgag gactgggttc aatacaagta cgagttggtg cccaaaaagg 600cagcatcaaa caccaataac actctgacca ttacttttga ctcaaaggta tgttaaattt 660tgggtttagt tcgatgtctg gcaattgtct tacgagaaac gtagggattg aaagacggat 720ccttgaactt caacttgatc agcctatttc ccccaactta caacaatcgg cccaatggcc 780taagaatcga cctggttgaa gctatggctg aactagaggg ggtaagctct tacaaatcaa 840ctttatcttt acgaagacta atgtgaaaac ttagaaattt ctgcggtttc caggcggtag 900cgatgtggaa ggtgtacaag ctccttactg gtataagtgg aatgaaacgg taggagatct 960caaggaccgt tatagtaggc ccagtgcatg gacgtacgaa gaaagcaatg gaattggctt 1020gattgagtac atgaattggt gtgatgacat ggggcttgag ccgagtgagt gtattccatt 1080cagcgtcaaa tccagtgttc taatcataca catcagttct tgccgtatgg gatggacatt 1140acctttcgaa cgaagtgata tcggaaaacg atttgcagcc atatatcgac gacaccctca 1200accaactgga attcctgatg ggtgccccag atacgccata tggtagttgg cgtgcgtctc 1260tgggctatcc gaagccgtgg acgattaact acgtcgagat tggaaacgaa gacaatctat 1320acgggggact agaaacatac atcgcctacc ggtttcaggc atattacgac gctataacag 1380ctaaatatcc ccatatgacg gtcatggaat ctttgacgga gatgcctggt ccggcggccg 1440ctgcaagcga ttaccatcaa tattctactc ctgatgggtt tgtttcccag ttcaactact 1500ttgatcagat gccagtcact aatagaacac tgaacggtat gaaaaccccc ccttttttaa 1560atatgctttt aatggtatta accatctttc ataggagaga ttgcaaccgt ttatccaaat 1620aatcctagta attcggtggc ctggggaagc ccattcccct tgtatccttg gtggattggg 1680tccgttgcag aagctgtttt cctaattggt gaagagagga attcgccaaa gataatcggt 1740gctagctacg tacggaattc tacttttcga gattttaaca ttggataaga aggactaacc 1800tcaatacagg ctccaatgtt cagaaatatc aacaattggc agtggtctcc aacactcatc 1860gcttttgacg ctgactcgtc gcgtacaagt cgttcaacaa gctggcatgt gatcaaggta 1920tgctaatttt cctcctcatt caaacccgca gatgtgagct aactttccga agcttctctc 1980gacaaacaaa atcacgcaaa atttacccac gacttggagt ggcggtgaca taggtccatt 2040atactgggta gctggacgaa acgacaatac aggatcgaac atattcaagg ccgctgttta 2100caacagcacc tcagacgtcc ctgtcaccgt tcaatttgca ggatgcaacg caaagagcgc 2160aaatttgacc atcttgtcat ccgacgatcc gaacgcatcg aactaccctg gggggcccga 2220agttgtgaag actgagatcc agtctgtcac tgcaaatgct catggagcat ttgagttcag 2280tctcccgaac ctaagtgtgg ctgttctcaa aacggagtaa 232022642PRTPenicillium funiculosum 22Met Gly Lys Met Trp His Ser Ile Leu Val Val Leu Gly Leu Leu Ser1 5 10 15Val Gly His Ala Ile Thr Ile Asn Val Ser Gln Ser Gly Gly Asn Lys 20 25 30Thr Ser Pro Leu Gln Tyr Gly Leu Met Phe Glu Asp Ile Asn His Gly 35 40 45Gly Asp Gly Gly Leu Tyr Ala Glu Leu Val Arg Asn Arg Ala Phe Gln 50 55 60Gly Ser Thr Val Tyr Pro Ala Asn Leu Asp Gly Tyr Asp Ser Val Asn65 70 75 80Gly Ala Ile Leu Ala Leu Gln Asn Leu Thr Asn Pro Leu Ser Pro Ser 85 90 95Met Pro Ser Ser Leu Asn Val Ala Lys Gly Ser Asn Asn Gly Ser Ile 100 105 110Gly Phe Ala Asn Glu Gly Trp Trp Gly Ile Glu Val Lys Pro Gln Arg 115 120 125Tyr Ala Gly Ser Phe Tyr Val Gln Gly Asp Tyr Gln Gly Asp Phe Asp 130 135 140Ile Ser Leu Gln Ser Lys Leu Thr Gln Glu Val Phe Ala Thr Ala Lys145 150 155 160Val Arg Ser Ser Gly Lys His Glu Asp Trp Val Gln Tyr Lys Tyr Glu 165 170 175Leu Val Pro Lys Lys Ala Ala Ser Asn Thr Asn Asn Thr Leu Thr Ile 180 185 190Thr Phe Asp Ser Lys Gly Leu Lys Asp Gly Ser Leu Asn Phe Asn Leu 195 200 205Ile Ser Leu Phe Pro Pro Thr Tyr Asn Asn Arg Pro Asn Gly Leu Arg 210 215 220Ile Asp Leu Val Glu Ala Met Ala Glu Leu Glu Gly Lys Phe Leu Arg225 230 235 240Phe Pro Gly Gly Ser Asp Val Glu Gly Val Gln Ala Pro Tyr Trp Tyr 245 250 255Lys Trp Asn Glu Thr Val Gly Asp Leu Lys Asp Arg Tyr Ser Arg Pro 260 265 270Ser Ala Trp Thr Tyr Glu Glu Ser Asn Gly Ile Gly Leu Ile Glu Tyr 275 280 285Met Asn Trp Cys Asp Asp Met Gly Leu Glu Pro Ile Leu Ala Val Trp 290 295 300Asp Gly His Tyr Leu Ser Asn Glu Val Ile Ser Glu Asn Asp Leu Gln305 310 315 320Pro Tyr Ile Asp Asp Thr Leu Asn Gln Leu Glu Phe Leu Met Gly Ala 325 330 335Pro Asp Thr Pro Tyr Gly Ser Trp Arg Ala Ser Leu Gly Tyr Pro Lys 340 345 350Pro Trp Thr Ile Asn Tyr Val Glu Ile Gly Asn Glu Asp Asn Leu Tyr 355 360 365Gly Gly Leu Glu Thr Tyr Ile Ala Tyr Arg Phe Gln Ala Tyr Tyr Asp 370 375 380Ala Ile Thr Ala Lys Tyr Pro His Met Thr Val Met Glu Ser Leu Thr385 390 395 400Glu Met Pro Gly Pro Ala Ala Ala Ala Ser Asp Tyr His Gln Tyr Ser 405 410 415Thr Pro Asp Gly Phe Val Ser Gln Phe Asn Tyr Phe Asp Gln Met Pro 420 425 430Val Thr Asn Arg Thr Leu Asn Gly Glu Ile Ala Thr Val Tyr Pro Asn 435 440 445Asn Pro Ser Asn Ser Val Ala Trp Gly Ser Pro Phe Pro Leu Tyr Pro 450 455 460Trp Trp Ile Gly Ser Val Ala Glu Ala Val Phe Leu Ile Gly Glu Glu465 470 475 480Arg Asn Ser Pro Lys Ile Ile Gly Ala Ser Tyr Ala Pro Met Phe Arg 485 490 495Asn Ile Asn Asn Trp Gln Trp Ser Pro Thr Leu Ile Ala Phe Asp Ala 500 505 510Asp Ser Ser Arg Thr Ser Arg Ser Thr Ser Trp His Val Ile Lys Leu 515 520 525Leu Ser Thr Asn Lys Ile Thr Gln Asn Leu Pro Thr Thr Trp Ser Gly 530 535 540Gly Asp Ile Gly Pro Leu Tyr Trp Val Ala Gly Arg Asn Asp Asn Thr545 550 555 560Gly Ser Asn Ile Phe Lys Ala Ala Val Tyr Asn Ser Thr Ser Asp Val 565 570 575Pro Val Thr Val Gln Phe Ala Gly Cys Asn Ala Lys Ser Ala Asn Leu 580 585 590Thr Ile Leu Ser Ser Asp Asp Pro Asn Ala Ser Asn Tyr Pro Gly Gly 595 600 605Pro Glu Val Val Lys Thr Glu Ile Gln Ser Val Thr Ala Asn Ala His 610 615 620Gly Ala Phe Glu Phe Ser Leu Pro Asn Leu Ser Val Ala Val Leu Lys625 630 635 640Thr Glu23739DNAAspergillus fumigatus 23atggtttctt tctcctacct gctgctggcg tgctccgcca ttggagctct ggctgccccc 60gtcgaacccg agaccacctc gttcaatgag actgctcttc atgagttcgc tgagcgcgcc 120ggcaccccaa gctccaccgg ctggaacaac ggctactact actccttctg gactgatggc 180ggcggcgacg tgacctacac caatggcgcc ggtggctcgt actccgtcaa ctggaggaac 240gtgggcaact ttgtcggtgg aaagggctgg aaccctggaa gcgctaggta ccgagctttg 300tcaacgtcgg atgtgcagac ctgtggctga cagaagtaga accatcaact acggaggcag 360cttcaacccc agcggcaatg gctacctggc tgtctacggc tggaccacca accccttgat 420tgagtactac gttgttgagt cgtatggtac atacaacccc ggcagcggcg gtaccttcag 480gggcactgtc aacaccgacg gtggcactta caacatctac acggccgttc gctacaatgc 540tccctccatc gaaggcacca agaccttcac ccagtactgg tctgtgcgca cctccaagcg 600taccggcggc actgtcacca tggccaacca cttcaacgcc tggagcagac tgggcatgaa 660cctgggaact cacaactacc agattgtcgc cactgagggt taccagagca gcggatctgc 720ttccatcact gtctactag 73924228PRTAspergillus fumigatus 24Met Val Ser Phe Ser Tyr Leu Leu Leu Ala Cys Ser Ala Ile Gly Ala1 5 10 15Leu Ala Ala Pro Val Glu Pro Glu Thr Thr Ser Phe Asn Glu Thr Ala 20 25 30Leu His Glu Phe Ala Glu Arg Ala Gly Thr Pro Ser Ser Thr Gly Trp 35 40 45Asn Asn Gly Tyr Tyr Tyr Ser Phe Trp Thr Asp Gly Gly Gly Asp Val 50 55 60Thr Tyr Thr Asn Gly Ala Gly Gly Ser Tyr Ser Val Asn Trp Arg Asn65 70 75 80Val Gly Asn Phe Val Gly Gly Lys Gly Trp Asn Pro Gly Ser Ala Arg 85 90 95Thr Ile Asn Tyr Gly Gly Ser Phe Asn Pro Ser Gly Asn Gly Tyr Leu 100 105 110Ala Val Tyr Gly Trp Thr Thr Asn Pro Leu Ile Glu Tyr Tyr Val Val 115 120 125Glu Ser Tyr Gly Thr Tyr Asn Pro Gly Ser Gly Gly Thr Phe Arg Gly 130 135 140Thr Val Asn Thr Asp Gly Gly Thr Tyr Asn Ile Tyr Thr Ala Val Arg145 150 155 160Tyr Asn Ala Pro Ser Ile Glu Gly Thr Lys Thr Phe Thr Gln Tyr Trp 165 170 175Ser Val Arg Thr Ser Lys Arg Thr Gly Gly Thr Val Thr Met Ala Asn 180 185 190His Phe Asn Ala Trp Ser Arg Leu Gly Met Asn Leu Gly Thr His Asn 195 200 205Tyr Gln Ile Val Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ala Ser 210 215 220Ile Thr Val Tyr225251002DNAAspergillus fumigatus 25atgatctcca tttcctcgct cagctttgga ctcgccgcta tcgccggcgc atatgctctt 60ccgagtgaca aatccgtcag cttagcggaa cgtcagacga tcacgaccag ccagacaggc 120acaaacaatg gctactacta ttccttctgg accaacggtg ccggatcagt gcaatataca 180aatggtgctg gtggcgaata tagtgtgacg tgggcgaacc agaacggtgg tgactttacc 240tgtgggaagg gctggaatcc agggagtgac cagtaggcaa cgcccgagaa ctatagaaga 300ggacgcaaag aaagcactaa actctctact agtgacatta ccttctctgg cagcttcaat 360ccttccggaa atgcttacct gtccgtgtat ggatggacta ccaaccccct agtcgaatac 420tacatcctcg agaactatgg cagttacaat cctggctcgg gcatgacgca caagggcacc 480gtcaccagcg atggatccac ctacgacatc tatgagcacc aacaggtcaa ccagccttcg 540atcgtcggca cggccacctt caaccaatac tggtccatcc gccaaaacaa gcgatccagc 600ggcacagtca ccaccgcgaa tcacttcaag gcctgggcta gtctggggat gaacctgggt 660acccataact atcagattgt ttccactgag ggatatgaga gcagcggtac ctcgaccatc 720actgtctcgt ctggtggttc ttcttctggt ggaagtggtg gcagctcgtc tactacttcc 780tcaggcagct cccctactgg tggctccggc agtgtaagtc ttcttccata tggttgtggc 840tttatgtgta ttctgactgt gatagtgctc tgctttgtgg ggccagtgcg gtggaattgg 900ctggtctggt cctacttgct gctcttcggg cacttgccag gtttcgaact cgtactactc 960ccagtgcttg tagtaccttc ttgcagggtt atatccaagt ga 100226286PRTAspergillus fumigatus 26Met Ile Ser Ile Ser Ser Leu Ser Phe Gly Leu Ala Ala Ile Ala Gly1 5 10 15Ala Tyr Ala Leu Pro Ser Asp Lys Ser Val Ser Leu Ala Glu Arg Gln 20 25 30Thr Ile Thr Thr Ser Gln Thr Gly Thr Asn Asn Gly Tyr Tyr Tyr Ser 35 40 45Phe Trp Thr Asn Gly Ala Gly Ser Val Gln Tyr Thr Asn Gly Ala Gly 50 55 60Gly Glu Tyr Ser Val Thr Trp Ala Asn Gln Asn Gly Gly Asp Phe Thr65 70 75 80Cys Gly Lys Gly Trp Asn Pro Gly Ser Asp His Asp Ile Thr Phe Ser 85 90 95Gly Ser Phe Asn Pro Ser Gly Asn Ala Tyr Leu Ser Val Tyr Gly Trp 100 105 110Thr Thr Asn Pro Leu Val Glu Tyr Tyr Ile Leu Glu Asn Tyr Gly Ser 115 120 125Tyr Asn Pro Gly Ser Gly Met Thr His Lys Gly Thr Val Thr Ser Asp 130 135 140Gly Ser Thr Tyr Asp Ile Tyr Glu His Gln Gln Val Asn Gln Pro Ser145 150 155 160Ile Val Gly Thr Ala Thr Phe Asn Gln Tyr Trp Ser Ile Arg Gln Asn 165 170 175Lys Arg Ser Ser Gly Thr Val Thr Thr Ala Asn His Phe Lys Ala Trp 180 185 190Ala Ser Leu Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val Ser 195 200 205Thr Glu Gly Tyr Glu Ser Ser Gly Thr Ser Thr Ile Thr Val Ser Ser 210 215 220Gly Gly Ser Ser Ser Gly Gly Ser Gly Gly Ser Ser Ser Thr Thr Ser225 230 235 240Ser Gly Ser Ser Pro Thr Gly Gly Ser Gly Ser Cys Ser Ala Leu Trp 245 250 255Gly Gln Cys Gly Gly Ile Gly Trp Ser Gly Pro Thr Cys Cys Ser Ser 260 265 270Gly Thr Cys Gln Val Ser Asn Ser Tyr Tyr Ser Gln Cys Leu 275 280 285271053DNAFusarium verticillioides 27atgcagctca agtttctgtc ttcagcattg ttgctgtctt tgaccggcaa ttgcgctgcg 60caagacacta atgatatccc tcctctgatc accgacctct ggtctgcgga tccctcggct 120catgttttcg agggcaaact ctgggtttac ccatctcacg acatcgaagc caatgtcgtc 180aacggcaccg gaggcgctca gtacgccatg agagattatc acacctattc catgaagacc 240atctatggaa aagatcccgt tatcgaccat ggcgtcgctc tgtcagtcga tgatgtccca 300tgggccaagc agcaaatgtg ggctcctgac gcagcttaca agaacggcaa atattatctc 360tacttccccg ccaaggataa agatgagatc ttcagaattg gagttgctgt ctccaacaag 420cccagcggtc ctttcaaggc cgacaagagc tggatccccg gtacttacag tatcgatcct 480gctagctatg tcgacactaa tggcgaggca tacctcatct ggggcggtat ctggggcggc 540cagcttcagg cctggcagga tcacaagacc tttaatgagt cgtggctcgg cgacaaagct 600gctcccaacg gcaccaacgc cctatctcct cagatcgcca agctaagcaa ggacatgcac 660aagatcaccg agacaccccg cgatctcgtc atcctggccc ccgagacagg caagcccctt 720caagcagagg acaataagcg acgatttttc gaggggccct gggttcacaa gcgcggcaag 780ctgtactacc tcatgtactc taccggcgac acgcacttcc tcgtctacgc gacttccaag 840aacatctacg gtccttatac ctatcagggc aagattctcg accctgttga tgggtggact 900acgcatggaa gtattgttga gtacaaggga cagtggtggt tgttctttgc ggatgcgcat 960acttctggaa aggattatct gagacaggtt aaggcgagga agatctggta tgacaaggat 1020ggcaagattt tgcttactcg tcctaagatt tag 105328350PRTFusarium verticillioides

