BR112015003085B1 - RECOMBINANT NUCLEIC ACID MOLECULE, RECOMBINANT EXPRESSION CONSTRUCT AND GENETICALLY ENGINEERED PROKARYOTIC HOST CELL - Google Patents

Abstract

MACROPHOMINA PHASEOLINE CELLULOSE AND/OR HEMICELLULOSE DEGRADATION ENZYMES AND USES THEREOF. The present invention provides Macrophomina phaseolina ("M. phaseolina") nucleotide sequences encoding proteins/enzymes with cellulolytic activity, including a cellulase activity, a Beta-endoglucanase, a cellobiohydrolase, a Beta-glucosidase, an Alpha- glucosidase, a xylanase, a mannanse, a Beta-xylosidase, an Alpha-xylosidase, a galactosidase, an arabinofuranosidase, an Alpha-fucosidases, a Beta-galactanase, an unsaturated Beta-glucuronyl hydrolase and/or oligomerase activity. Vectors, expression constructs and host cells comprising and/or consisting of the nucleotide sequences of the enzyme genes are also provided. The invention further provides methods for producing the enzymes and methods for modifying the enzymes to improve their desirable characteristics. The enzymes of the invention can be used in a variety of, but not limited to, pharmaceutical, agricultural, food and feed processing, biofuels, energy efficiency and industrial contexts. These enzymes are also useful for the complete hydrolysis of lignocellulosic biomass into simple sugar which can then be fermented into liquid fuels and chemical feed stocks.

Description

RELATED ORDERS

[1] This application claims the priority benefit of United States Provisional Patent Application Serial Number 61/683908, filed August 16, 2012; the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[2] The present invention relates to cellulose and/or hemicellulose degradation enzymes derived from a fungus M. phaseolina. The invention provides polypeptides having any cellulolytic activity, including a cellulase activity, an endoglycanase, a cellobiohydrolase, a β-glucosidase, an α-glucosidase, a xylanase, a mannanase, a β-xylosidase, an arabinofuranosidase, an α-fucosidase, an unsaturated β-glucuronyl hydrolase and/or oligomerase activity, polynucleotides encoding the foregoing polypeptides, and methods for preparing and using the foregoing polynucleotides and polypeptides. The invention provides enzymes for the bioconversion of cellulose residues into fermentable sugars and these sugars can be used as a chemical feedstock for the production of ethanol and fuels, including biofuels such as bioethanol, biopropanol, biobutanol and biodiesel. Polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and food processing, biofuels, energy efficiency and industrial contexts. The invention also provides compositions or manufactures comprising enzyme mixtures having at least one enzyme of the present invention. The invention also relates to nucleic acid, vector and host cell constructs comprising the polynucleotides as well as methods for making and using the polypeptides that are used in cellulose and/or hemicellulose degradation.

FUNDAMENTALS OF THE INVENTION

[3] Cellulose consists of a linear chain of β 1-4 linked D-glucose residues having a molecular structure as shown in Figure 1. These long linear glucose chains are tightly packed together in microfibrils and are non-covalently linked together by hemicelluloses (Kolpak FJ, Blackwell J. Determination of the structure of cellulose II. Macromolecules 1976; 9:273-278; Carpita NC, Gibeaut DM. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J 1993; 3:1-30). Cellulose forms crystalline, insoluble microfibrils in plant cell walls that are recalcitrant to enzymatic hydrolysis. This recalcitrance is the bottleneck in cellulosic ethanol production (Himmel M, Ding S, Johnson D, Adney W, Nimlos M, Brady JW, Foust TD. Biomass recalcitrance: Engineering plants and enzymes for biofuels production. Science 2007; 315: 804) . On the other hand, hemicelluloses is the most complex group of non-starch polysaccharides and consists of polymers of xylose, arabinose, galactose, mannose, which are often highly branched and connected to another cell wall structure.