28Met Gln Leu Lys Phe Leu Ser Ser Ala Leu Leu Leu Ser Leu Thr Gly1 5 10 15Asn Cys Ala Ala Gln Asp Thr Asn Asp Ile Pro Pro Leu Ile Thr Asp 20 25 30Leu Trp Ser Ala Asp Pro Ser Ala His Val Phe Glu Gly Lys Leu Trp 35 40 45Val Tyr Pro Ser His Asp Ile Glu Ala Asn Val Val Asn Gly Thr Gly 50 55 60Gly Ala Gln Tyr Ala Met Arg Asp Tyr His Thr Tyr Ser Met Lys Thr65 70 75 80Ile Tyr Gly Lys Asp Pro Val Ile Asp His Gly Val Ala Leu Ser Val 85 90 95Asp Asp Val Pro Trp Ala Lys Gln Gln Met Trp Ala Pro Asp Ala Ala 100 105 110Tyr Lys Asn Gly Lys Tyr Tyr Leu Tyr Phe Pro Ala Lys Asp Lys Asp 115 120 125Glu Ile Phe Arg Ile Gly Val Ala Val Ser Asn Lys Pro Ser Gly Pro 130 135 140Phe Lys Ala Asp Lys Ser Trp Ile Pro Gly Thr Tyr Ser Ile Asp Pro145 150 155 160Ala Ser Tyr Val Asp Thr Asn Gly Glu Ala Tyr Leu Ile Trp Gly Gly 165 170 175Ile Trp Gly Gly Gln Leu Gln Ala Trp Gln Asp His Lys Thr Phe Asn 180 185 190Glu Ser Trp Leu Gly Asp Lys Ala Ala Pro Asn Gly Thr Asn Ala Leu 195 200 205Ser Pro Gln Ile Ala Lys Leu Ser Lys Asp Met His Lys Ile Thr Glu 210 215 220Thr Pro Arg Asp Leu Val Ile Leu Ala Pro Glu Thr Gly Lys Pro Leu225 230 235 240Gln Ala Glu Asp Asn Lys Arg Arg Phe Phe Glu Gly Pro Trp Val His 245 250 255Lys Arg Gly Lys Leu Tyr Tyr Leu Met Tyr Ser Thr Gly Asp Thr His 260 265 270Phe Leu Val Tyr Ala Thr Ser Lys Asn Ile Tyr Gly Pro Tyr Thr Tyr 275 280 285Gln Gly Lys Ile Leu Asp Pro Val Asp Gly Trp Thr Thr His Gly Ser 290 295 300Ile Val Glu Tyr Lys Gly Gln Trp Trp Leu Phe Phe Ala Asp Ala His305 310 315 320Thr Ser Gly Lys Asp Tyr Leu Arg Gln Val Lys Ala Arg Lys Ile Trp 325 330 335Tyr Asp Lys Asp Gly Lys Ile Leu Leu Thr Arg Pro Lys Ile 340 345 350291031DNAPenicillium funiculosum 29atgagtcgca gcatccttcc gtacgcctct gttttcgccc tcctgggcgg ggctatcgcc 60gaaccgtttt tggttctcaa tagcgatttt cccgatccca gtctcataga gacatccagc 120ggatactatg cattcggtac caccggaaac ggagtcaatg cgcaggttgc ttcttcacca 180gactttaata cctggacttt gctttccggc acagatgccc tcccgggacc atttccgtca 240tgggtagctt cgtctccaca aatctgggcg ccagatgttt tggttaaggt atgttcttat 300ggaataacag ttttaggagt aggtcagcca ggatattgac aaaattataa taggccgatg 360gtacctatgt catgtacttt tcggcatctg ctgcgagtga ctcgggcaaa cactgcgttg 420gtgccgcaac tgcgacctca ccggaaggac cttacacccc ggtcgatagc gctgttgcct 480gtccattaga ccagggagga gctattgatg ccaatggatt tattgacacc gacggcacta 540tatacgttgt atacaaaatt gatggaaaca gtctagacgg tgatggaacc acacatccta 600cccccatcat gcttcaacaa atggaggcag acggaacaac cccaaccggc agcccaatcc 660aactcattga ccgatccgac ctcgacggac ctttgatcga ggctcctagt ttgctcctct 720ccaatggaat ctactacctc agtttctctt ccaactacta caacactaat tactacgaca 780cttcatacgc ctatgcctcg tcgattactg gtccttggac caaacaatct gcgccttatg 840cacccttgtt ggttactgga accgagacta gcaatgacgg cgcattgagc gcccctggtg 900gtgccgattt ctccgtcgat ggcaccaaga tgttgttcca cgcaaacctc aatggacaag 960atatctcggg cggacgcgcc ttatttgctg cgtcaattac tgaggccagc gatgtggtta 1020cattgcagta g 103130321PRTPenicillium funiculosum 30Met Ser Arg Ser Ile Leu Pro Tyr Ala Ser Val Phe Ala Leu Leu Gly1 5 10 15Gly Ala Ile Ala Glu Pro Phe Leu Val Leu Asn Ser Asp Phe Pro Asp 20 25 30Pro Ser Leu Ile Glu Thr Ser Ser Gly Tyr Tyr Ala Phe Gly Thr Thr 35 40 45Gly Asn Gly Val Asn Ala Gln Val Ala Ser Ser Pro Asp Phe Asn Thr 50 55 60Trp Thr Leu Leu Ser Gly Thr Asp Ala Leu Pro Gly Pro Phe Pro Ser65 70 75 80Trp Val Ala Ser Ser Pro Gln Ile Trp Ala Pro Asp Val Leu Val Lys 85 90 95Ala Asp Gly Thr Tyr Val Met Tyr Phe Ser Ala Ser Ala Ala Ser Asp 100 105 110Ser Gly Lys His Cys Val Gly Ala Ala Thr Ala Thr Ser Pro Glu Gly 115 120 125Pro Tyr Thr Pro Val Asp Ser Ala Val Ala Cys Pro Leu Asp Gln Gly 130 135 140Gly Ala Ile Asp Ala Asn Gly Phe Ile Asp Thr Asp Gly Thr Ile Tyr145 150 155 160Val Val Tyr Lys Ile Asp Gly Asn Ser Leu Asp Gly Asp Gly Thr Thr 165 170 175His Pro Thr Pro Ile Met Leu Gln Gln Met Glu Ala Asp Gly Thr Thr 180 185 190Pro Thr Gly Ser Pro Ile Gln Leu Ile Asp Arg Ser Asp Leu Asp Gly 195 200 205Pro Leu Ile Glu Ala Pro Ser Leu Leu Leu Ser Asn Gly Ile Tyr Tyr 210 215 220Leu Ser Phe Ser Ser Asn Tyr Tyr Asn Thr Asn Tyr Tyr Asp Thr Ser225 230 235 240Tyr Ala Tyr Ala Ser Ser Ile Thr Gly Pro Trp Thr Lys Gln Ser Ala 245 250 255Pro Tyr Ala Pro Leu Leu Val Thr Gly Thr Glu Thr Ser Asn Asp Gly 260 265 270Ala Leu Ser Ala Pro Gly Gly Ala Asp Phe Ser Val Asp Gly Thr Lys 275 280 285Met Leu Phe His Ala Asn Leu Asn Gly Gln Asp Ile Ser Gly Gly Arg 290 295 300Ala Leu Phe Ala Ala Ser Ile Thr Glu Ala Ser Asp Val Val Thr Leu305 310 315 320Gln312186DNAFusarium verticillioides 31atggttcgct tcagttcaat cctagcggct gcggcttgct tcgtggctgt tgagtcagtc 60aacatcaagg tcgacagcaa gggcggaaac gctactagcg gtcaccaata tggcttcctt 120cacgaggttg gtattgacac accactggcg atgattggga tgctaacttg gagctaggat 180atcaacaatt ccggtgatgg tggcatctac gctgagctca tccgcaatcg tgctttccag 240tacagcaaga aataccctgt ttctctatct ggctggagac ccatcaacga tgctaagctc 300tccctcaacc gtctcgacac tcctctctcc gacgctctcc ccgtttccat gaacgtgaag 360cctggaaagg gcaaggccaa ggagattggt ttcctcaacg agggttactg gggaatggat 420gtcaagaagc aaaagtacac tggctctttc tgggttaagg gcgcttacaa gggccacttt 480acagcttctt tgcgatctaa ccttaccgac gatgtctttg gcagcgtcaa ggtcaagtcc 540aaggccaaca agaagcagtg ggttgagcat gagtttgtgc ttactcctaa caagaatgcc 600cctaacagca acaacacttt tgctatcacc tacgatccca aggtgagtaa caatcaaaac 660tgggacgtga tgtatactga caatttgtag ggcgctgatg gagctcttga cttcaacctc 720attagcttgt tccctcccac ctacaagggc cgcaagaacg gtcttcgagt tgatcttgcc 780gaggctctcg aaggtctcca ccccgtaagg tttaccgtct cacgtgtatc gtgaacagtc 840gctgacttgt agaaaagagc ctgctgcgct tccccggtgg taacatgctc gagggcaaca 900ccaacaagac ctggtgggac tggaaggata ccctcggacc tctccgcaac cgtcctggtt 960tcgagggtgt ctggaactac cagcagaccc atggtcttgg aatcttggag tacctccagt 1020gggctgagga catgaacctt gaaatcagta ggttctataa aattcagtga cggttatgtg 1080catgctaaca gatttcagtt gtcggtgtct acgctggcct ctccctcgac ggctccgtca 1140cccccaagga ccaactccag cccctcatcg acgacgcgct cgacgagatc gaattcatcc 1200gaggtcccgt cacttcaaag tggggaaaga agcgcgctga gctcggccac cccaagcctt 1260tcagactctc ctacgttgaa gtcggaaacg aggactggct cgctggttat cccactggct 1320ggaactctta caaggagtac cgcttcccca tgttcctcga ggctatcaag aaagctcacc 1380ccgatctcac cgtcatctcc tctggtgctt ctattgaccc cgttggtaag aaggatgctg 1440gtttcgatat tcctgctcct ggaatcggtg actaccaccc ttaccgcgag cctgatgttc 1500ttgttgagga gttcaacctg tttgataaca ataagtatgg tcacatcatt ggtgaggttg 1560cttctaccca ccccaacggt ggaactggct ggagtggtaa ccttatgcct tacccctggt 1620ggatctctgg tgttggcgag gccgtcgctc tctgcggtta tgagcgcaac gccgatcgta 1680ttcccggaac attctacgct cctatcctca agaacgagaa ccgttggcag tgggctatca 1740ccatgatcca attcgccgcc gactccgcca tgaccacccg ctccaccagc tggtatgtct 1800ggtcactctt cgcaggccac cccatgaccc atactctccc caccaccgcc gacttcgacc 1860ccctctacta cgtcgctggt aagaacgagg acaagggaac tcttatctgg aagggtgctg 1920cgtataacac caccaagggt gctgacgttc ccgtgtctct gtccttcaag ggtgtcaagc 1980ccggtgctca agctgagctt actcttctga ccaacaagga gaaggatcct tttgcgttca 2040atgatcctca caagggcaac aatgttgttg atactaagaa gactgttctc aaggccgatg 2100gaaagggtgc tttcaacttc aagcttccta acctgagcgt cgctgttctt gagaccctca 2160agaagggaaa gccttactct agctag 218632660PRTFusarium verticillioides 32Met Val Arg Phe Ser Ser Ile Leu Ala Ala Ala Ala Cys Phe Val Ala1 5 10 15Val Glu Ser Val Asn Ile Lys Val Asp Ser Lys Gly Gly Asn Ala Thr 20 25 30Ser Gly His Gln Tyr Gly Phe Leu His Glu Asp Ile Asn Asn Ser Gly 35 40 45Asp Gly Gly Ile Tyr Ala Glu Leu Ile Arg Asn Arg Ala Phe Gln Tyr 50 55 60Ser Lys Lys Tyr Pro Val Ser Leu Ser Gly Trp Arg Pro Ile Asn Asp65 70 75 80Ala Lys Leu Ser Leu Asn Arg Leu Asp Thr Pro Leu Ser Asp Ala Leu 85 90 95Pro Val Ser Met Asn Val Lys Pro Gly Lys Gly Lys Ala Lys Glu Ile 100 105 110Gly Phe Leu Asn Glu Gly Tyr Trp Gly Met Asp Val Lys Lys Gln Lys 115 120 125Tyr Thr Gly Ser Phe Trp Val Lys Gly Ala Tyr Lys Gly His Phe Thr 130 135 140Ala Ser Leu Arg Ser Asn Leu Thr Asp Asp Val Phe Gly Ser Val Lys145 150 155 160Val Lys Ser Lys Ala Asn Lys Lys Gln Trp Val Glu His Glu Phe Val 165 170 175Leu Thr Pro Asn Lys Asn Ala Pro Asn Ser Asn Asn Thr Phe Ala Ile 180 185 190Thr Tyr Asp Pro Lys Gly Ala Asp Gly Ala Leu Asp Phe Asn Leu Ile 195 200 205Ser Leu Phe Pro Pro Thr Tyr Lys Gly Arg Lys Asn Gly Leu Arg Val 210 215 220Asp Leu Ala Glu Ala Leu Glu Gly Leu His Pro Ser Leu Leu Arg Phe225 230 235 240Pro Gly Gly Asn Met Leu Glu Gly Asn Thr Asn Lys Thr Trp Trp Asp 245 250 255Trp Lys Asp Thr Leu Gly Pro Leu Arg Asn Arg Pro Gly Phe Glu Gly 260 265 270Val Trp Asn Tyr Gln Gln Thr His Gly Leu Gly Ile Leu Glu Tyr Leu 275 280 285Gln Trp Ala Glu Asp Met Asn Leu Glu Ile Ile Val Gly Val Tyr Ala 290 295 300Gly Leu Ser Leu Asp Gly Ser Val Thr Pro Lys Asp Gln Leu Gln Pro305 310 315 320Leu Ile Asp Asp Ala Leu Asp Glu Ile Glu Phe Ile Arg Gly Pro Val 325 330 335Thr Ser Lys Trp Gly Lys Lys Arg Ala Glu Leu Gly His Pro Lys Pro 340 345 350Phe Arg Leu Ser Tyr Val Glu Val Gly Asn Glu Asp Trp Leu Ala Gly 355 360 365Tyr Pro Thr Gly Trp Asn Ser Tyr Lys Glu Tyr Arg Phe Pro Met Phe 370 375 380Leu Glu Ala Ile Lys Lys Ala His Pro Asp Leu Thr Val Ile Ser Ser385 390 395 400Gly Ala Ser Ile Asp Pro Val Gly Lys Lys Asp Ala Gly Phe Asp Ile 405 410 415Pro Ala Pro Gly Ile Gly Asp Tyr His Pro Tyr Arg Glu Pro Asp Val 420 425 430Leu Val Glu Glu Phe Asn Leu Phe Asp Asn Asn Lys Tyr Gly His Ile 435 440 445Ile Gly Glu Val Ala Ser Thr His Pro Asn Gly Gly Thr Gly Trp Ser 450 455 460Gly Asn Leu Met Pro Tyr Pro Trp Trp Ile Ser Gly Val Gly Glu Ala465 470 475 480Val Ala Leu Cys Gly Tyr Glu Arg Asn Ala Asp Arg Ile Pro Gly Thr 485 490 495Phe Tyr Ala Pro Ile Leu Lys Asn Glu Asn Arg Trp Gln Trp Ala Ile 500 505 510Thr Met Ile Gln Phe Ala Ala Asp Ser Ala Met Thr Thr Arg Ser Thr 515 520 525Ser Trp Tyr Val Trp Ser Leu Phe Ala Gly His Pro Met Thr His Thr 530 535 540Leu Pro Thr Thr Ala Asp Phe Asp Pro Leu Tyr Tyr Val Ala Gly Lys545 550 555 560Asn Glu Asp Lys Gly Thr Leu Ile Trp Lys Gly Ala Ala Tyr Asn Thr 565 570 575Thr Lys Gly Ala Asp Val Pro Val Ser Leu Ser Phe Lys Gly Val Lys 580 585 590Pro Gly Ala Gln Ala Glu Leu Thr Leu Leu Thr Asn Lys Glu Lys Asp 595 600 605Pro Phe Ala Phe Asn Asp Pro His Lys Gly Asn Asn Val Val Asp Thr 610 615 620Lys Lys Thr Val Leu Lys Ala Asp Gly Lys Gly Ala Phe Asn Phe Lys625 630 635 640Leu Pro Asn Leu Ser Val Ala Val Leu Glu Thr Leu Lys Lys Gly Lys 645 650 655Pro Tyr Ser Ser 660332312DNAChaetomium globosum 33atggcgcccc tttcgcttcg ggccctctcg ctgctcgcgc tcacaggagc cgcagccgcg 60gtgaccctat cggtcgcgaa ctctggcggt aatgatacgt ctccgtacat gtatggcatc 120atgttcgagg acatcaatca gagcggtgac ggcgggctgt aagttctgtc gcggcttccc 180ctgacaagct tgcatgatgc ttaactaaag tccttaggta cgccgagctg attcgcaacc 240gagccttcca taatagctcc ctccaggcct ggaccgccgt gggggacagc actctcgagg 300tcgtaacctc tgcaccgtta tcggatgccc tgcctcgctc ggtcaaggtc acgagtggaa 360agggcaaggc gggcttgaag aatgccggct actggggaat ggacgtccag aagaccgaca 420agtatagcgg cagcttctac tcgtacggcg cctacgacgg aaagtttacc ctctctctgg 480tgtcggacat cacaaatgag accctggcca ccaccaagat caagtccagg tcggtggagc 540atgcctggac cgagcacaag ttcgagcttc tcccgaccaa gagcgcggcg aacagcaaca 600acagcttcgt gctggagttc cgcccctgcc accagacgga gctccagttc aacctcatca 660gcttgttccc gccgacgtat aagaacaggc ccaacggcat gcgccgagag ctcatggaga 720agctcgcaga cctcaagccc agtttccttc ggattccagg aggcaacaac ctgtaagtgc 780ttccggcgaa actagcagta gttgcctgag agacactaat ctcagcgaac aacagcgagg 840gcaactatgc tggcaactac tggaactggt caagcacact tggcccgctg accgaccggc 900ccggtcgtga cggcgtgtgg acgtacgcca acacggacgg catcgggctg gtcgagtaca 960tgcactgggc cgaggacctc gacgtggagg ttgtgctggc ggtcgccgca ggcctgtacc 1020tgaacggcga tgtggtcccg gaggaggagc tgcacgtctt cgtggaggat gcgctgaacg 1080agctcgagtt cctcatgggc gacgtctcga ccccttgggg cgcgcgccgc gctaagctcg 1140gctaccccaa gccgtggaac atcaagttcg tcgaggtcgg caacgaggac aacctgtggg 1200gcggcctcga ctcgtacaag agctaccggc tgaagacttt ctacgacgcc atcaaggcga 1260agtaccccga catctccatc ttttcgtcga ccgacgagtt tgtgtacaag gagtcgggcc 1320aggactacca caagtacacc cggccggact actccgtgtc ccagttcgac ctgtttgaca 1380actgggccga cggccacccc atcatcatcg gagagtgagt gaacggcgac ccccacctcc 1440ccctaacgcg ggatcgcgag ctgatagatc accccaggta tgcgaccatc cagaacaaca 1500cgggcaagct cgaggacacg gactgggacg cgcccaagaa caagtggtcc aactggatcg 1560gctccgtcgc cgaggccgtc ttcatcctcg gagccgagcg caacggcgac cgggtctggg 1620gcaccacctt tgcgccgatc ctccagaacc tcaacagcta ccaatgggct gtaagtacat 1680acatacatac cgcaccccca accccaaccc ccccaaagcg cacctccacc cacccaccca 1740aacacaccac aactacctag ctaacccgcc acacaaacaa acagcccgac ctaatctcct 1800tcaccgccaa cccggccgac accacgccca gcgtctcgta cccgatcatc cagctgctcg 1860cctcgcaccg catcacgcac accctccccg tcagcagcgc cgacgccttc ggcccggcct 1920actgggtggc cggtcgcggc gccgacgacg gctcgtacat cctcaaggcg gccgtgtaca 1980acagcacggg gggtgcggat gtaccggtga gggtgcagtt tgaggcgggg ggtggtggtg 2040gtggtggtgg tggtggtggt ggtggtggtg gtgatgggaa ggggaagggt aaagggaagg 2100gaggggaggg tggtgagggt gtgaagaagg gtgaccgcgc gcagttgacc gtgttgacgg 2160cgccggaggg gccctgggcg cataatacgc cggagaataa gggggcggtc aagacgacag 2220tgacgacgtt gaaggccggg aggggtgggg tgtttgagtt tagtctgccg gatttgtcgg 2280tggcggtgtt ggtggtggag ggggagaagt ga 231234670PRTChaetomium globosum 34Met Ala Pro Leu Ser Leu Arg Ala Leu Ser Leu Leu Ala Leu Thr Gly1 5 10 15Ala Ala Ala Ala Val Thr Leu Ser Val Ala Asn Ser Gly Gly Asn Asp 20 25 30Thr Ser Pro Tyr Met Tyr Gly Ile Met Phe Glu Asp Ile Asn Gln Ser 35 40 45Gly Asp Gly Gly Leu Tyr Ala Glu Leu Ile Arg Asn Arg Ala Phe His 50 55 60Asn Ser Ser Leu Gln Ala Trp Thr Ala Val Gly Asp Ser Thr Leu Glu65 70 75 80Val Val Thr Ser Ala Pro Leu Ser Asp Ala Leu Pro Arg Ser Val Lys 85 90 95Val Thr Ser Gly Lys Gly Lys Ala Gly Leu Lys Asn Ala Gly Tyr Trp 100 105 110Gly Met Asp Val Gln Lys Thr Asp Lys Tyr Ser Gly Ser Phe Tyr Ser 115 120 125Tyr Gly Ala Tyr Asp Gly Lys Phe Thr Leu Ser Leu Val Ser Asp Ile 130 135 140Thr Asn Glu Thr Leu Ala Thr Thr Lys Ile Lys Ser Arg Ser Val Glu145 150 155 160His Ala Trp Thr Glu His Lys Phe Glu Leu Leu Pro Thr Lys Ser Ala 165 170 175Ala Asn Ser Asn Asn Ser Phe Val Leu Glu Phe Arg Pro Cys His Gln