[4] Cellulose, the most abundant biological material in the world, is a vast, renewable resource that could help meet the world's energy needs. But the production of fermentable sugars from biomass using cellulolytic enzymes is still not able to compete economically due to the inefficiency of currently used cellulolytic enzymes. There is a need for research directed towards increasing the effectiveness of cellulolytic enzymes that can be used to generate fermentable sugar from lignocellulosic materials at reduced cost. For complete digestion of cellulose into glucose, cellulase systems require three classes of enzymes, β-1,4-endoglycanases (EGL), exoglycanases/cellobiohydrolases (CBH) and β-glucosidase (BGL). During the hydrolysis process, endoglycanase first randomly cleaves different regions of crystalline cellulose, producing chain ends. Cellobiohydrolases then sequentially release cellobiose from the end of the cellulose polymer. Finally, β-glucosidase breaks the bonds between the two glucose sugars of cellobiose to produce glucose monomers (Figure 2). This synergistic effect of these enzymes makes possible the hydrolysis of cellulose into glucose (Wood TM. Synergism between enzyme components of Penicillium pinophilum cellulase in solubilizing hydrogen bond ordered cellulose. J Biochem 1989; 260:37-43; Wood TM, McRae ST. The cellulase of Trichoderma koningii: purification and properties of some endoglycanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase. J Biochem 1978;171:61-9; Wood TM, McRae ST. Synergism between enzyme involved in the solubilization of the native cellulose Adv Chem Ser 1979; 18:181-210). That is, at least three types of enzymes, eg endoglycanases, cellobiohydrolases and β-glucosidase and their synergistic effects are necessary for the complete digestion of cellulose. For each of these three enzymes there are different structural variants that perform the same function. Many other enzymes are required to digest hemicelluloses to sugar monomer including xylanase, xylosidase, arabinofuranosidase, mannanase, galactosidase and glucuronidase.

[5] It is an object of the present invention to provide isolated polypeptides having cellulolytic activity and isolated nucleic acid sequences encoding the polypeptides for improving the conversion of cellulosic materials to fermentable sugar.

SUMMARY OF THE INVENTION

[6] Among other things, the present invention discloses a polynucleotide molecule encoding cellulolytic enzyme which is derived from a fungus M. phaseolina. The current invention also relates to the use of the fungus M. phaseolina in the degradation of cellulosic materials.

[7] The invention provides polypeptides having cellulolytic activity, including endoglycanase, cellobiohydrolase, β-glucosidase, α-glucosidase, glycanases, α-glycan lyase, α-xylosidase, β-xylosidase, b-glucuronyl hydrolase d-4,5 activity -unsaturated, amyloglycosidase, mannosidase, α-fucosidase, arabinosidase, xylanase, mannanase, β-galactosidase, β-galactanase, arabinofuranosidase, and/or oligomerase, and the nucleic acids encoding each polypeptide, and methods for preparing and using each one of said polypeptides.

[8] The main object of the present invention is to disclose the sets of nucleotide sequences that encode β-1,4-endoglycanase (SEQ ID NOs. 1, 2, 4, 5, 7, 8, 10, 11, 13, 14 , 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46 and 47), cellobiohydrolase (SEQ ID NOs. 49, 50, 52, 53, 55 and 56), β-glucosidase (SEQ ID NOs. 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77 , 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115 and 116), α-glucosidase (SEQ ID NOs. 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136 and 137), Exo-1,3-β-glucanase (SEQ ID NOs. 139, 140, 142, 143, 145, 146, 148, 149, 151, 152, 154, 155, 157, 158, 160, 161, 163 and 164), α-glycan lyase (SEQ ID NOs 166, 167, 169 and 170), α-xylosidase (SEQ ID NOs. 172, 173, 175 and 176), d-4,5-unsaturated b-glucuronyl hydrolase (SEQ ID NOs. 178, 179, 181, 182 , 184, 185, 187, 188, 190 and 191), amyloglycosidase (SEQ ID NOs. 193, 194, 196 and 197), α-1,2-mannosidase (SEQ ID NOs. 199, 200, 202, 203, 205, 206, 208, 209, 211, 212, 214 and 215), α-1,3-glucanase (SEQ ID NOs. 217, 218, 220, 221, 223, 224, 226 , 227, 229, 230, 232, 233, 235, 236, 238, 239, 241, 242, 244, 245, 247, 248, 250 and 251), α-fucosidase (SEQ ID NOs. 253, 254, 256 and 257), xylan 1,4-β-Xylosidase (SEQ ID NOs. 259, 260, 262, 263, 265, 266, 268, 269, 271, 272, 274, 275, 277, 278, 280, 281, 283, 284, 286, 287, 289 and 290), endo-1,5-α-arabinosidase (SEQ ID NOs. 292, 293, 295, 296, 298, 299, 301 and 302), Endo-1,4-β- xylanase (SEQ ID NOs. 304, 305, 307, 308, 310, 311, 313 and 314), α-arabinofuranosidase (SEQ ID NOs. 316, 317, 319, 320, 322, 323, 325, 326, 328, 329 , 331 and 332), β-galactosidase (SEQ ID NOs. 334, 335, 337, 338, 340, 341, 343, 344, 346, 347, 349 and 350), Endo-1,4-β-galactanase (SEQ ID NOs. 352, 353, 355, 356, 358 and 359), Endo-1,6-β-glucanase (SEQ ID NOs. 361 and 362) and endo-β-1,4-mannanase (SEQ ID NOs. 364 and 365) of the fungi M. phaseolina. For each gene of the invention, an open reading frame (ORF) sequence was manually derived from the respective genomic sequence by deleting predicted intron sequences and processing exon sequences together. Vectors, constructs/expression vectors, and host cells comprising the enzyme genes are also included.

[9] In another object, the invention provides polypeptide sequences deduced from the ORF sequences of genes. Based on the conservation of the sequence presented between M. phaseolina genes of the invention and their homologues in other fungi, it is concluded that the polypeptides encoded by these M. phaseolina genes present enzymatic activities similar to their homologues. The polypeptide sequences of the invention correspond to those of β-1,4-endoglycanase (SEQ ID NOs. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 and 48), cellobiohydrolase (SEQ ID NOs. 51, 54 and 57), β-glucosidase (SEQ ID NOs. 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96 , 99, 102, 105, 108, 111, 114 and 117), α-glucosidase (SEQ ID NOs. 120, 123, 126, 129, 132, 135 and 138), Exo-1,3-β-glucanase (SEQ ID NOs 141, 144, 147, 150, 153, 156, 159, 162 and 165), α-glycan lyase (SEQ ID NOs 168 and 171), α-xylosidase (SEQ ID NOs 174 and 177), b d-4,5-unsaturated -glucuronyl hydrolase (SEQ ID NOs. 180, 183, 186, 189 and 192), amyloglycosidase (SEQ ID NOs. 195 and 198), α-1,2-mannosidase (SEQ ID NOs. 201 , 204, 207, 210, 213 and 216), α-1,3-glycanase (SEQ ID NOs. 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249 and 252), α -fucosidase (SEQ ID NOs. 255 and 258), xylan 1,4-β-Xylosidase (SEQ ID NOs. 261, 264, 267, 270, 273, 276, 279, 282, 285, 288 and 291), endo- 1,5-α-arabinosidase (SEQ ID NOs. 294, 297, 300 and 303), Endo-1,4-β-xylanase (SEQ ID NOs. 306, 309, 312 and 315), α-arabinofuranosidase (SEQ ID NOs. 318, 321, 324, 327, 330 and 333), β-galactosidase (SEQ ID NOs. 336, 339, 342, 345, 348, and 351 ), Endo-1,4-β-galactanase (SEQ ID NOs. 354, 357 and 360), Endo-1,6-β-glucanase (SEQ ID NOs. 363) and endo-β-1,4-mannanase ( SEQ ID NOs. 366). The present invention also relates to isolated polynucleotides comprising any complement of the nucleotide sequences described above.

[10] In one aspect, the enzymes of the invention have an increased catalytic rate to enhance the cellulose hydrolysis process. This increased catalytic rate leads to increased efficiency in the production of fermentable sugars, which can be used by microorganisms to produce ethanol. The invention provides industrial applications (e.g. ethanol biomass) using enzymes of the invention having decreased costs in processes of converting biomass to ethanol. Thus, the invention provides efficient processes for producing bioethanol from any cellulosic material.