180 185 190Thr Glu Leu Gln Phe Asn Leu Ile Ser Leu Phe Pro Pro Thr Tyr Lys 195 200 205Asn Arg Pro Asn Gly Met Arg Arg Glu Leu Met Glu Lys Leu Ala Asp 210 215 220Leu Lys Pro Ser Phe Leu Arg Ile Pro Gly Gly Asn Asn Leu Glu Gly225 230 235 240Asn Tyr Ala Gly Asn Tyr Trp Asn Trp Ser Ser Thr Leu Gly Pro Leu 245 250 255Thr Asp Arg Pro Gly Arg Asp Gly Val Trp Thr Tyr Ala Asn Thr Asp 260 265 270Gly Ile Gly Leu Val Glu Tyr Met His Trp Ala Glu Asp Leu Asp Val 275 280 285Glu Val Val Leu Ala Val Ala Ala Gly Leu Tyr Leu Asn Gly Asp Val 290 295 300Val Pro Glu Glu Glu Leu His Val Phe Val Glu Asp Ala Leu Asn Glu305 310 315 320Leu Glu Phe Leu Met Gly Asp Val Ser Thr Pro Trp Gly Ala Arg Arg 325 330 335Ala Lys Leu Gly Tyr Pro Lys Pro Trp Asn Ile Lys Phe Val Glu Val 340 345 350Gly Asn Glu Asp Asn Leu Trp Gly Gly Leu Asp Ser Tyr Lys Ser Tyr 355 360 365Arg Leu Lys Thr Phe Tyr Asp Ala Ile Lys Ala Lys Tyr Pro Asp Ile 370 375 380Ser Ile Phe Ser Ser Thr Asp Glu Phe Val Tyr Lys Glu Ser Gly Gln385 390 395 400Asp Tyr His Lys Tyr Thr Arg Pro Asp Tyr Ser Val Ser Gln Phe Asp 405 410 415Leu Phe Asp Asn Trp Ala Asp Gly His Pro Ile Ile Ile Gly Glu Tyr 420 425 430Ala Thr Ile Gln Asn Asn Thr Gly Lys Leu Glu Asp Thr Asp Trp Asp 435 440 445Ala Pro Lys Asn Lys Trp Ser Asn Trp Ile Gly Ser Val Ala Glu Ala 450 455 460Val Phe Ile Leu Gly Ala Glu Arg Asn Gly Asp Arg Val Trp Gly Thr465 470 475 480Thr Phe Ala Pro Ile Leu Gln Asn Leu Asn Ser Tyr Gln Trp Ala Pro 485 490 495Asp Leu Ile Ser Phe Thr Ala Asn Pro Ala Asp Thr Thr Pro Ser Val 500 505 510Ser Tyr Pro Ile Ile Gln Leu Leu Ala Ser His Arg Ile Thr His Thr 515 520 525Leu Pro Val Ser Ser Ala Asp Ala Phe Gly Pro Ala Tyr Trp Val Ala 530 535 540Gly Arg Gly Ala Asp Asp Gly Ser Tyr Ile Leu Lys Ala Ala Val Tyr545 550 555 560Asn Ser Thr Gly Gly Ala Asp Val Pro Val Arg Val Gln Phe Glu Ala 565 570 575Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asp 580 585 590Gly Lys Gly Lys Gly Lys Gly Lys Gly Gly Glu Gly Gly Glu Gly Val 595 600 605Lys Lys Gly Asp Arg Ala Gln Leu Thr Val Leu Thr Ala Pro Glu Gly 610 615 620Pro Trp Ala His Asn Thr Pro Glu Asn Lys Gly Ala Val Lys Thr Thr625 630 635 640Val Thr Thr Leu Lys Ala Gly Arg Gly Gly Val Phe Glu Phe Ser Leu 645 650 655Pro Asp Leu Ser Val Ala Val Leu Val Val Glu Gly Glu Lys 660 665 670351002DNAFusarium verticillioides 35atgcgtcttc tatcgtttcc cagccatctc ctcgtggcct tcctaaccct caaagaggct 60tcatccctcg ccctcagcaa acgggatagc cctgtcctcc ccggcctctg ggcggacccc 120aacatcgcca tcgtcgacaa gacatactac atcttcccta ccaccgacgg tttcgaaggc 180tggggcggca acgtcttcta ctggtggaaa tcaaaagatc tcgtatcatg gacaaagagc 240gacaagccat tccttactct caatggtacg aatggcaacg ttccctgggc tacaggtaat 300gcctgggctc ctgctttcgc tgctcgcgga ggcaagtatt acttctacca tagtgggaat 360aatccctctg tgagtgatgg gcataagagt attggtgcgg cggtggctga tcatcctgag 420gggccgtgga aggcacagga taagccgatg atcaagggaa cttctgatga ggagattgtc 480agcaaccagg ctatcgatcc cgctgccttt gaagaccctg agactggaaa gtggtatatc 540tactggggaa acggtgtccc cattgtcgca gagctcaacg acgacatggt ctctctcaaa 600gcaggctggc acaaaatcac aggtcttcag aatttccgcg agggtctttt cgtcaactat 660cgcgatggaa catatcatct gacatactct atcgacgata cgggctcaga gaactatcgc 720gttgggtacg ctacggcgga taaccccatt ggaccttgga catatcgtgg tgttcttctg 780gagaaggacg aatcgaaggg cattcttgct acgggacata actccatcat caacattcct 840ggaacggatg agtggtatat cgcgtatcat cgcttccata ttcccgatgg aaatgggtat 900aatagggaga ctacgattga tagggtaccc atcgacaagg atacgggttt gtttggaaag 960gttacgccga ctttgcagag tgttgatcct aggcctttgt ag 100236333PRTFusarium verticillioides 36Met Arg Leu Leu Ser Phe Pro Ser His Leu Leu Val Ala Phe Leu Thr1 5 10 15Leu Lys Glu Ala Ser Ser Leu Ala Leu Ser Lys Arg Asp Ser Pro Val 20 25 30Leu Pro Gly Leu Trp Ala Asp Pro Asn Ile Ala Ile Val Asp Lys Thr 35 40 45Tyr Tyr Ile Phe Pro Thr Thr Asp Gly Phe Glu Gly Trp Gly Gly Asn 50 55 60Val Phe Tyr Trp Trp Lys Ser Lys Asp Leu Val Ser Trp Thr Lys Ser65 70 75 80Asp Lys Pro Phe Leu Thr Leu Asn Gly Thr Asn Gly Asn Val Pro Trp 85 90 95Ala Thr Gly Asn Ala Trp Ala Pro Ala Phe Ala Ala Arg Gly Gly Lys 100 105 110Tyr Tyr Phe Tyr His Ser Gly Asn Asn Pro Ser Val Ser Asp Gly His 115 120 125Lys Ser Ile Gly Ala Ala Val Ala Asp His Pro Glu Gly Pro Trp Lys 130 135 140Ala Gln Asp Lys Pro Met Ile Lys Gly Thr Ser Asp Glu Glu Ile Val145 150 155 160Ser Asn Gln Ala Ile Asp Pro Ala Ala Phe Glu Asp Pro Glu Thr Gly 165 170 175Lys Trp Tyr Ile Tyr Trp Gly Asn Gly Val Pro Ile Val Ala Glu Leu 180 185 190Asn Asp Asp Met Val Ser Leu Lys Ala Gly Trp His Lys Ile Thr Gly 195 200 205Leu Gln Asn Phe Arg Glu Gly Leu Phe Val Asn Tyr Arg Asp Gly Thr 210 215 220Tyr His Leu Thr Tyr Ser Ile Asp Asp Thr Gly Ser Glu Asn Tyr Arg225 230 235 240Val Gly Tyr Ala Thr Ala Asp Asn Pro Ile Gly Pro Trp Thr Tyr Arg 245 250 255Gly Val Leu Leu Glu Lys Asp Glu Ser Lys Gly Ile Leu Ala Thr Gly 260 265 270His Asn Ser Ile Ile Asn Ile Pro Gly Thr Asp Glu Trp Tyr Ile Ala 275 280 285Tyr His Arg Phe His Ile Pro Asp Gly Asn Gly Tyr Asn Arg Glu Thr 290 295 300Thr Ile Asp Arg Val Pro Ile Asp Lys Asp Thr Gly Leu Phe Gly Lys305 310 315 320Val Thr Pro Thr Leu Gln Ser Val Asp Pro Arg Pro Leu 325 330371695DNAFusarium verticillioides 37atgctcttct cgctcgttct tcctaccctt gcctttcaag ccagcctggc gctcggcgat 60acatccgtta ctgtcgacac cagccagaaa ctccaggtca tcgatggctt tggtgtctca 120gaagcctacg gccacgccaa acaattccaa aacctcggtc ctggaccaca gaaagagggc 180ctcgatcttc tcttcaacac tacaaccggc gcaggcttat ccatcatccg aaacaagatc 240ggctgcgacg cctccaactc catcaccagc accaacaccg acaacccaga taagcaggct 300gtttaccatt ttgacggcga tgatgatggt caggtatggt ttagcaaaca ggccatgagc 360tatggtgtag atactatcta cgctaatgct tggtctgcgc ctgtatacat gaagtcagcc 420cagagtatgg gccgtctctg cggtacacct ggtgtgtcgt gctcctctgg agattggaga 480catcgttacg ttgagatgat agctgagtac ctctcctact acaagcaggc tggcatccca 540gtgtcgcacg ttggattcct caatgagggt gacggctcgg actttatgct ctcaactgcc 600gaacaggctg cagatgtcat tcctcttcta cacagcgctt tgcagtccaa gggccttggc 660gatatcaaga tgacgtgctg tgataacatc ggttggaagt cacagatgga ctataccgcc 720aagctggctg agcttgaggt ggagaagtat ctatctgtca tcacatccca cgagtactcc 780agcagcccca accagcctat gaacactaca ttgccaacct ggatgtccga gggagctgcc 840aatgaccagg catttgccac agcgtggtac gtcaacggcg gttccaacga aggtttcaca 900tgggcagtca agatcgcaca aggcatcgtc aatgccgacc tctcagcgta tatctactgg 960gagggcgttg agaccaacaa caaggggtct ctatctcacg tcatcgacac ggacggtacc 1020aagtttacca tatcctcgat tctctgggcc attgctcact ggtcgcgcca tattcgccct 1080ggtgcgcata gactttcgac ttcaggtgtt gtgcaagata cgattgttgg tgcgtttgag 1140aacgttgatg gcagtgtcgt catggtgctc accaactctg gcactgctgc tcagactgtg 1200gacctgggtg tttcgggaag tagcttctca acagctcagg ctttcacttc ggatgctgag 1260gcgcagatgg tcgataccaa ggtgactctg tccgacggtc gtgtcaaggt tacggtcccg 1320gtgcacggtg tcgtcactgt gaagctcaca acagcaaaaa gctccaaacc ggtctcaact 1380gctgtttctg cgcaatctgc ccccactcca actagtgtta agcacacctt gactcaccag 1440aagacttctt caacaacact ctcgaccgcc aaggccccaa cctccactca gactacctct 1500gtagttgagt cagccaaggc ggtgaaatac cctgtccccc ctgtagcatc caagggatcc 1560tcgaagagtg ctcccaagaa gggtaccaag aagaccacta cgaagaaggg ctcccaccaa 1620tcgcacaagg cgcatagtgc tactcatcgt cgatgccgcc atggaagtta ccgtcgtggc 1680cactgcacca actaa 169538537PRTFusarium verticillioides 38Met Leu Phe Ser Leu Val Leu Pro Thr Leu Ala Phe Gln Ala Ser Leu1 5 10 15Ala Leu Gly Asp Thr Ser Val Thr Val Asp Thr Ser Gln Lys Leu Gln 20 25 30Val Ile Asp Gly Phe Gly Val Ser Glu Ala Tyr Gly His Ala Lys Gln 35 40 45Phe Gln Asn Leu Gly Pro Gly Pro Gln Lys Glu Gly Leu Asp Leu Leu 50 55 60Phe Asn Thr Thr Thr Gly Ala Gly Leu Ser Ile Ile Arg Asn Lys Ile65 70 75 80Gly Cys Asp Ala Ser Asn Ser Ile Thr Ser Thr Asn Thr Asp Asn Pro 85 90 95Asp Lys Gln Ala Val Tyr His Phe Asp Gly Asp Asp Asp Gly Gln Ser 100 105 110Ala Gln Ser Met Gly Arg Leu Cys Gly Thr Pro Gly Val Ser Cys Ser 115 120 125Ser Gly Asp Trp Arg His Arg Tyr Val Glu Met Ile Ala Glu Tyr Leu 130 135 140Ser Tyr Tyr Lys Gln Ala Gly Ile Pro Val Ser His Val Gly Phe Leu145 150 155 160Asn Glu Gly Asp Gly Ser Asp Phe Met Leu Ser Thr Ala Glu Gln Ala 165 170 175Ala Asp Val Ile Pro Leu Leu His Ser Ala Leu Gln Ser Lys Gly Leu 180 185 190Gly Asp Ile Lys Met Thr Cys Cys Asp Asn Ile Gly Trp Lys Ser Gln 195 200 205Met Asp Tyr Thr Ala Lys Leu Ala Glu Leu Glu Val Glu Lys Tyr Leu 210 215 220Ser Val Ile Thr Ser His Glu Tyr Ser Ser Ser Pro Asn Gln Pro Met225 230 235 240Asn Thr Thr Leu Pro Thr Trp Met Ser Glu Gly Ala Ala Asn Asp Gln 245 250 255Ala Phe Ala Thr Ala Trp Tyr Val Asn Gly Gly Ser Asn Glu Gly Phe 260 265 270Thr Trp Ala Val Lys Ile Ala Gln Gly Ile Val Asn Ala Asp Leu Ser 275 280 285Ala Tyr Ile Tyr Trp Glu Gly Val Glu Thr Asn Asn Lys Gly Ser Leu 290 295 300Ser His Val Ile Asp Thr Asp Gly Thr Lys Phe Thr Ile Ser Ser Ile305 310 315 320Leu Trp Ala Ile Ala His Trp Ser Arg His Ile Arg Pro Gly Ala His 325 330 335Arg Leu Ser Thr Ser Gly Val Val Gln Asp Thr Ile Val Gly Ala Phe 340 345 350Glu Asn Val Asp Gly Ser Val Val Met Val Leu Thr Asn Ser Gly Thr 355 360 365Ala Ala Gln Thr Val Asp Leu Gly Val Ser Gly Ser Ser Phe Ser Thr 370 375 380Ala Gln Ala Phe Thr Ser Asp Ala Glu Ala Gln Met Val Asp Thr Lys385 390 395 400Val Thr Leu Ser Asp Gly Arg Val Lys Val Thr Val Pro Val His Gly 405 410 415Val Val Thr Val Lys Leu Thr Thr Ala Lys Ser Ser Lys Pro Val Ser 420 425 430Thr Ala Val Ser Ala Gln Ser Ala Pro Thr Pro Thr Ser Val Lys His 435 440 445Thr Leu Thr His Gln Lys Thr Ser Ser Thr Thr Leu Ser Thr Ala Lys 450 455 460Ala Pro Thr Ser Thr Gln Thr Thr Ser Val Val Glu Ser Ala Lys Ala465 470 475 480Val Lys Tyr Pro Val Pro Pro Val Ala Ser Lys Gly Ser Ser Lys Ser 485 490 495Ala Pro Lys Lys Gly Thr Lys Lys Thr Thr Thr Lys Lys Gly Ser His 500 505 510Gln Ser His Lys Ala His Ser Ala Thr His Arg Arg Cys Arg His Gly 515 520 525Ser Tyr Arg Arg Gly His Cys Thr Asn 530 53539948DNAFusarium verticillioides 39atgtggaaac tcctcgtcag cggtcttgtc gccgtcgcgt ccctcagcgg cgtgaacgct 60gcttatccta accctggtcc cgtcaccggc gatactcgtg ttcacgaccc tacggttgtc 120aagactccca gcggtggata cttgctggct catactggcg ataacgtttc gctcaagact 180tcttctgatc gaactgcttg gaaggatgca ggtgctgttt tccccaacgg tgcgccttgg 240actacgcagt acaccaaggg cgacaagaac ctctgggccc ctgatatctc ctaccacaac 300ggccagtact atctgtacta ctccgcctct tccttcggtc agcgtacctc tgccattttt 360ctcgctacca gcaagaccgg tgcatccggc tcgtggacca accaaggcgt cgtcgtcgag 420tccaacaaca acaacgacta caatgccatt gacggaaatc tctttgtcga ctctgatgga 480aaatggtggc tctccttcgg ctctttctgg tccggcatca agctcatcca actcgacccc 540aagaccggca agcgcaccgg ctcaagcatg tactccctcg ccaaacgcga cgcctccgtc 600gaaggcgccg tcgaggctcc gttcatcacc aaacgcggaa gcacctacta cctctgggtg 660tcgttcgaca agtgttgcca gggcgctgct agcacgtacc gtgtcatggt tggacggtcg 720agcagcatta ctggtcctta tgttgacaag gctggtaagc agatgatgtc tggtggagga 780acggagatta tggctagtca cggatctatt catggaccgg gacataatgc tgttttcact 840gataacgatg cggacgttct tgtctatcat tactacgata acgctggcac agcgctgttg 900ggcatcaact tgctcagata tgacaatggc tggcctgttg cttattag 94840315PRTFusarium verticillioides 40Met Trp Lys Leu Leu Val Ser Gly Leu Val Ala Val Ala Ser Leu Ser1 5 10 15Gly Val Asn Ala Ala Tyr Pro Asn Pro Gly Pro Val Thr Gly Asp Thr 20 25 30Arg Val His Asp Pro Thr Val Val Lys Thr Pro Ser Gly Gly Tyr Leu 35 40 45Leu Ala His Thr Gly Asp Asn Val Ser Leu Lys Thr Ser Ser Asp Arg 50 55 60Thr Ala Trp Lys Asp Ala Gly Ala Val Phe Pro Asn Gly Ala Pro Trp65 70 75 80Thr Thr Gln Tyr Thr Lys Gly Asp Lys Asn Leu Trp Ala Pro Asp Ile 85 90 95Ser Tyr His Asn Gly Gln Tyr Tyr Leu Tyr Tyr Ser Ala Ser Ser Phe 100 105 110Gly Gln Arg Thr Ser Ala Ile Phe Leu Ala Thr Ser Lys Thr Gly Ala 115 120 125Ser Gly Ser Trp Thr Asn Gln Gly Val Val Val Glu Ser Asn Asn Asn 130 135 140Asn Asp Tyr Asn Ala Ile Asp Gly Asn Leu Phe Val Asp Ser Asp Gly145 150 155 160Lys Trp Trp Leu Ser Phe Gly Ser Phe Trp Ser Gly Ile Lys Leu Ile 165 170 175Gln Leu Asp Pro Lys Thr Gly Lys Arg Thr Gly Ser Ser Met Tyr Ser 180 185 190Leu Ala Lys Arg Asp Ala Ser Val Glu Gly Ala Val Glu Ala Pro Phe 195 200 205Ile Thr Lys Arg Gly Ser Thr Tyr Tyr Leu Trp Val Ser Phe Asp Lys 210 215 220Cys Cys Gln Gly Ala Ala Ser Thr Tyr Arg Val Met Val Gly Arg Ser225 230 235 240Ser Ser Ile Thr Gly Pro Tyr Val Asp Lys Ala Gly Lys Gln Met Met 245 250 255Ser Gly Gly Gly Thr Glu Ile Met Ala Ser His Gly Ser Ile His Gly 260 265 270Pro Gly His Asn Ala Val Phe Thr Asp Asn Asp Ala Asp Val Leu Val 275 280 285Tyr His Tyr Tyr Asp Asn Ala Gly Thr Ala Leu Leu Gly Ile Asn Leu 290 295 300Leu Arg Tyr Asp Asn Gly Trp Pro Val Ala Tyr305 310 315411352DNATrichoderma reesei 41atgaaagcaa acgtcatctt gtgcctcctg gcccccctgg tcgccgctct ccccaccgaa 60accatccacc tcgaccccga gctcgccgct ctccgcgcca acctcaccga gcgaacagcc 120gacctctggg accgccaagc ctctcaaagc atcgaccagc tcatcaagag aaaaggcaag 180ctctactttg gcaccgccac cgaccgcggc ctcctccaac gggaaaagaa cgcggccatc 240atccaggcag acctcggcca ggtgacgccg gagaacagca tgaagtggca gtcgctcgag 300aacaaccaag gccagctgaa ctggggagac gccgactatc tcgtcaactt tgcccagcaa 360aacggcaagt cgatacgcgg ccacactctg atctggcact cgcagctgcc tgcgtgggtg 420aacaatatca acaacgcgga tactctgcgg caagtcatcc gcacccatgt ctctactgtg 480gttgggcggt acaagggcaa gattcgtgct tgggtgagtt ttgaacacca catgcccctt 540ttcttagtcc gctcctcctc ctcttggaac ttctcacagt tatagccgta tacaacattc 600gacaggaaat ttaggatgac aactactgac tgacttgtgt gtgtgatggc gataggacgt 660ggtcaatgaa atcttcaacg aggatggaac gctgcgctct tcagtctttt ccaggctcct 720cggcgaggag tttgtctcga ttgcctttcg tgctgctcga gatgctgacc cttctgcccg 780tctttacatc aacgactaca atctcgaccg cgccaactat ggcaaggtca acgggttgaa 840gacttacgtc tccaagtgga tctctcaagg agttcccatt gacggtattg gtgagccacg 900acccctaaat gtcccccatt agagtctctt tctagagcca aggcttgaag ccattcaggg