[11] In another aspect, the compositions and methods of the invention are used in the enzymatic digestion of biomass and may comprise the use of many different enzymes, including cellulases and hemicellulases.

[12] In another aspect, compositions used to practice the invention include a "cellulase blend" which is a mixture of at least three different cellulase enzymes such as endoglycanase, cellobiohydrolase and β-glucosidase for the complete digestion of cellulose for the production of glucose monomer.

[13] In another aspect, compositions used to practice the invention can include mixtures of enzymes, including xylanases, xylosidases, cellobiohydrolases, and/or arabinofuranosidases or other enzymes that can digest hemicellulose to monomer sugars.

[14] In another aspect, the endoglycanases of the invention are used in the food industry, for example, for cooking and processing of fruit and vegetables, depletion of agricultural residues, in the manufacture of animal feed, in the production of pulp and paper, in the manufacture of of textiles and domestic and industrial cleaning products.

[15] In another aspect, the present invention also provides the molecular biology and genetic information of the genes and enzymes established in the main object to be exploited/used for the regulation, cellulose conversion and/or degradation of hemicelluloses for the production of valuable products .

[16] In another aspect, in order to facilitate the in vitro production of cellulose degradation polypeptides and/or hemicelluloses, the present invention also includes an expression construct capable of expressing the polypeptide containing at least 70% sequential amino acids as established in SEQ ID NOs. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 2 25, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 3 00, Preferably, the expression construct inserted DNA or cDNA with nucleotide sequence as set forth in SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 2 32, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 3 07, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364.

[17] Yet another aspect of the present invention discloses a recombinant gene construct comprising a polynucleotide template having the nucleotide sequence as set forth in SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 2 32, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 3 07, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364, wherein the polynucleotide template is expressible in a host cell to produce an enzyme that degrades cellulose and/or hemicellulose. Preferably, the recombinant gene construct further comprises an operably linked promoter region to enhance expression of the polynucleotide template.

[18] According to one of the preferred embodiments of the present invention, the M. phaseolina fungi are strain ms6. The isolated polypeptide is also preferably derived from this strain.

[19] Yet another aspect of the present invention is to provide a viable and potentially commercial way to isolate cellulose and/or hemicellulose degradation enzyme from M. phaseolina to meet the growing global demand to digest cellulose and/or hemicelluloses from any source, including all biological sources, such as plant biomass, wood or wood processing by-products, in the manufacture of textiles and in household and industrial cleaning products, and/or in the processing of biomass residues to produce fermentable sugar.

[20] Another aspect of the invention is directed to the use of converting cellulosic and/or hemicellulosic substrates into fermentable sugars and successive production of fuel alcohol.

[21] Any or all of these utilities are capable of being developed into a kit for commercialization as research products or as supplies for industrial uses. Kits may comprise polynucleotides and/or polypeptides corresponding to one or more M. phaseolina genes of the invention, antibodies, and/or other reagents.

[22] A person skilled in the art will readily appreciate that the present invention is well adapted to realize the objects and obtain the purposes and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended to be limitations on the scope of the invention.

[23] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

[24] The accompanying figures, which are incorporated into and constitute a part of this specification, illustrate embodiments of the present invention, and, together with the description, serve to explain the principles of the invention.

[25] Figure 1 Shows the molecular structure of cellulose.

[26] Figure 2 Shows the conversion of cellulose to glucose using enzymatic digestion.

[27] Figure 3 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 1 and the M line is the DNA molecular weight ladder.

[28] Figure 4 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 4 and line M is the DNA molecular weight ladder.

[29] Figure 5 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 7 and line M is the DNA molecular weight ladder.

[30] Figure 6 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 10 and line M is the DNA molecular weight ladder.

[31] Figure 7 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 13 and line M is the DNA molecular weight ladder.

[32] Figure 8 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 16 and line M is the DNA molecular weight ladder.

[33] Figure 9 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 19 and line M is the DNA molecular weight ladder.

[34] Figure 10 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 22 and line M is the DNA molecular weight ladder.

[35] Figure 11 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 25 and line M is the DNA molecular weight ladder.

[36] Figure 12 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 28 and line M is the DNA molecular weight ladder.

[37] Figure 13 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 31 and line M is the DNA molecular weight ladder.

[38] Figure 14 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 34 and line M is the DNA molecular weight ladder.

[39] Figure 15 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 37 and line M is the DNA molecular weight ladder.

[40] Figure 16 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-1,4-endoglycanase polynucleotides from SEQ ID NO. 46 and the M line is the DNA molecular weight ladder.

[41] Figure 17 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is cellobiohydrolase polynucleotides of SEQ ID NO. 49 and the M line is the DNA molecular weight ladder.

[42] Figure 18 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is cellobiohydrolase polynucleotides of SEQ ID NO. 52 and line M is the DNA molecular weight ladder.

[43] Figure 19 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is cellobiohydrolase polynucleotides of SEQ ID NO. 55 and the M line is the DNA molecular weight ladder.

[44] Figure 20 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glycosidase polynucleotides from SEQ ID NO. 58 and the M line is the DNA molecular weight ladder.

[45] Figure 21 is the agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides from SEQ ID NO. 61 and the M line is the DNA molecular weight ladder.

[46] Figure 22 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glycosidase polynucleotides from SEQ ID NO. 64 and the M line is the DNA molecular weight ladder.

[47] Figure 23 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glycosidase polynucleotides from SEQ ID NO. 67 and the M line is the DNA molecular weight ladder.

[48] Figure 24 is agarose gel image of electrophoresis showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides of SEQ ID NO. 70 and the M line is the DNA molecular weight ladder.

[49] Figure 25 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides from SEQ ID NO. 73 and the M line is the DNA molecular weight ladder.

[50] Figure 26 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glycosidase polynucleotides from SEQ ID NO. 76 and the M line is the DNA molecular weight ladder.

[51] Figure 27 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides from SEQ ID NO. 79 and the M line is the DNA molecular weight ladder.

[52] Figure 28 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides from SEQ ID NO. 82 and the M line is the DNA molecular weight ladder.

[53] Figure 29 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides from SEQ ID NO. 85 and the M line is the DNA molecular weight ladder.

[54] Figure 30 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides from SEQ ID NO. 91 and the M line is the DNA molecular weight ladder.

[55] Figure 31 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glycosidase polynucleotides from SEQ ID NO. 94 and the M line is the DNA molecular weight ladder.

[56] Figure 32 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides from SEQ ID NO. 97 and the M line is the DNA molecular weight ladder.

[57] Figure 33 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glycosidase polynucleotides from SEQ ID NO. 100 and the M line is the DNA molecular weight ladder.

[58] Figure 34 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glycosidase polynucleotides from SEQ ID NO. 103 and the M line is the DNA molecular weight ladder.

[59] Figure 35 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides from SEQ ID NO. 106 and the M line is the DNA molecular weight ladder.

[60] Figure 36 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glycosidase polynucleotides from SEQ ID NO. 109 and the M line is the DNA molecular weight ladder.

[61] Figure 37 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glycosidase polynucleotides from SEQ ID NO. 112 and the M line is the DNA molecular weight ladder.

[62] Figure 38 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-glucosidase polynucleotides of SEQ ID NO. 115 and the M line is the DNA molecular weight ladder.

[63] Figure 39 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-glycosidase polynucleotides from SEQ ID NO. 118 and the M line is the DNA molecular weight ladder.

[64] Figure 40 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-glycosidase polynucleotides from SEQ ID NO. 121 and the M line is the DNA molecular weight ladder.

[65] Figure 41 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-glycosidase polynucleotides from SEQ ID NO. 124 and the M line is the DNA molecular weight ladder.

[66] Figure 42 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-glycosidase polynucleotides from SEQ ID NO. 127 and the M line is the DNA molecular weight ladder.

[67] Figure 43 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-glycosidase polynucleotides from SEQ ID NO. 133 and the M line is the DNA molecular weight ladder.

[68] Figure 44 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-glycosidase polynucleotides from SEQ ID NO. 136 and the M line is the DNA molecular weight ladder.