960actgacacga gagccttctc tacaggaagc cagtcccatc tcagcggcgg cggaggctct 1020ggtacgctgg gtgcgctcca gcagctggca acggtacccg tcaccgagct ggccattacc 1080gagctggaca ttcagggggc accgacgacg gattacaccc aagttgttca agcatgcctg 1140agcgtctcca agtgcgtcgg catcaccgtg tggggcatca gtgacaaggt aagttgcttc 1200ccctgtctgt gcttatcaac tgtaagcagc aacaactgat gctgtctgtc tttacctagg 1260actcgtggcg tgccagcacc aaccctcttc tgtttgacgc aaacttcaac cccaagccgg 1320catataacag cattgttggc atcttacaat ag 135242347PRTTrichoderma reesei 42Met Lys Ala Asn Val Ile Leu Cys Leu Leu Ala Pro Leu Val Ala Ala1 5 10 15Leu Pro Thr Glu Thr Ile His Leu Asp Pro Glu Leu Ala Ala Leu Arg 20 25 30Ala Asn Leu Thr Glu Arg Thr Ala Asp Leu Trp Asp Arg Gln Ala Ser 35 40 45Gln Ser Ile Asp Gln Leu Ile Lys Arg Lys Gly Lys Leu Tyr Phe Gly 50 55 60Thr Ala Thr Asp Arg Gly Leu Leu Gln Arg Glu Lys Asn Ala Ala Ile65 70 75 80Ile Gln Ala Asp Leu Gly Gln Val Thr Pro Glu Asn Ser Met Lys Trp 85 90 95Gln Ser Leu Glu Asn Asn Gln Gly Gln Leu Asn Trp Gly Asp Ala Asp 100 105 110Tyr Leu Val Asn Phe Ala Gln Gln Asn Gly Lys Ser Ile Arg Gly His 115 120 125Thr Leu Ile Trp His Ser Gln Leu Pro Ala Trp Val Asn Asn Ile Asn 130 135 140Asn Ala Asp Thr Leu Arg Gln Val Ile Arg Thr His Val Ser Thr Val145 150 155 160Val Gly Arg Tyr Lys Gly Lys Ile Arg Ala Trp Asp Val Val Asn Glu 165 170 175Ile Phe Asn Glu Asp Gly Thr Leu Arg Ser Ser Val Phe Ser Arg Leu 180 185 190Leu Gly Glu Glu Phe Val Ser Ile Ala Phe Arg Ala Ala Arg Asp Ala 195 200 205Asp Pro Ser Ala Arg Leu Tyr Ile Asn Asp Tyr Asn Leu Asp Arg Ala 210 215 220Asn Tyr Gly Lys Val Asn Gly Leu Lys Thr Tyr Val Ser Lys Trp Ile225 230 235 240Ser Gln Gly Val Pro Ile Asp Gly Ile Gly Ser Gln Ser His Leu Ser 245 250 255Gly Gly Gly Gly Ser Gly Thr Leu Gly Ala Leu Gln Gln Leu Ala Thr 260 265 270Val Pro Val Thr Glu Leu Ala Ile Thr Glu Leu Asp Ile Gln Gly Ala 275 280 285Pro Thr Thr Asp Tyr Thr Gln Val Val Gln Ala Cys Leu Ser Val Ser 290 295 300Lys Cys Val Gly Ile Thr Val Trp Gly Ile Ser Asp Lys Asp Ser Trp305 310 315 320Arg Ala Ser Thr Asn Pro Leu Leu Phe Asp Ala Asn Phe Asn Pro Lys 325 330 335Pro Ala Tyr Asn Ser Ile Val Gly Ile Leu Gln 340 34543222PRTTrichoderma reesei 43Met Val Ser Phe Thr Ser Leu Leu Ala Ala Ser Pro Pro Ser Arg Ala1 5 10 15Ser Cys Arg Pro Ala Ala Glu Val Glu Ser Val Ala Val Glu Lys Arg 20 25 30Gln Thr Ile Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr Ser 35 40 45Tyr Trp Asn Asp Gly His Gly Gly Val Thr Tyr Thr Asn Gly Pro Gly 50 55 60Gly Gln Phe Ser Val Asn Trp Ser Asn Ser Gly Asn Phe Val Gly Gly65 70 75 80Lys Gly Trp Gln Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser Gly 85 90 95Ser Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser 100 105 110Arg Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr 115 120 125Asn Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp Gly 130 135 140Ser Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile145 150 155 160Ile Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn His 165 170 175Arg Ser Ser Gly Ser Val Asn Thr Ala Asn His Phe Asn Ala Trp Ala 180 185 190Gln Gln Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val 195 200 205Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Val Ser 210 215 22044797PRTTrichoderma reesei 44Met Val Asn Asn Ala Ala Leu Leu Ala Ala Leu Ser Ala Leu Leu Pro1 5 10 15Thr Ala Leu Ala Gln Asn Asn Gln Thr Tyr Ala Asn Tyr Ser Ala Gln 20 25 30Gly Gln Pro Asp Leu Tyr Pro Glu Thr Leu Ala Thr Leu Thr Leu Ser 35 40 45Phe Pro Asp Cys Glu His Gly Pro Leu Lys Asn Asn Leu Val Cys Asp 50 55 60Ser Ser Ala Gly Tyr Val Glu Arg Ala Gln Ala Leu Ile Ser Leu Phe65 70 75 80Thr Leu Glu Glu Leu Ile Leu Asn Thr Gln Asn Ser Gly Pro Gly Val 85 90 95Pro Arg Leu Gly Leu Pro Asn Tyr Gln Val Trp Asn Glu Ala Leu His 100 105 110Gly Leu Asp Arg Ala Asn Phe Ala Thr Lys Gly Gly Gln Phe Glu Trp 115 120 125Ala Thr Ser Phe Pro Met Pro Ile Leu Thr Thr Ala Ala Leu Asn Arg 130 135 140Thr Leu Ile His Gln Ile Ala Asp Ile Ile Ser Thr Gln Ala Arg Ala145 150 155 160Phe Ser Asn Ser Gly Arg Tyr Gly Leu Asp Val Tyr Ala Pro Asn Val 165 170 175Asn Gly Phe Arg Ser Pro Leu Trp Gly Arg Gly Gln Glu Thr Pro Gly 180 185 190Glu Asp Ala Phe Phe Leu Ser Ser Ala Tyr Thr Tyr Glu Tyr Ile Thr 195 200 205Gly Ile Gln Gly Gly Val Asp Pro Glu His Leu Lys Val Ala Ala Thr 210 215 220Val Lys His Phe Ala Gly Tyr Asp Leu Glu Asn Trp Asn Asn Gln Ser225 230 235 240Arg Leu Gly Phe Asp Ala Ile Ile Thr Gln Gln Asp Leu Ser Glu Tyr 245 250 255Tyr Thr Pro Gln Phe Leu Ala Ala Ala Arg Tyr Ala Lys Ser Arg Ser 260 265 270Leu Met Cys Ala Tyr Asn Ser Val Asn Gly Val Pro Ser Cys Ala Asn 275 280 285Ser Phe Phe Leu Gln Thr Leu Leu Arg Glu Ser Trp Gly Phe Pro Glu 290 295 300Trp Gly Tyr Val Ser Ser Asp Cys Asp Ala Val Tyr Asn Val Phe Asn305 310 315 320Pro His Asp Tyr Ala Ser Asn Gln Ser Ser Ala Ala Ala Ser Ser Leu 325 330 335Arg Ala Gly Thr Asp Ile Asp Cys Gly Gln Thr Tyr Pro Trp His Leu 340 345 350Asn Glu Ser Phe Val Ala Gly Glu Val Ser Arg Gly Glu Ile Glu Arg 355 360 365Ser Val Thr Arg Leu Tyr Ala Asn Leu Val Arg Leu Gly Tyr Phe Asp 370 375 380Lys Lys Asn Gln Tyr Arg Ser Leu Gly Trp Lys Asp Val Val Lys Thr385 390 395 400Asp Ala Trp Asn Ile Ser Tyr Glu Ala Ala Val Glu Gly Ile Val Leu 405 410 415Leu Lys Asn Asp Gly Thr Leu Pro Leu Ser Lys Lys Val Arg Ser Ile 420 425 430Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Thr Gln Met Gln Gly Asn 435 440 445Tyr Tyr Gly Pro Ala Pro Tyr Leu Ile Ser Pro Leu Glu Ala Ala Lys 450 455 460Lys Ala Gly Tyr His Val Asn Phe Glu Leu Gly Thr Glu Ile Ala Gly465 470 475 480Asn Ser Thr Thr Gly Phe Ala Lys Ala Ile Ala Ala Ala Lys Lys Ser 485 490 495Asp Ala Ile Ile Tyr Leu Gly Gly Ile Asp Asn Thr Ile Glu Gln Glu 500 505 510Gly Ala Asp Arg Thr Asp Ile Ala Trp Pro Gly Asn Gln Leu Asp Leu 515 520 525Ile Lys Gln Leu Ser Glu Val Gly Lys Pro Leu Val Val Leu Gln Met 530 535 540Gly Gly Gly Gln Val Asp Ser Ser Ser Leu Lys Ser Asn Lys Lys Val545 550 555 560Asn Ser Leu Val Trp Gly Gly Tyr Pro Gly Gln Ser Gly Gly Val Ala 565 570 575Leu Phe Asp Ile Leu Ser Gly Lys Arg Ala Pro Ala Gly Arg Leu Val 580 585 590Thr Thr Gln Tyr Pro Ala Glu Tyr Val His Gln Phe Pro Gln Asn Asp 595 600 605Met Asn Leu Arg Pro Asp Gly Lys Ser Asn Pro Gly Gln Thr Tyr Ile 610 615 620Trp Tyr Thr Gly Lys Pro Val Tyr Glu Phe Gly Ser Gly Leu Phe Tyr625 630 635 640Thr Thr Phe Lys Glu Thr Leu Ala Ser His Pro Lys Ser Leu Lys Phe 645 650 655Asn Thr Ser Ser Ile Leu Ser Ala Pro His Pro Gly Tyr Thr Tyr Ser 660 665 670Glu Gln Ile Pro Val Phe Thr Phe Glu Ala Asn Ile Lys Asn Ser Gly 675 680 685Lys Thr Glu Ser Pro Tyr Thr Ala Met Leu Phe Val Arg Thr Ser Asn 690 695 700Ala Gly Pro Ala Pro Tyr Pro Asn Lys Trp Leu Val Gly Phe Asp Arg705 710 715 720Leu Ala Asp Ile Lys Pro Gly His Ser Ser Lys Leu Ser Ile Pro Ile 725 730 735Pro Val Ser Ala Leu Ala Arg Val Asp Ser His Gly Asn Arg Ile Val 740 745 750Tyr Pro Gly Lys Tyr Glu Leu Ala Leu Asn Thr Asp Glu Ser Val Lys 755 760 765Leu Glu Phe Glu Leu Val Gly Glu Glu Val Thr Ile Glu Asn Trp Pro 770 775 780Leu Glu Glu Gln Gln Ile Lys Asp Ala Thr Pro Asp Ala785 790 79545744PRTTrichoderma reesei 45Met Arg Tyr Arg Thr Ala Ala Ala Leu Ala Leu Ala Thr Gly Pro Phe1 5 10 15Ala Arg Ala Asp Ser His Ser Thr Ser Gly Ala Ser Ala Glu Ala Val 20 25 30Val Pro Pro Ala Gly Thr Pro Trp Gly Thr Ala Tyr Asp Lys Ala Lys 35 40 45Ala Ala Leu Ala Lys Leu Asn Leu Gln Asp Lys Val Gly Ile Val Ser 50 55 60Gly Val Gly Trp Asn Gly Gly Pro Cys Val Gly Asn Thr Ser Pro Ala65 70 75 80Ser Lys Ile Ser Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly 85 90 95Val Arg Tyr Ser Thr Gly Ser Thr Ala Phe Thr Pro Gly Val Gln Ala 100 105 110Ala Ser Thr Trp Asp Val Asn Leu Ile Arg Glu Arg Gly Gln Phe Ile 115 120 125Gly Glu Glu Val Lys Ala Ser Gly Ile His Val Ile Leu Gly Pro Val 130 135 140Ala Gly Pro Leu Gly Lys Thr Pro Gln Gly Gly Arg Asn Trp Glu Gly145 150 155 160Phe Gly Val Asp Pro Tyr Leu Thr Gly Ile Ala Met Gly Gln Thr Ile 165 170 175Asn Gly Ile Gln Ser Val Gly Val Gln Ala Thr Ala Lys His Tyr Ile 180 185 190Leu Asn Glu Gln Glu Leu Asn Arg Glu Thr Ile Ser Ser Asn Pro Asp 195 200 205Asp Arg Thr Leu His Glu Leu Tyr Thr Trp Pro Phe Ala Asp Ala Val 210 215 220Gln Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn Thr225 230 235 240Thr Trp Ala Cys Glu Asp Gln Tyr Thr Leu Gln Thr Val Leu Lys Asp 245 250 255Gln Leu Gly Phe Pro Gly Tyr Val Met Thr Asp Trp Asn Ala Gln His 260 265 270Thr Thr Val Gln Ser Ala Asn Ser Gly Leu Asp Met Ser Met Pro Gly 275 280 285Thr Asp Phe Asn Gly Asn Asn Arg Leu Trp Gly Pro Ala Leu Thr Asn 290 295 300Ala Val Asn Ser Asn Gln Val Pro Thr Ser Arg Val Asp Asp Met Val305 310 315 320Thr Arg Ile Leu Ala Ala Trp Tyr Leu Thr Gly Gln Asp Gln Ala Gly 325 330 335Tyr Pro Ser Phe Asn Ile Ser Arg Asn Val Gln Gly Asn His Lys Thr 340 345 350Asn Val Arg Ala Ile Ala Arg Asp Gly Ile Val Leu Leu Lys Asn Asp 355 360 365Ala Asn Ile Leu Pro Leu Lys Lys Pro Ala Ser Ile Ala Val Val Gly 370 375 380Ser Ala Ala Ile Ile Gly Asn His Ala Arg Asn Ser Pro Ser Cys Asn385 390 395 400Asp Lys Gly Cys Asp Asp Gly Ala Leu Gly Met Gly Trp Gly Ser Gly 405 410 415Ala Val Asn Tyr Pro Tyr Phe Val Ala Pro Tyr Asp Ala Ile Asn Thr 420 425 430Arg Ala Ser Ser Gln Gly Thr Gln Val Thr Leu Ser Asn Thr Asp Asn 435 440 445Thr Ser Ser Gly Ala Ser Ala Ala Arg Gly Lys Asp Val Ala Ile Val 450 455 460Phe Ile Thr Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Glu Gly Asn465 470 475 480Ala Gly Asp Arg Asn Asn Leu Asp Pro Trp His Asn Gly Asn Ala Leu 485 490 495Val Gln Ala Val Ala Gly Ala Asn Ser Asn Val Ile Val Val Val His 500 505 510Ser Val Gly Ala Ile Ile Leu Glu Gln Ile Leu Ala Leu Pro Gln Val 515 520 525Lys Ala Val Val Trp Ala Gly Leu Pro Ser Gln Glu Ser Gly Asn Ala 530 535 540Leu Val Asp Val Leu Trp Gly Asp Val Ser Pro Ser Gly Lys Leu Val545 550 555 560Tyr Thr Ile Ala Lys Ser Pro Asn Asp Tyr Asn Thr Arg Ile Val Ser 565 570 575Gly Gly Ser Asp Ser Phe Ser Glu Gly Leu Phe Ile Asp Tyr Lys His 580 585 590Phe Asp Asp Ala Asn Ile Thr Pro Arg Tyr Glu Phe Gly Tyr Gly Leu 595 600 605Ser Tyr Thr Lys Phe Asn Tyr Ser Arg Leu Ser Val Leu Ser Thr Ala 610 615 620Lys Ser Gly Pro Ala Thr Gly Ala Val Val Pro Gly Gly Pro Ser Asp625 630 635 640Leu Phe Gln Asn Val Ala Thr Val Thr Val Asp Ile Ala Asn Ser Gly 645 650 655Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Thr Tyr Pro Ser 660 665 670Ser Ala Pro Arg Thr Pro Pro Lys Gln Leu Arg Gly Phe Ala Lys Leu 675 680 685Asn Leu Thr Pro Gly Gln Ser Gly Thr Ala Thr Phe Asn Ile Arg Arg 690 695 700Arg Asp Leu Ser Tyr Trp Asp Thr Ala Ser Gln Lys Trp Val Val Pro705 710 715 720Ser Gly Ser Phe Gly Ile Ser Val Gly Ala Ser Ser Arg Asp Ile Arg 725 730 735Leu Thr Ser Thr Leu Ser Val Ala 740462031DNAPodospora anserina 46atgatccacc tcaagccagc cctcgcggcg ttgttggcgc tgtcgacgca atgtgtggct 60attgatttgt ttgtcaagtc ttcggggggg aataagacga ctgatatcat gtatggtctt 120atgcacgagg atatcaacaa ctccggcgac ggcggcatct acgccgagct aatctccaac 180cgcgcgttcc aagggagtga gaagttcccc tccaacctcg acaactggag ccccgtcggt 240ggcgctaccc ttacccttca gaagcttgcc aagccccttt cctctgcgtt gccttactcc 300gtcaatgttg ccaaccccaa ggagggcaag ggcaagggca aggacaccaa ggggaagaag 360gttggcttgg ccaatgctgg gttttggggt atggatgtca agaggcagaa gtacactggt 420agcttccacg ttactggtga gtacaagggt gactttgagg ttagcttgcg cagcgcgatt 480accggggaga cctttggcaa gaaggtggtg aagggtggga gtaagaaggg gaagtggacc 540gagaaggagt ttgagttggt gcctttcaag gatgcgccca acagcaacaa cacctttgtt 600gtgcagtggg atgccgaggg cgcaaaggac ggatctttgg atctcaactt gatcagcttg 660ttccctccga cattcaaggg aaggaagaat gggctgagaa ttgatcttgc gcagacgatg 720gttgagctca agccgacctt cttgcgcttc cccggtggca acatgctcga gggtaacacc 780ttggacactt ggtggaagtg gtacgagacc attggccctc tgaaggatcg cccgggcatg 840gctggtgtct gggagtacca gcaaaccctt ggcttgggtc tggtcgagta catggagtgg 900gccgatgaca tgaacttgga gcccattgtc ggtgtcttcg ctggtcttgc cctcgatggc 960tcgttcgttc ccgaatccga gatgggatgg gtcatccaac aggctctcga cgaaatcgag 1020ttcctcactg gcgatgctaa gaccaccaaa tggggtgccg tccgcgcgaa gcttggtcac 1080cccaagcctt ggaaggtcaa gtgggttgag atcggtaacg aggattggct tgccggacgc 1140cctgctggct tcgagtcgta catcaactac cgcttcccca tgatgatgaa ggccttcaac 1200gaaaagtacc ccgacatcaa gatcatcgcc tcgccctcca tcttcgacaa catgacaatc 1260cccgcgggtg ctgccggtga tcaccacccg tacctgactc ccgatgagtt cgttgagcga 1320ttcgccaagt tcgataactt gagcaaggat aacgtgacgc tcatcggcga ggctgcgtcg 1380acgcatccta acggtggtat cgcttgggag ggagatctca tgcccttgcc ttggtggggc 1440ggcagtgttg ctgaggctat cttcttgatc agcactgaga gaaacggtga caagatcatc 1500ggtgctactt acgcgcctgg tcttcgcagc ttggaccgct ggcaatggag catgacctgg 1560gtgcagcatg ccgccgaccc ggccctcacc actcgctcga ccagttggta tgtctggaga 1620atcctcgccc accacatcat ccgtgagacg ctcccggtcg atgccccggc cggcaagccc