[69] Figure 45 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is exo-1,3-β-glycanase polynucleotides from SEQ ID NO. 139 and the M line is the DNA molecular weight ladder.

[70] Figure 46 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is exo-1,3-β-glycanase polynucleotides from SEQ ID NO. 142 and the M line is the DNA molecular weight ladder.

[71] Figure 47 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is exo-1,3-β-glycanase polynucleotides from SEQ ID NO. 145 and the M line is the DNA molecular weight ladder.

[72] Figure 48 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is exo-1,3-β-glycanase polynucleotides from SEQ ID NO. 148 and the M line is the DNA molecular weight ladder.

[73] Figure 49 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is exo-1,3-β-glycanase polynucleotides from SEQ ID NO. 151 and the M line is the DNA molecular weight ladder.

[74] Figure 50 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is exo-1,3-β-glycanase polynucleotides from SEQ ID NO. 154 and the M line is the DNA molecular weight ladder.

[75] Figure 51 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is exo-1,3-β-glycanase polynucleotides from SEQ ID NO. 157 and the M line is the DNA molecular weight ladder.

[76] Figure 52 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is exo-1,3-β-glycanase polynucleotides from SEQ ID NO. 160 and the M line is the DNA molecular weight ladder.

[77] Figure 53 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is exo-1,3-β-glycanase polynucleotides from SEQ ID NO. 163 and the M line is the DNA molecular weight ladder.

[78] Figure 54 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-glycan lyase polynucleotides from SEQ ID NO. 166 and the M line is the DNA molecular weight ladder.

[79] Figure 55 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-xylosidase polynucleotides from SEQ ID NO. 175 and the M line is the DNA molecular weight ladder.

[80] Figure 56 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is d-4,5-unsaturated b-glucuronyl hydrolase polynucleotides of SEQ ID NO. 178 and the M line is the DNA molecular weight ladder.

[81] Figure 57 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is b-glucuronyl hydrolase d-4,5-unsaturated polynucleotides of SEQ ID NO. 181 and the M line is the DNA molecular weight ladder.

[82] Figure 58 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is b-glucuronyl hydrolase d-4,5-unsaturated polynucleotides from SEQ ID NO. 184 and the M line is the DNA molecular weight ladder.

[83] Figure 59 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is b-glucuronyl hydrolase d-4,5-unsaturated polynucleotides of SEQ ID NO. 187 and the M line is the DNA molecular weight ladder.

[84] Figure 60 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is b-glucuronyl hydrolase d-4,5-unsaturated polynucleotides of SEQ ID NO. 190 and the M line is the DNA molecular weight ladder.

[85] Figure 61 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is 1,4-α-glycosidase glycan polynucleotides from SEQ ID NO. 193 and the M line is the DNA molecular weight ladder.

[86] Figure 62 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is glycan 1,4-α-glucosidase polynucleotides from SEQ ID NO. 196 and the M line is the DNA molecular weight ladder.

[87] Figure 63 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,2-mannosidase polynucleotides from SEQ ID NO. 202 and the M line is the DNA molecular weight ladder.

[88] Figure 64 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,2-mannosidase polynucleotides from SEQ ID NO. 205 and the M line is the DNA molecular weight ladder.

[89] Figure 65 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,2-mannosidase polynucleotides from SEQ ID NO. 208 and the M line is the DNA molecular weight ladder.

[90] Figure 66 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,2-mannosidase polynucleotides from SEQ ID NO. 211 and the M line is the DNA molecular weight ladder.

[91] Figure 67 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,2-mannosidase polynucleotides from SEQ ID NO. 214 and the M line is the DNA molecular weight ladder.

[92] Figure 68 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 220 and the M line is the DNA molecular weight ladder.

[93] Figure 69 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 223 and the M line is the DNA molecular weight ladder.

[94] Figure 70 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 226 and the M line is the DNA molecular weight ladder.

[95] Figure 71 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 229 and the M line is the DNA molecular weight ladder.

[96] Figure 72 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 232 and the M line is the DNA molecular weight ladder.