1680aactttgacc ctctgttcta cgttgccgga aagagcgaga gtggcaccgg tatcttcaag 1740gctgccgtct acaactcgac tgaatcgatc ccggtgtcgt tgaagtttga tggtctcaac 1800gagggagcgg ttgccaactt gacggtgctt actgggccgg aggatccgta tggatacaac 1860gaccccttca ctggtatcaa tgttgtcaag gagaagacca ccttcatcaa ggccggaaag 1920ggcggcaagt tcaccttcac cctgccgggc ttgagtgttg ctgtgttgga gacggccgac 1980gcggtcaagg gtggcaaggg aaagggcaag ggcaagggaa agggtaactg a 2031472031DNAArtificial Sequencesynthesized codon optimized cDNA for Pa51A 47atgatccacc tcaagcccgc cctcgccgcc ctcctcgccc tcagcaccca atgcgtcgcc 60atcgacctct tcgtcaagag cagcggcggc aacaagacca ccgacatcat gtacggcctc 120atgcacgagg acatcaacaa cagcggcgac ggcggcatct acgccgagct gatcagcaac 180cgcgccttcc agggcagcga gaagttcccc agcaacctcg acaactggtc ccccgtcggc 240ggcgccaccc tcaccctcca gaagctcgcc aagcccctgt cctctgccct cccctactcc 300gtcaacgtcg ccaaccccaa ggagggtaag ggtaagggca aggacaccaa gggcaagaag 360gtcggcctcg ccaacgccgg cttttggggc atggacgtca agcgccagaa atacaccggc 420agcttccacg tcaccggcga gtacaagggc gacttcgagg tcagcctccg cagcgccatt 480accggcgaga ccttcggcaa gaaggtcgtc aagggcggca gcaagaaggg caagtggacc 540gagaaggagt tcgagctggt ccccttcaag gacgccccca acagcaacaa caccttcgtc 600gtccagtggg acgccgaggg cgccaaggac ggcagcctcg acctcaacct catcagcctc 660ttcccgccca ccttcaaggg ccgcaagaac ggcctccgca tcgacctcgc ccagaccatg 720gtcgagctga agcccacctt cctccgcttt cccggcggca acatgctcga gggcaacacc 780ctcgacacct ggtggaagtg gtacgagacc atcggccccc tgaaggaccg ccctggcatg 840gccggcgtct gggagtacca gcagacgctg ggcctcggcc tggtcgagta catggagtgg 900gccgacgaca tgaacctcga gcccatcgtc ggcgtctttg ctggcctggc cctggatggc 960agctttgtcc ccgagagcga gatgggctgg gtcatccagc aggctctcga tgagatcgag 1020ttcctcaccg gcgacgccaa gaccaccaag tggggcgccg tccgcgccaa gctcggccac 1080cctaagccct ggaaggtcaa atgggtcgag atcggcaacg aggactggct cgccggccga 1140cctgccggct tcgagagcta catcaactac cgcttcccca tgatgatgaa ggccttcaac 1200gagaaatacc ccgacatcaa gatcattgcc agcccctcca tcttcgacaa catgaccatt 1260ccagccggtg ctgccggtga ccaccacccc tacctcaccc ccgacgaatt tgtcgagcgc 1320ttcgccaagt tcgacaacct cagcaaggac aacgtcaccc tcattggcga ggccgccagc 1380acccacccca acggcggcat tgcctgggag ggcgacctca tgcccctgcc ctggtggggc 1440ggcagcgtcg ccgaggccat cttcctcatc agcaccgagc gcaacggcga caagatcatc 1500ggcgccacct acgcccctgg cctccgatct ctcgaccgct ggcagtggag catgacctgg 1560gtccagcacg ccgccgaccc tgccctcacc acccgcagca ccagctggta cgtctggcgc 1620atcctcgccc accacatcat tcgcgagacc ctccccgtcg acgcccccgc cggcaagccc 1680aacttcgacc ccctcttcta cgtcgctggc aagtcggaga gcggcaccgg catcttcaag 1740gccgccgtct acaacagcac cgagagcatc cccgtcagcc tcaagttcga cggcctcaac 1800gagggcgccg tcgccaacct caccgtcctc accggccccg aggaccccta cggctacaac 1860gaccccttca ccggcatcaa cgtcgtcaag gaaaagacca ccttcatcaa ggccggcaag 1920ggcggcaagt tcacctttac cctccccggc ctctctgtcg ccgtcctcga gaccgccgac 1980gccgtgaagg gtggcaaggg aaagggaaag ggcaagggta agggtaacta a 2031481020DNAArtificial Sequenceconstructed GZ43A sequence 48atgtatcgga agttggccgt catctcggcc ttcttggcca cagctcgtgc taccaacgac 60gactgtcctc tcatcactag tagatggact gcggatcctt cggctcatgt ctttaacgac 120accttgtggc tctacccgtc tcatgacatc gatgctggat ttgagaatga tcctgatgga 180ggccagtacg ccatgagaga ttaccatgtc tactctatcg acaagatcta cggttccctg 240ccggtcgatc acggtacggc cctgtcagtg gaggatgtcc cctgggcctc tcgacagatg 300tgggctcctg acgctgccca caagaacggc aaatactacc tatacttccc tgccaaagac 360aaggatgata tcttcagaat cggcgttgct gtctcaccaa cccccggcgg accattcgtc 420cccgacaaga gttggatccc tcacactttc agcatcgacc ccgccagttt cgtcgatgat 480gatgacagag cctacttggc atggggtggt atcatgggtg gccagcttca acgatggcag 540gataagaaca agtacaacga atctggcact gagccaggaa acggcaccgc tgccttgagc 600cctcagattg ccaagctgag caaggacatg cacactctgg cagagaagcc tcgcgacatg 660ctcattcttg accccaagac tggcaagccg ctcctttctg aggatgaaga ccgacgcttc 720ttcgaaggac cctggattca caagcgcaac aagatttact acctcaccta ctctactggc 780acaacccact atcttgtcta tgcgacttca aagaccccct atggtcctta cacctaccag 840ggcagaattc tggagccagt tgatggctgg actactcact ctagtatcgt caagtaccag 900ggtcagtggt ggctatttta tcacgatgcc aagacatctg gcaaggacta tcttcgccag 960gtaaaggcta agaagatttg gtacgatagc aaaggaaaga tcttgacaaa gaagccttga 1020491038DNAArtificial Sequenceconstructed Gz43A sequence 49atgtatcgga agttggccgt catctcggcc ttcttggcca cagctcgtgc tcaagacact 60aatgacattc ctcccctgat caccgacctc tggtccgcag atccctcggc tcatgttttc 120gaaggcaagc tctgggttta cccatctcac gacatcgaag ccaatgttgt caacggcaca 180ggaggcgctc aatacgccat gagggattac catacctact ccatgaagag catctatggt 240aaagatcccg ttgtcgacca cggcgtcgct ctctcagtcg atgacgttcc ctgggcgaag 300cagcaaatgt gggctcctga cgcagctcat aagaacggca aatattatct gtacttcccc 360gccaaggaca aggatgagat cttcagaatt ggagttgctg tctccaacaa gcccagcggt 420cctttcaagg ccgacaagag ctggatccct ggcacgtaca gtatcgatcc tgctagctac 480gtcgacactg ataacgaggc ctacctcatc tggggcggta tctggggcgg ccagctccaa 540gcctggcagg ataaaaagaa ctttaacgag tcgtggattg gagacaaggc tgctcctaac 600ggcaccaatg ccctatctcc tcagatcgcc aagctaagca aggacatgca caagatcacc 660gaaacacccc gcgatctcgt cattctcgcc cccgagacag gcaagcctct tcaggctgag 720gacaacaagc gacgattctt cgagggccct tggatccaca agcgcggcaa gctttactac 780ctcatgtact ccaccggtga tacccacttc cttgtctacg ctacttccaa gaacatctac 840ggtccttata cctaccgggg caagattctt gatcctgttg atgggtggac tactcatgga 900agtattgttg agtataaggg acagtggtgg cttttctttg ctgatgcgca tacgtctggt 960aaggattacc ttcgacaggt gaaggcgagg aagatctggt atgacaagaa cggcaagatc 1020ttgcttcacc gtccttag 1038501920DNAArtificial Sequencesynthesized codon optimized Pf51A sequence 50atgtaccgga agctcgccgt gatcagcgcc ttcctggcga ctgctcgcgc catcaccatc 60aacgtcagcc agagcggcgg caacaagacc agcccgctcc agtacggcct catgttcgag 120gacatcaacc acggcggcga cggcggcctc tacgccgagc tggtccggaa ccgggccttc 180cagggcagca ccgtctaccc ggccaacctc gacggctacg actcggtgaa cggcgcgatt 240ctcgcgctcc agaacctcac caacccgctc agcccgagca tgccctcgtc gctgaacgtc 300gccaagggct cgaacaacgg cagcatcggc ttcgccaacg aggggtggtg gggcatcgag 360gtcaagccgc agcggtacgc cggcagcttc tacgtccagg gcgactacca gggcgacttc 420gacatcagcc tccagagcaa gctcacccag gaggtcttcg cgacggcgaa ggtccggtcg 480agcggcaagc acgaggactg ggtccagtac aagtacgagc tggtcccgaa gaaggccgcc 540agcaacacca acaacaccct caccatcacc ttcgacagca agggcctcaa ggacggcagc 600ctcaacttca acctcatcag cctcttcccg ccgacctaca acaaccggcc gaacggcctc 660cggatcgacc tcgtcgaggc catggcggag ctggagggca agttcctccg cttccccggc 720ggctcggacg tggagggcgt ccaggccccg tactggtaca agtggaacga gaccgtcggc 780gacctcaagg accgctactc gcgcccgagc gcctggacct acgaggagag caacggcatc 840ggcctcatcg agtacatgaa ctggtgcgac gacatgggcc tcgagccgat cctcgccgtc 900tgggacggcc actacctcag caacgaggtc atcagcgaga acgacctcca gccgtacatc 960gacgacaccc tcaaccagct cgagttcctc atgggcgccc cggacactcc ctacgggtct 1020tggagggcta gcctcggcta cccgaagccg tggaccatca actacgtcga gatcggcaac 1080gaggacaacc tctacggcgg cctcgagacc tacatcgcct accggttcca ggcctactac 1140gacgccatca ccgccaagta cccgcacatg accgtcatgg agagcctcac cgagatgccc 1200ggccccgctg ccgcggcgtc ggactaccac cagtactcga cgcccgacgg cttcgtcagc 1260cagttcaact acttcgacca gatgccggtc accaaccgca cgctgaacgg cgagatcgcc 1320accgtctacc ccaacaaccc gagcaactcg gtggcgtggg gcagcccgtt cccgctctac 1380ccgtggtgga tcgggtccgt ggctgaggcc gtcttcctca tcggcgagga gcggaacagc 1440ccgaagatca tcggcgccag ctacgccccc atgttccgca acattaacaa ctggcagtgg 1500agcccgaccc tgatcgcctt cgacgccgac agcagccgga cgtcgcgctc tacttcctgg 1560cacgtcatca agctcctcag caccaacaag atcacccaga acctgcccac gacgtggtct 1620gggggggaca tcggcccgct ctactgggtc gccggccgga acgacaacac cggcagcaac 1680atcttcaagg ccgccgtcta caacagcacc agcgacgtcc cggtcaccgt ccagttcgcc 1740ggctgcaacg ccaagagcgc caacctcacc atcctctcgt cggacgaccc caacgccagc 1800aactacccgg gcggccccga ggtcgtcaag accgagatcc agagcgtcac cgccaacgcc 1860cacggcgcct tcgagttcag cctcccgaac ctgtcggtgg ctgtgctgaa gacggagtag 19205117PRTTrichomonas reesei 51Met Tyr Arg Lys Leu Ala Val Ile Ser Ala Phe Leu Ala Thr Ala Arg1 5 10 15Ala5223DNAArtificial Sequencesynthetic primer 52cacccatgct gctcaatctt cag 235323DNAArtificial Sequencesynthetic primer 53ttacgcagac ttggggtctt gag 235424DNAArtificial Sequencesynthetic primer 54caccatgtgg ctgacctccc catt 245528DNAArtificial Sequencesynthetic primer 55ttagctaaac tgccaccagt tgaagttg 285626DNAArtificial Sequencesynthetic primer 56caccatgcgc ttctcttggc tattgt 265727DNAArtificial Sequencesynthetic primer 57ctacaattct gatttcacaa aaacacc 275822DNAArtificial Sequencesynthetic primer 58caccatgcag ctcaagtttc tg 225922DNAArtificial Sequencesynthetic primer 59ctaaatctta ggacgagtaa gc 226029DNAArtificial Sequencesynthetic primer 60caccatggtt cgcttcagtt caatcctag 296121DNAArtificial Sequencesynthetic primer 61ctagctagag taaggctttc c 216227DNAArtificial Sequencesynthetic primer 62caccatgcac tacgctaccc tcaccac 276322DNAArtificial Sequencesynthetic primer 63tcaagtagag gggctgctca cc 226427DNAArtificial Sequencesynthetic primer 64caccatgaaa ctctctagct acctctg 276525DNAArtificial Sequencesynthetic primer 65ctacgaaact gtgacagtca cgttg 256630DNAArtificial Sequencesynthetic primer 66caccatgctc ttctcgctcg ttcttcctac 306721DNAArtificial Sequencesynthetic primer 67ttagttggtg cagtggccac g 216829DNAArtificial Sequencesynthetic primer 68caccatgaat cctttatctc tcggccttg 296923DNAArtificial Sequencesynthetic primer 69cagccctcat agtcgtcttc ttc 237024DNAArtificial Sequencesynthetic primer 70caccatgcgt cttctatcgt ttcc 247120DNAArtificial Sequencesynthetic primer 71ctacaaaggc ctaggatcaa 207227DNAArtificial Sequencesynthetic primer 72caccatgcac tacgctaccc tcaccac 277322DNAArtificial Sequencesynthetic primer 73tcaagtagag gggctgctca cc 227426DNAArtificial Sequencesynthetic primer 74caccatgaag gtatactggc tcgtgg 267525DNAArtificial Sequencesynthetic primer 75ctatgcagct gtgaaagact caacc 257625DNAArtificial Sequencesynthetic primer 76caccatgtgg aaactcctcg tcagc 257725DNAArtificial Sequencesynthetic primer 77ctaataagca acaggccagc cattg 257833DNAArtificial Sequencesynthetic primer 78caccatgctt cagcgatttg cttatatttt acc 337928DNAArtificial Sequencesynthetic primer 79ttatgcgaac tgccaataat caaagttg 288025DNAArtificial Sequencesynthetic primer 80caccatgtac cggaagctcg ccgtg 258124DNAArtificial Sequencesynthetic primer 81ctactccgtc ttcagcacag ccac 248240DNAArtificial Sequencesynthetic primer 82ccgcggccgc accatggttt ctttctccta cctgctgctg 408342DNAArtificial Sequencesynthetic primer 83ccggcgcgcc cttactagta gacagtgatg gaagcagatc cg 428438DNAArtificial Sequencesynthetic primer 84ccgcggccgc accatgatct ccatttcctc gctcagct 388543DNAArtificial Sequencesynthetic primer 85ccggcgcgcc cttatcactt ggatataacc ctgcaagaag gta 438625DNAArtificial Sequencesynthetic primer 86caccatggca gctccaagtt tatcc 258720DNAArtificial Sequencesynthetic primer 87tcagtagctc gggaccactc 208828DNAArtificial Sequencesynthetic primer 88caccatgaga tatagaacag ctgccgct 288940DNAArtificial Sequencesynthetic primer 89cgaccgccct gcggagtctt gcccagtggt cccgcgacag 409040DNAArtificial Sequencesynthetic primer 90ctgtcgcggg accactgggc aagactccgc agggcggtcg 409120DNAArtificial Sequencesynthetic primer 91cctacgctac cgacagagtg 209220DNAArtificial Sequencesynthetic primer 92gtctagactg gaaacgcaac 209321DNAArtificial Sequencesynthetic primer 93gagttgtgaa gtcggtaatc c 219435DNAArtificial Sequencesynthetic primer 94caccatgaaa gcaaacgtca tcttgtgcct cctgg 359543DNAArtificial Sequencesynthetic primer 95ctattgtaag atgccaacaa tgctgttata tgccggcttg ggg 439621DNAArtificial Sequencesynthetic primer 96gagttgtgaa gtcggtaatc c 219718DNAArtificial Sequencesynthetic primer 97cacgaagagc ggcgattc 189823DNAArtificial Sequencesynthetic primer 98cacccatgct gctcaatctt cag 239923DNAArtificial Sequencesynthetic primer 99ttacgcagac ttggggtctt gag 2310020DNAArtificial Sequencesynthetic primer 100gcttgagtgt atcgtgtaag 2010121DNAArtificial Sequencesynthetic primer 101gcaacggcaa agccccactt c 2110232DNAArtificial Sequencesynthetic primer 102gtagcggccg cctcatctca tctcatccat cc 3210324DNAArtificial Sequencesynthetic primer 103caccatgcag ctcaagtttc tgtc 2410432DNAArtificial Sequencesynthetic primer 104ggttactagt caactgcccg ttctgtagcg ag 3210529DNAArtificial Sequencesynthetic primer 105catgcgatcg cgacgttttg gtcaggtcg 2910640DNAArtificial Sequencesynthetic primer 106gacagaaact tgagctgcat ggtgtgggac aacaagaagg 4010729DNAArtificial Sequencesynthetic primer 107caccatggtt cgcttcagtt caatcctag 2910822DNAArtificial Sequencesynthetic primer 108gtggctagaa gatatccaac ac 2210929DNAArtificial Sequencesynthetic primer 109catgcgatcg cgacgttttg gtcaggtcg 2911039DNAArtificial Sequencesynthetic primer 110gaactgaagc gaaccatggt gtgggacaac aagaaggac 3911121DNAArtificial Sequencesynthetic primer 111gtagttatgc gcatgctaga c 2111222DNAArtificial Sequencesynthetic primer 112gtggctagaa gatatccaac ac 22113504PRTGeobacillus stearothermophilus 113Met Lys Val Val Asn Val Pro Ser Asn Gly Arg Glu Lys Phe Lys Lys1 5 10 15Asn Trp Lys Phe Cys Val Gly Thr Gly Arg Leu Gly Leu Ala Leu Gln 20 25 30Lys Glu Tyr Leu Asp His Leu Lys Leu Val Gln Glu Lys Ile Gly Phe 35 40 45Arg Tyr Ile Arg Gly His Gly Leu Leu Ser Asp Asp Val Gly Ile Tyr 50 55 60Arg Glu Val Glu Ile Asp Gly Glu Met Lys Pro Phe Tyr Asn Phe Thr65 70 75 80Tyr Ile Asp Arg Ile Val Asp Ser Tyr Leu Ala Leu Asn Ile Arg Pro 85 90 95Phe Ile Glu Phe Gly Phe Met Pro Lys Ala Leu Ala Ser Gly Asp Gln 100 105 110Thr Val Phe Tyr Trp Lys Gly Asn Val Thr Pro Pro Lys Asp Tyr Asn 115 120 125Lys Trp Arg Asp Leu Ile Val Ala Val Val Ser His Phe Ile Glu Arg 130 135 140Tyr Gly Ile Glu Glu Val Arg Thr Trp Leu Phe Glu Val Trp Asn Glu145 150 155 160Pro Asn Leu Val Asn Phe Trp Lys Asp Ala Asn Lys Gln Glu Tyr Phe 165 170 175Lys Leu Tyr Glu Val Thr Ala Arg Ala Val Lys Ser Val Asp Pro His 180 185 190Leu Gln Val Gly Gly Pro Ala Ile Cys Gly Gly Ser Asp Glu Trp Ile 195 200 205Thr Asp Phe Leu His Phe Cys Ala Glu Arg Arg Val Pro Val Asp Phe 210 215 220Val Ser Arg His Ala Tyr Thr Ser Lys Ala Pro His Lys Lys Thr Phe225 230 235 240Glu Tyr Tyr Tyr Gln Glu Leu Glu Leu Glu Pro Pro Glu Asp Met Leu 245 250 255Glu Gln Phe Lys Thr Val Arg Ala Leu Ile Arg Gln Ser Pro Phe Pro 260 265 270His Leu Pro Leu His Ile Thr Glu Tyr Asn Thr Ser Tyr Ser Pro Ile 275 280 285Asn Pro Val His Asp Thr Ala Leu Asn Ala Ala Tyr Ile Ala Arg Ile 290 295 300Leu Ser Glu Gly Gly Asp Tyr Val Asp Ser Phe Ser Tyr Trp Thr Phe305 310 315 320Ser Asp Val Phe Glu Glu Met Asp Val Pro Lys Ala Leu Phe His Gly 325 330 335Gly Phe Gly Leu Val Ala Leu His Ser Ile Pro Lys Pro Thr Phe His 340

345 350Ala Phe Thr Phe Phe Asn Ala Leu Gly Asp Glu Leu Leu Tyr Arg Asp 355 360 365Gly Glu Met Ile Val Thr Arg Arg Lys Asp Gly Ser Ile Ala Ala Val 370 375 380Leu Trp Asn Leu Val Met Glu Lys Gly Glu Gly Leu Thr Lys Glu Val385 390 395 400Gln Leu Val Ile Pro Val Ser Phe Ser Ala Val Phe Ile Lys Arg Gln 405 410 415Ile Val Asn Glu Gln Tyr Gly Asn Ala Trp Arg Val Trp Lys Gln Met 420 425 430Gly Arg Pro Arg Phe Pro Ser Arg Gln Ala Val Glu Thr Leu Pro Ser 435 440 445Ala Gln Pro His Val Met Thr Glu Gln Arg Arg Ala Thr Asp Gly Val 450 455 460Ile His Leu Ser Ile Val Leu Ser Lys Asn Glu Val Thr Leu Ile Glu465 470 475 480Ile Glu Gln Val Arg Asp Glu Thr Ser Thr Tyr Val Gly Leu Asp Asp 485 490 495Gly Glu Ile Thr Ser Tyr Ser Ser 500114500PRTThermoanaerobacterium saccharolyticum 114Met Ile Lys Val Arg Val Pro Asp Phe Ser Asp Lys Lys Phe Ser Asp1 5 10 15Arg Trp Arg Tyr Cys Val Gly Thr Gly Arg Leu Gly Leu Ala Leu Gln 20 25 30Lys Glu Tyr Ile Glu Thr Leu Lys Tyr Val Lys Glu Asn Ile Asp Phe 35 40 45Lys Tyr Ile Arg Gly His Gly Leu Leu Cys Asp Asp Val Gly Ile Tyr 50 55 60Arg Glu Asp Val Val Gly Asp Glu Val Lys Pro Phe Tyr Asn Phe Thr65 70 75 80Tyr Ile Asp Arg Ile Phe Asp Ser Phe Leu Glu Ile Gly Ile Arg Pro 85 90 95Phe Val Glu Ile Gly Phe Met Pro Lys Lys Leu Ala Ser Gly Thr Gln 100 105 110Thr Val Phe Tyr Trp Glu Gly Asn Val Thr Pro Pro Lys Asp Tyr Glu 115 120 125Lys Trp Ser Asp Leu Val Lys Ala Val Leu His His Phe Ile Ser Arg 130 135 140Tyr Gly Ile Glu Glu Val Leu Lys Trp Pro Phe Glu Ile Trp Asn Glu145 150 155 160Pro Asn Leu Lys Glu Phe Trp Lys Asp Ala Asp Glu Lys Glu Tyr Phe 165 170 175Lys Leu Tyr Lys Val Thr Ala Lys Ala Ile Lys Glu Val Asn Glu Asn 180 185 190Leu Lys Val Gly Gly Pro Ala Ile Cys Gly Gly Ala Asp Tyr Trp Ile 195 200 205Glu Asp Phe Leu Asn Phe Cys Tyr Glu Glu Asn Val Pro Val Asp Phe 210 215 220Val Ser Arg His Ala Thr Thr Ser Lys Gln Gly Glu Tyr Thr Pro His225 230 235 240Leu Ile Tyr Gln Glu Ile Met Pro Ser Glu Tyr Met Leu Asn Glu Phe 245 250 255Lys Thr Val Arg Glu Ile Ile Lys Asn Ser His Phe Pro Asn Leu Pro 260 265 270Phe His Ile Thr Glu Tyr Asn Thr Ser Tyr Ser Pro Gln Asn Pro Val 275 280 285His Asp Thr Pro Phe Asn Ala Ala Tyr Ile Ala Arg Ile Leu Ser Glu 290 295 300Gly Gly Asp Tyr Val Asp Ser Phe Ser Tyr Trp Thr Phe Ser Asp Val305 310 315 320Phe Glu Glu Arg Asp Val Pro Arg Ser Gln Phe His Gly Gly Phe Gly 325 330 335Leu Val Ala Leu Asn Met Ile Pro Lys Pro Thr Phe Tyr Thr Phe Lys 340 345 350Phe Phe Asn Ala Met Gly Glu Glu Met Leu Tyr Arg Asp Glu His Met 355 360 365Leu Val Thr Arg Arg Asp Asp Gly Ser Val Ala Leu Ile Ala Trp Asn 370 375 380Glu Val Met Asp Lys Thr Glu Asn Pro Asp Glu Asp Tyr Glu Val Glu385 390 395 400Ile Pro Val Arg Phe Arg Asp Val Phe Ile Lys Arg Gln Leu Ile Asp 405 410 415Glu Glu His Gly Asn Pro Trp Gly Thr Trp Ile His Met Gly Arg Pro 420 425 430Arg Tyr Pro Ser Lys Glu Gln Val Asn Thr Leu Arg Glu Val Ala Lys 435 440 445Pro Glu Ile Met Thr Ser Gln Pro Val Ala Asn Asp Gly Tyr Leu Asn 450 455 460Leu Lys Phe Lys Leu Gly Lys Asn Ala Val Val Leu Tyr Glu Leu Thr465 470 475 480Glu Arg Ile Asp Glu Ser Ser Thr Tyr Ile Gly Leu Asp Asp Ser Lys 485 490 495Ile Asn Gly Tyr 500115302PRTPenicillium simplicissimum 115Gln Ala Ser Val Ser Ile Asp Ala Lys Phe Lys Ala His Gly Lys Lys1 5 10 15Tyr Leu Gly Thr Ile Gly Asp Gln Tyr Thr Leu Thr Lys Asn Thr Lys 20 25 30Asn Pro Ala Ile Ile Lys Ala Asp Phe Gly Gln Leu Thr Pro Glu Asn 35 40 45Ser Met Lys Trp Asp Ala Thr Glu Pro Asn Arg Gly Gln Phe Thr Phe 50 55 60Ser Gly Ser Asp Tyr Leu Val Asn Phe Ala Gln Ser Asn Gly Lys Leu65 70 75 80Ile Arg Gly His Thr Leu Val Trp His Ser Gln Leu Pro Gly Trp Val 85 90 95Ser Ser Ile Thr Asp Lys Asn Thr Leu Ile Ser Val Leu Lys Asn His 100 105 110Ile Thr Thr Val Met Thr Arg Tyr Lys Gly Lys Ile Tyr Ala Trp Asp 115 120 125Val Leu Asn Glu Ile Phe Asn Glu Asp Gly Ser Leu Arg Asn Ser Val 130 135 140Phe Tyr Asn Val Ile Gly Glu Asp Tyr Val Arg Ile Ala Phe Glu Thr145 150 155 160Ala Arg Ser Val Asp Pro Asn Ala Lys Leu Tyr Ile Asn Asp Tyr Asn 165 170 175Leu Asp Ser Ala Gly Tyr Ser Lys Val Asn Gly Met Val Ser His Val 180 185 190Lys Lys Trp Leu Ala Ala Gly Ile Pro Ile Asp Gly Ile Gly Ser Gln 195 200 205Thr His Leu Gly Ala Gly Ala Gly Ser Ala Val Ala Gly Ala Leu Asn 210 215 220Ala Leu Ala Ser Ala Gly Thr Lys Glu Ile Ala Ile Thr Glu Leu Asp225 230 235 240Ile Ala Gly Ala Ser Ser Thr Asp Tyr Val Asn Val Val Asn Ala Cys 245 250 255Leu Asn Gln Ala Lys Cys Val Gly Ile Thr Val Trp Gly Val Ala Asp 260 265 270Pro Asp Ser Trp Arg Ser Ser Ser Ser Pro Leu Leu Phe Asp Gly Asn 275 280 285Tyr Asn Pro Lys Ala Ala Tyr Asn Ala Ile Ala Asn Ala Leu 290 295 300116329PRTThermoascus aurantiacus 116Met Val Arg Pro Thr Ile Leu Leu Thr Ser Leu Leu Leu Ala Pro Phe1 5 10 15Ala Ala Ala Ser Pro Ile Leu Glu Glu Arg Gln Ala Ala Gln Ser Val 20 25 30Asp Gln Leu Ile Lys Ala Arg Gly Lys Val Tyr Phe Gly Val Ala Thr 35 40 45Asp Gln Asn Arg Leu Thr Thr Gly Lys Asn Ala Ala Ile Ile Gln Ala 50 55 60Asp Phe Gly Gln Val Thr Pro Glu Asn Ser Met Lys Trp Asp Ala Thr65 70 75 80Glu Pro Ser Gln Gly Asn Phe Asn Phe Ala Gly Ala Asp Tyr Leu Val 85 90 95Asn Trp Ala Gln Gln Asn Gly Lys Leu Ile Arg Gly His Thr Leu Val 100 105 110Trp His Ser Gln Leu Pro Ser Trp Val Ser Ser Ile Thr Asp Lys Asn 115 120 125Thr Leu Thr Asn Val Met Lys Asn His Ile Thr Thr Leu Met Thr Arg 130 135 140Tyr Lys Gly Lys Ile Arg Ala Trp Asp Val Val Asn Glu Ala Phe Asn145 150 155 160Glu Asp Gly Ser Leu Arg Gln Thr Val Phe Leu Asn Val Ile Gly Glu 165 170 175Asp Tyr Ile Pro Ile Ala Phe Gln Thr Ala Arg Ala Ala Asp Pro Asn 180 185 190Ala Lys Leu Tyr Ile Asn Asp Tyr Asn Leu Asp Ser Ala Ser Tyr Pro 195 200 205Lys Thr Gln Ala Ile Val Asn Arg Val Lys Gln Trp Arg Ala Ala Gly 210 215 220Val Pro Ile Asp Gly Ile Gly Ser Gln Thr His Leu Ser Ala Gly Gln225 230 235 240Gly Ala Gly Val Leu Gln Ala Leu Pro Leu Leu Ala Ser Ala Gly Thr 245 250 255Pro Glu Val Ala Ile Thr Glu Leu Asp Val Ala Gly Ala Ser Pro Thr 260 265 270Asp Tyr Val Asn Val Val Asn Ala Cys Leu Asn Val Gln Ser Cys Val 275 280 285Gly Ile Thr Val Trp Gly Val Ala Asp Pro Asp Ser Trp Arg Ala Ser 290 295 300Thr Thr Pro Leu Leu Phe Asp Gly Asn Phe Asn Pro Lys Pro Ala Tyr305 310 315 320Asn Ala Ile Val Gln Asp Leu Gln Gln 325117485PRTFusarium verticillioides 117Met Asn Pro Leu Ser Leu Gly Leu Ala Ala Leu Ser Leu Leu Gly Tyr1 5 10 15Val Gly Val Asn Phe Val Ala Ala Phe Pro Thr Asp Ser Asn Ser Gly 20 25 30Ser Glu Val Leu Ile Ser Val Asn Gly His Val Lys His Gln Glu Leu 35 40 45Asp Gly Phe Gly Ala Ser Gln Ala Phe Gln Arg Ala Glu Asp Ile Leu 50 55 60Gly Lys Asp Gly Leu Ser Lys Glu Gly Thr Gln His Val Leu Asp Leu65 70 75 80Leu Phe Ser Lys Asp Ile Gly Ala Gly Phe Ser Ile Leu Arg Asn Gly 85 90 95Ile Gly Ser Ser Asn Ser Ser Asp Lys Asn Phe Met Asn Ser Ile Glu 100 105 110Pro Phe Ser Pro Gly Ser Pro Gly Ala Lys Pro His Tyr Val Trp Asp 115 120 125Gly Tyr Asp Ser Gly Gln Leu Thr Val Ala Gln Glu Ala Phe Lys Arg 130 135 140Gly Leu Lys Phe Leu Tyr Gly Asp Ala Trp Ser Ala Pro Gly Tyr Met145 150 155 160Lys Thr Asn His Asp Glu Asn Asn Gly Gly Tyr Leu Cys Gly Val Thr 165 170 175Gly Ala Ala Cys Ala Ser Gly Asp Trp Lys Gln Ala Tyr Ala Asp Tyr 180 185 190Leu Leu Gln Trp Val Glu Phe Tyr Arg Lys Ser Gly Val Lys Val Thr 195 200 205Asn Leu Gly Phe Leu Asn Glu Pro Gln Phe Ala Ala Pro Tyr Ala Gly 210 215 220Met Leu Ser Asn Gly Thr Gln Ala Ala Asp Phe Ile Arg Val Leu Gly225 230 235 240Lys Thr Ile Arg Lys Arg Gly Ile His Asp Leu Thr Ile Ala Cys Cys 245 250 255Asp Gly Glu Gly Trp Asp Leu Gln Glu Asp Met Met Ala Gly Leu Thr 260 265 270Ala Gly Pro Asp Pro Ala Ile Asn Tyr Leu Ser Val Val Thr Gly His 275 280 285Gly Tyr Val Ser Pro Pro Asn His Pro Leu Ser Thr Thr Lys Lys Thr 290 295 300Trp Leu Thr Glu Trp Ala Asp Leu Thr Gly Gln Phe Thr Pro Tyr Thr305 310 315 320Phe Tyr Asn Asn Ser Gly Gln Gly Glu Gly Met Thr Trp Ala Gly Arg 325 330 335Ile Gln Thr Ala Leu Val Asp Ala Asn Val Ser Gly Phe Leu Tyr Trp 340 345 350Ile Gly Ala Glu Asn Ser Thr Thr Asn Ser Ala Leu Ile Asn Met Ile 355 360 365Gly Asp Lys Val Ile Pro Ser Lys Arg Phe Trp Ala Phe Ala Ser Phe 370 375 380Ser Arg Phe Ala Arg Pro Gly Ala Arg Arg Ile Glu Ala Thr Ser Ser385 390 395 400Val Pro Leu Val Thr Val Ser Ser Phe Leu Asn Thr Asp Gly Thr Val 405 410 415Ala Thr Gln Val Leu Asn Asn Asp Thr Val Ala His Ser Val Gln Leu 420 425 430Val Val Ser Gly Thr Gly Arg Asn Pro His Ser Leu Lys Pro Phe Leu 435 440 445Thr Asp Asn Ser Asn Asp Leu Thr Ala Leu Lys His Leu Lys Ala Thr 450 455 460Gly Lys Gly Ser Phe Gln Thr Thr Ile Pro Pro Arg Ser Leu Val Ser465 470 475 480Phe Val Thr Asp Phe 485

Claims

1. A non-naturally occurring composition comprising: a. a first polypeptide or a variant thereof having .beta.-xylosidase activity, L-.alpha.-arabinofuranosidase activity, or both .beta.-xylosidase activity and L-.alpha.-arabinofuranosidase activity, selected from a Fv43D, Fv3A, Pf43A, Fv43E, Fv39A, Fv43A, Fv43B, Pa51A, Fo43A, Gz43A, Trichoderma reesei Bxl1, Fv51A, Af43A, or Pf51A polypeptide; and b. a second polypeptide or a variant thereof having xylanase activity, selected from a Trichoderma xylanase or an Aspergillus xylanase; wherein the composition is capable of converting 60% or more of the hemicellulose xylan from a biomass into xylose.

2-4. (canceled)

5. The composition of claim 1, wherein the first polypeptide is selected from a Fv43D, Fv3A, Pf43A, Fv43E, Fv39A, Fo43A, Gz43A, or T. reesei Bxl1, or Fv43A, further comprising a third polypeptide or a variant thereof having L-.alpha.-arabinofuranosidase activity, selected from a Fv51A, Af43A, Fv43B, Pa51A or Pf51A polypeptide.

6. (canceled)

7. The composition of claim 1, further comprising a third polypeptide having .beta.-xylosidase activity, wherein the first polypeptide is an Fv3A polypeptide or an Fv43A polypeptide, and the third polypeptide is an Fv43D, Pa51A, Gz43A, Trichoderma reesei Bxl1, Pf43A, Fv43E, Fv39A, Fo43A, or Fv43B polypeptide.

8. (canceled)

9. The composition of claim 1, wherein, if present: (a) the Fv43D polypeptide has at least 90% sequence identity to SEQ ID NO:28, or to residues (i) 20-341, (ii) 21-350, (iii) 107-341, or (iv) 107-350 of SEQ ID NO:28; (b) the Fv3A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2, or to residues (i) 24-766, (ii) 73-321, (iii) 73-394, (iv) 395-622, (v) 24-622, or (vi) 73-622 of SEQ ID NO:2; (c) the Pf43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4, or to residues (i) 21-445, (ii) 21-301, (iii) 21-323, (iv) 21-444, (v) 302-444; (vi) 302-445, (vii) 324-444, or (viii) 324-445 of SEQ ID NO:4; (d) the Fv43E polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6, or to residues (i) 19-530, (ii) 29-530, (iii) 19-300, or (iv) 29-300 of SEQ ID NO:6: (e) the Fv39A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:8, or to residues (i) 20-439, (ii) 20-291, (iii) 145-291, or 145-439 of SEQ ID NO:8: (f) the Fv43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:10, or to residues (i) 23-449, (ii) 23-302, (iii) 23-320, (iv) 23-448, (v) 303-448, or (vi) 321-449 of SEQ ID NO:10; (g) the Fv43B polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:12, or to residues (i) 17-574, (ii) 27-574, (iii) 17-303, or (iv) 27-303 of SEQ ID NO:12; (h) the Pa51A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:14, or to residues (i) 21-676, (ii) 21-652, (iii) 469-652, or (iv) 469-676 of SEQ ID NO:14; (i) the Fo43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:18, or to residues (i) 21-341, (ii) 107-341, (iii) 21-348, or (iv) 107-348 of SEQ ID NO:18; (j) the Gz43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:16, or to residues (i) 19-340, (ii) 53-340, (iii) 19-383, or (iv) 53-383 of SEQ ID NO:16: (k) the Trichoderma reesei Bxl1 polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:44; (l) the Fv51A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:32, or to residues (i) 21-660, (ii) 21-645, (iii) 450-645, or (iv) 450-660 of SEQ ID NO:32; (m) the Af43A polypeptide has at least 90% sequence identity to SEQ ID NO:20, or to residues (i) 15-558, or (ii) 15-295 of SEQ ID NO:20; or (n) the Pf51A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:22, or to residues (i) 21-632, (ii) 461-632, (iii) 21-642, or (iv) 461-642 of SEQ ID NO:22.

10-22. (canceled)

23. The composition of claim 1, wherein, if present: (a) the Fv43D polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:27, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:27, or to a fragment thereof; (b) the Fv3A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:1, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:1, or to a fragment thereof; (c) the Pf43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:3, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:3, or to a fragment thereof; (d) the Fv43E polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:5, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:5, or to a fragment thereof; (e) the Fv39A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:7, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:7, or to a fragment thereof; (f) the Fv43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:9, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:9, or to a fragment thereof; (g) the Fv43B polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:11, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:11, or to a fragment thereof; (h) the Pa51A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:13, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:13, or to a fragment thereof; (i) the Fo43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:17, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:17, or to a fragment thereof; (j) the Gz43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:15, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:15, or to a fragment thereof; (k) the Fv51A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:31, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:31, or to a fragment thereof; (l) the Af43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:19, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:19, or to a fragment thereof; or (m) the Pf51A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:21, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:21, or to a fragment thereof.

24-36. (canceled)

37. The composition of claim 1, wherein the Trichoderma xylanase is a Trichoderma reesei Xyn3 polypeptide, having at least 90% sequence identity to SEQ ID NO:42, or to residues 17-347 of SEQ ID NO:42, or is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:41, or by a nucleic acid that is capable of hybridizing under high stringency conditions to SEQ ID NO:41, or to a fragment thereof; or a Trichoderma reesei Xyn2 polypeptide, having at least 90% sequence identity to SEQ ID NO:43, or to residues 33-222 of SEQ ID NO:43.

38-40. (canceled)

41. The composition of claim 1, wherein the Aspergillus xylanase is an AfuXyn2 polypeptide, having at least 90% sequence identity to SEQ ID NO:24, or to residues 19-228 of SEQ ID NO:24, or is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:23, or by a nucleic acid that is capable of hybridizing under high stringency to SEQ ID NO:23, or to a fragment thereof; or an AfuXyn5 polypeptide, having at least 90% sequence identity to SEQ ID NO:26, or to residues 20-313 of SEQ ID NO:26, or is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:25, or by a nucleic acid that is capable of hybridizing under stringent conditions to a complement of SEQ ID NO:25, or to a fragment thereof.

42-45. (canceled)

46. The composition of claim 1, further comprising one or more polypeptides having cellulase activity.

47. The composition of claim 6, wherein the one or more polypeptides having cellulase activity are independently components of a whole cellulase.

48. The composition of claim 7, wherein the whole cellulase is a .beta.-glucosidase-enriched whole cellulase.

49. The composition of claim 48, wherein the whole cellulase is a filamentous fungal whole cellulase.

50. The composition of claim 48, wherein the filamentous fungal whole cellulase is a preparation from an Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Scytalidium, Thielavia, Chrysosporium, Phanerochaete, Tolypocladium, or Trichoderma species.

51. The composition of claim 48, wherein the whole cellulase is a Chrysosporium lucknowense, Phanerochaete chrysosporium, Trichoderma reesei, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma viride, or Pencillium funiculosum whole cellulase.

52. The composition of claim 46, wherein the one or more polypeptides having cellulase activity are selected from a .beta.-glucosidase, an endoglucanase, and a cellobiohydrolase.

53. The composition of claim 52, wherein the .beta.-glucosidase is a Trichoderma reesei Bgl1 polypeptide, having at least 90% sequence identity to SEQ ID NO:45, or to residues 32-744 of SEQ ID NO:45, which constitutes up to 50 wt. % of the total weight of cellulase enzymes in the composition.

54-55. (canceled)

56. The composition of claim 53, wherein the .beta.-glucosidase polypeptide constitutes 2 wt. % to 50 wt. % of the total weight of proteins in the composition.

57. The composition of claim 47, wherein the one or more polypeptides having cellulase activity constitute 30 wt. % to 80 wt. % of the total weight of proteins in the composition.

58. The composition of claim 57, wherein the one or more polypeptides having cellulase activity together are capable of achieving at least 0.00005 fraction product per mg protein per gram of phosphoric acid swollen cellulose (PASC) as determined by a calcofluor assay.

59. The composition of claim 1, further comprising one or more accessory proteins, selected from mannanases, galactanases, arabinases, ligninases, amyloases, glucuronidases, proteases, esterases, lipases, xyloglucanases, glycoside hydrolase Family 61 polypeptides, CIP1, CIP2, CIP1-like proteins, CIP2-like proteins, cellobiose dehydrogenases, manganese peroxidases, swollenin, and expansins.

60-61. (canceled)

62. The composition of claim 59, wherein the one or more accessory proteins constitute 1 wt. % to 50 wt. % of the total weight of proteins in the composition.

63. (canceled)

64. The composition of claim 1, capable of converting 70% or more of the hemicellulose xylan from a biomass into xylose.

65. The composition of claim 64, capable of converting 80% or more of the hemicellulose xylan from a biomass into xylose.

66. The composition of claim 1, wherein the biomass is corncob, corn stover, corn fiber, switchgrass, sorghum, paper, pulp, or sugarcane bagasse.

67. The composition of claim 1, wherein the biomass is Miscanthus.

68. The composition of claim 1, further comprising a biomass.

69. The composition of claim 68, wherein the combined weight of polypeptides having xylanase activity is 1 g to 40 g per 1 kg of hemicellulose in the biomass.

70. The composition of claim 69, wherein the combined weight of polypeptides having xylanase activity is 0.5 g to 40 g per 1 kg of hemicellulose in the biomass.

71. The composition of claim 70, wherein the combined weight of polypetpides having xylanase activity is 0.5 g to 20 g per 1 kg of hemicellulose in the biomass.

72. The composition of claim 71, wherein the combined weight of polypeptides having xylanase activity is 2 g to 20 g per 1 kg of hemicellulose in the biomass.

73. The composition of claim 68, wherein the combined weight of polypeptides having .beta.-xylosidase activity is 1 g to 50 g per 1 kg of hemicellulose in the biomass.

74. The composition of claim 73, wherein the combined weight of polypeptides having .beta.-xylosidase activity is 0.5 g to 50 g per 1 kg of hemicellulose in the biomass.

75. The composition of claim 74, wherein the combined weight of polypeptides having .beta.-xylosidase activity is 0.5 g to 40 g per 1 kg of hemicellulose in the biomass.

76. The composition of claim 75, wherein the combined weight of polypeptides having .beta.-xylosidase activity is 2 g to 40 g per 1 kg of hemicellulose in the biomass.

77. The composition of claim 68, wherein the combined weight of polypeptides having L-.alpha.-arabinofuranosidase activity, if present, is 0.5 g to 20 g per 1 kg of hemicellulose in the biomass.

78. The composition of claim 77, wherein the combined weight of polypetpides having L-.alpha.-arabinofuranosidase activity, if present, is 0.2 g to 20 g per 1 kg of hemicellulose in the biomass.

79. The composition of claim 68 further comprising one or more polypeptides having cellulase activity, wherein the combined weight of polypeptides having cellulase activity, if present, is 1 g to 100 g per 1 kg of cellulose in the biomass.

80. A fermentation broth comprising the composition of claim 1.

81. The fermentation broth of claim 80, which is the fermentation broth of a filamentous fungus.

82. The fermentation broth of claim 81, wherein the filamentous fungus is a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, Phanerochaete, or Chrysosporium.

83. The fermentation broth of claim 82, wherein the filamentous fungus is a Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereas, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

84-86. (canceled)

87. The fermentation broth of claim 80, which is a cell-free fermentation broth.

88. A method of converting biomass to sugars, comprising contacting the biomass with a non-naturally occurring composition comprising: (a) a first polypeptide or a variant thereof having .beta.-xylosidase activity, L-.alpha.-arabinofuranosidase activity, or both .beta.-xylosidase activity and L-.alpha.-arabinofuranosidase activity, selected from a Fv43D, Fv3A, Pf43A, Fv43E, Fv39A, Fv43A, Fv43B, Pa51A, Fo43A, Gz43A, Trichoderma reesei Bxl1, Fv51A, Af43A, or Pf51A polypeptide; and (b) a second polypeptide or a variant thereof having xylanase activity, selected from a Trichoderma xylanase or an Aspergillus xylanase.

89. A saccharification process comprising treating a material comprising hemicellulose with a non-naturally occurring composition or a fermentation broth comprising: (a) a first polypeptide or a variant thereof having .beta.-xylosidase activity, L-.alpha.-arabinofuranosidase activity, or both .beta.-xylosidase activity and L-.alpha.-arabinofuranosidase activity, selected from a Fv43D, Fv3A, Pf43A, Fv43E, Fv39A, Fv43A, Fv43B, Pa51A, Fo43A, Gz43A, Trichoderma reesei Bxl1, Fv51A, Af43A, or Pf51A polypeptide; and (b) a second polypeptide or a variant thereof having xylanase activity, selected from a Trichoderma xylanase or an Aspergillus xylanase.

90. The process of claim 89, wherein the material comprising hemicellulose is corncob, corn stover, corn fiber, switchgrass, sorghum, paper, pulp, Miscanthus, or sugarcane bagasse.

91. (canceled)

92. The process of claim 89, which yields at least 60% xylose from hemicellulose xylan of the material comprising hemicellulose.

93. The process of claim 89, further comprising recovering monosaccharides.

94-119. (canceled)

120. A host cell engineered to recombinantly express a polypeptide: (a) having .beta.-xylosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:28, or to the amino acid sequence corresponding to residues (i) 20-341, (ii) 21-350, (iii) 107-341, or (iv) 107-350 of SEQ ID NO:28; (b) having .beta.-xylosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4, or to the amino acid sequence corresponding to residues (i) 21-445, (ii) 21-301, (iii) 21-323, (iv) 21-444, (v) 302-444, (vi) 302-445, (vii) 324-444, or (viii) 324-445 of SEQ ID NO:4; (c) having .beta.-xylosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6, or to the amino acid sequence corresponding to residues (i) 19-530, (ii) 29-530, (iii) 19-300, or (iv) 29-300 of SEQ ID NO:6; (d) having .beta.-xylosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:8, or to the amino acid sequence corresponding to residues (i) 20-439, (ii) 20-291, (iii) 145-291, or (iv) 145-439 of SEQ ID NO:8; (e) having .beta.-xylosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:10, or to the amino acid sequence corresponding to residues (i) 23-449, (ii) 23-302, (iii) 23-320, (iv) 23-448, (v) 303-448, (vi) 303-449, (vii) 321-448, or (viii) 321-449 of SEQ ID NO:10; (f) having .beta.-xylosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2, or to the amino acid sequence corresponding to residues (i) 24-766, (ii) 73-321, (iii) 73-394, (iv) 395-622, (v) 24-622, or (iv) 73-622 of SEQ ID NO:2; (g) having .beta.-xylosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:16, or to the amino acid sequence corresponding to residues (i) 19-340, (ii) 53-340, (iii) 19-383, or (iv) 53-383 of SEQ ID NO:16; (h) having .beta.-xylosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:18, or to the amino acid sequence corresponding to residues (i) 21-341, (ii) 107-341, (iii) 21-348, or (iv) 107-348 of SEQ ID NO:18; (i) having .beta.-xylosidase and/or L-.alpha.-arabinofuranosidase activities, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:12, or to the amino acid sequence corresponding to residues (i) 17-574, (ii) 27-574, (iii) 17-303, or (iv) 27-303 of SEQ ID NO:12; (j) having .beta.-xylosidase and/or L-.alpha.-arabinofuranosidase activities, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:14, or to the amino acid sequence corresponding to residues (i) 21-676, (ii) 21-652, (iii) 469-652, or (iv) 469-676 of SEQ ID NO:14; (k) having L-.alpha.-arabinofuranosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20, or to the amino acid sequence corresponding to residues (i) 15-558, or (ii) 15-295 of SEQ ID NO:20; (l) having L-.alpha.-arabinofuranosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:22, or to the amino acid sequence corresponding to residues (i) 21-632, (ii) 461-632, (iii) 21-642, or (iv) 461-642 of SEQ ID NO:22; or (m) having L-.alpha.-arabinofuranosidase activity, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:32, or to the amino acid sequence corresponding to residues (i) 21-660, (ii) 21-645, (iii) 450-645, or (iv) 450-660 of SEQ ID NO:32.

121. The host cell of claim 120, which is a cell of a filamentous fungus.

122. The host cell of claim 121, which is a cell of a Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, Phanerochaete, or Chrysosporium.

123. The host cell of claim 122, which is a cell of a Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

124-125. (canceled)

126. The host cell of claim 120, which is a cell of a bacterium.

127. The host cell of claim 126, wherein the bacterium is a Streptomyces, a Thermomonospora, a Bacillus, or a Cellulomonas.

128. A method of producing the composition of claim 1.

129. (canceled)

130. The process of claim 89, wherein the material comprising hemicellulose further comprises cellulose.

131. The process of claim 89, wherein the amount of polypeptides having xylanase activity is 1 g to 40 g per kg of hemicellulose in the material.

132. The process of claim 131, wherein the amount of polypeptides having xylanase activity is 0.5 g to 40 g per kg of hemicellulose in the material.

134. The process of claim 89, wherein the amount of polypeptides having .beta.-xylosidase activity is 1 g to 50 g per kg of hemicellulose in the material.

135. The process of claim 134, wherein the amount of polypeptide having .beta.-xylosidase activity is 0.5 g to 50 g per kg of hemicellulose in the material.

136. The process of claim 89, wherein the amount of polypeptides having L-.alpha.-arabinofuranosidase activity is 0.5 g to 20 g per kg of hemicellulose in the material.

137. The process of claim 136, wherein the amount of polypeptides having L-.alpha.-arabinofuranosidase activity is 0.2 g to 20 g per kg of hemicellulose in the material.

138. The process of claim 130, wherein the amount of polypeptides having cellulase activity is 1 g to 100 g per kg of cellulose in the material comprising hemicellulose.

139. The process of claim 130, wherein the amount of polypeptides having .beta.-glucosidase activity is 50% or less of the total weight of polypeptides having cellulase activity.

140. The composition of claim 5, wherein, if present: (a) the Fv43D polypeptide has at least 90% sequence identity to SEQ ID NO:28, or to residues (i) 20-341, (ii) 21-350, (iii) 107-341, or (iv) 107-350 of SEQ ID NO:28; (b) the Fv3A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2, or to residues (i) 24-766, (ii) 73-321, (iii) 73-394, (iv) 395-622, (v) 24-622, or (vi) 73-622 of SEQ ID NO:2; (c) the Pf43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4, or to residues (i) 21-445, (ii) 21-301, (iii) 21-323, (iv) 21-444, (v) 302-444; (vi) 302-445, (vii) 324-444, or (viii) 324-445 of SEQ ID NO:4; (d) the Fv43E polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6, or to residues (i) 19-530, (ii) 29-530, (iii) 19-300, or (iv) 29-300 of SEQ ID NO:6; (e) the Fv39A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:8, or to residues (i) 20-439, (ii) 20-291, (iii) 145-291, or 145-439 of SEQ ID NO:8; (f) the Fv43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:10, or to residues (i) 23-449, (ii) 23-302, (iii) 23-320, (iv) 23-448, (v) 303-448, or (vi) 321-449 of SEQ ID NO:10; (g) the Fv43B polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:12, or to residues (i) 17-574, (ii) 27-574, (iii) 17-303, or (iv) 27-303 of SEQ ID NO:12; (h) the Pa51A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:14, or to residues (i) 21-676, (ii) 21-652, (iii) 469-652, or (iv) 469-676 of SEQ ID NO:14; (i) the Fo43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:18, or to residues (i) 21-341, (ii) 107-341, (iii) 21-348, or (iv) 107-348 of SEQ ID NO:18; (j) the Gz43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:16, or to residues (i) 19-340, (ii) 53-340, (iii) 19-383, or (iv) 53-383 of SEQ ID NO:16; (k) the Trichoderma reesei Bxl1 polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:44; (l) the Fv51A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:32, or to residues (i) 21-660, (ii) 21-645, (iii) 450-645, or (iv) 450-660 of SEQ ID NO:32; (m) the Af43A polypeptide has at least 90% sequence identity to SEQ ID NO:20, or to residues (i) 15-558, or (ii) 15-295 of SEQ ID NO:20; or (n) the Pf51A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:22, or to residues (i) 21-632, (ii) 461-632, (iii) 21-642, or (iv) 461-642 of SEQ ID NO:22.

141. The composition of claim 7, wherein, if present: (a) the Fv43D polypeptide has at least 90% sequence identity to SEQ ID NO:28, or to residues (i) 20-341, (ii) 21-350, (iii) 107-341, or (iv) 107-350 of SEQ ID NO:28; (b) the Fv3A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2, or to residues (i) 24-766, (ii) 73-321, (iii) 73-394, (iv) 395-622, (v) 24-622, or (vi) 73-622 of SEQ ID NO:2; (c) the Pf43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4, or to residues (i) 21-445, (ii) 21-301, (iii) 21-323, (iv) 21-444, (v) 302-444; (vi) 302-445, (vii) 324-444, or (viii) 324-445 of SEQ ID NO:4; (d) the Fv43E polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6, or to residues (i) 19-530, (ii) 29-530, (iii) 19-300, or (iv) 29-300 of SEQ ID NO:6; (e) the Fv39A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:8, or to residues (i) 20-439, (ii) 20-291, (iii) 145-291, or 145-439 of SEQ ID NO:8; (f) the Fv43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:10, or to residues (i) 23-449, (ii) 23-302, (iii) 23-320, (iv) 23-448, (v) 303-448, or (vi) 321-449 of SEQ ID NO:10; (g) the Fv43B polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:12, or to residues (i) 17-574, (ii) 27-574, (iii) 17-303, or (iv) 27-303 of SEQ ID NO:12; (h) the Pa51A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:14, or to residues (i) 21-676, (ii) 21-652, (iii) 469-652, or (iv) 469-676 of SEQ ID NO:14; (i) the Fo43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:18, or to residues (i) 21-341, (ii) 107-341, (iii) 21-348, or (iv) 107-348 of SEQ ID NO:18; (j) the Gz43A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:16, or to residues (i) 19-340, (ii) 53-340, (iii) 19-383, or (iv) 53-383 of SEQ ID NO:16; (k) the Trichoderma reesei Bxl1 polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:44; (l) the Fv51A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:32, or to residues (i) 21-660, (ii) 21-645, (iii) 450-645, or (iv) 450-660 of SEQ ID NO:32; (m) the Af43A polypeptide has at least 90% sequence identity to SEQ ID NO:20, or to residues (i) 15-558, or (ii) 15-295 of SEQ ID NO:20; or (n) the Pf51A polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:22, or to residues (i) 21-632, (ii) 461-632, (iii) 21-642, or (iv) 461-642 of SEQ ID NO:22.

142. The composition of claim 5, wherein, if present: (a) the Fv43D polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:27, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:27, or to a fragment thereof; (b) the Fv3A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:1, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:1, or to a fragment thereof; (c) the Pf43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:3, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:3, or to a fragment thereof; (d) the Fv43E polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:5, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:5, or to a fragment thereof; (e) the Fv39A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:7, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:7, or to a fragment thereof; (f) the Fv43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:9, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:9, or to a fragment thereof; (g) the Fv43B polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:11, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:11, or to a fragment thereof; (h) the Pa51A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:13, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:13, or to a fragment thereof; (i) the Fo43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:17, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:17, or to a fragment thereof; (j) the Gz43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:15, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:15, or to a fragment thereof; (k) the Fv51A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:31, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:31, or to a fragment thereof; (l) the Af43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:19, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:19, or to a fragment thereof; or (m) the Pf51A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:21, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:21, or to a fragment thereof.

143. The composition of claim 7, wherein, if present: (a) the Fv43D polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:27, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:27, or to a fragment thereof; (b) the Fv3A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:1, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:1, or to a fragment thereof; (c) the Pf43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:3, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:3, or to a fragment thereof; (d) the Fv43E polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:5, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:5, or to a fragment thereof; (e) the Fv39A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:7, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:7, or to a fragment thereof; (f) the Fv43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:9, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:9, or to a fragment thereof; (g) the Fv43B polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:11, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:11, or to a fragment thereof; (h) the Pa51A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:13, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:13, or to a fragment thereof; (i) the Fo43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:17, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:17, or to a fragment thereof; (j) the Gz43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:15, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:15, or to a fragment thereof; (k) the Fv51A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:31, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:31, or to a fragment thereof; (l) the Af43A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:19, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:19, or to a fragment thereof; or (m) the Pf51A polypeptide is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO:21, or by a nucleic acid that is capable of hybridizing, under high stringency conditions, to SEQ ID NO:21, or to a fragment thereof.