[97] Figure 73 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 235 and the M line is the DNA molecular weight ladder.

[98] Figure 74 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 238 and the M line is the DNA molecular weight ladder.

[99] Figure 75 is an agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 241 and the M line is the DNA molecular weight ladder.

[100] Figure 76 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 247 and the M line is the DNA molecular weight ladder.

[101] Figure 77 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is α-1,3-glycanase polynucleotides from SEQ ID NO. 250 and the M line is the DNA molecular weight ladder.

[102] Figure 78 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is α-fucosidase polynucleotides of SEQ ID NO. 253 and the M line is the DNA molecular weight ladder.

[103] Figure 79 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is α-fucosidase polynucleotides from SEQ ID NO. 256 and the M line is the DNA molecular weight ladder.

[104] Figure 80 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is xylan β-1,4-xylosidase polynucleotides from SEQ ID NO. 259 and the M line is the DNA molecular weight ladder.

[105] Figure 81 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is xylan β-1,4-xylosidase polynucleotides from SEQ ID NO. 265 and the M line is the DNA molecular weight ladder.

[106] Figure 82 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is xylan β-1,4-xylosidase polynucleotides from SEQ ID NO. 268 and the M line is the DNA molecular weight ladder.

[107] Figure 83 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is xylan β-1,4-xylosidase polynucleotides from SEQ ID NO. 271 and the M line is the DNA molecular weight ladder.

[108] Figure 84 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is xylan β-1,4-xylosidase polynucleotides from SEQ ID NO. 274 and the M line is the DNA molecular weight ladder.

[109] Figure 85 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is xylan β-1,4-xylosidase polynucleotides from SEQ ID NO. 277 and the M line is the DNA molecular weight ladder.

[110] Figure 86 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is xylan β-1,4-xylosidase polynucleotides from SEQ ID NO. 280 and the M line is the DNA molecular weight ladder.

[111] Figure 87 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is xylan β-1,4-xylosidase polynucleotides from SEQ ID NO. 283 and the M line is the DNA molecular weight ladder.

[112] Figure 88 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is endo-1,5-α-arabinosidase polynucleotides from SEQ ID NO. 292 and the M line is the DNA molecular weight ladder.

[113] Figure 89 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is endo-1,5-α-arabinosidase polynucleotides from SEQ ID NO. 295 and the M line is the DNA molecular weight ladder.

[114] Figure 90 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is endo-1,5-α-arabinosidase polynucleotides from SEQ ID NO. 298 and the M line is the DNA molecular weight ladder.

[115] Figure 91 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is endo-1,4-β-xylanase polynucleotides from SEQ ID NO. 304 and the M line is the DNA molecular weight ladder.

[116] Figure 92 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is endo-1,4-β-xylanase polynucleotides from SEQ ID NO. 307 and the M line is the DNA molecular weight ladder.

[117] Figure 93 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is endo-1,4-β-xylanase polynucleotides from SEQ ID NO. 310 and the M line is the DNA molecular weight ladder.

[118] Figure 94 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is endo-1,4-β-xylanase polynucleotides from SEQ ID NO. 313 and the M line is the DNA molecular weight ladder.

[119] Figure 95 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-arabinofuranosidase polynucleotides from SEQ ID NO. 316 and the M line is the DNA molecular weight ladder.

[120] Figure 96 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-arabinofuranosidase polynucleotides from SEQ ID NO. 319 and the M line is the DNA molecular weight ladder.

[121] Figure 97 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-arabinofuranosidase polynucleotides from SEQ ID NO. 322 and the M line is the DNA molecular weight ladder.

[122] Figure 98 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-arabinofuranosidase polynucleotides from SEQ ID NO. 325 and the M line is the DNA molecular weight ladder.

[123] Figure 99 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-arabinofuranosidase polynucleotides from SEQ ID NO. 328 and the M line is the DNA molecular weight ladder.

[124] Figure 100 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is α-arabinofuranosidase polynucleotides from SEQ ID NO. 331 and the M line is the DNA molecular weight ladder.

[125] Figure 101 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is β-galactosidase polynucleotides of SEQ ID NO. 337 and the M line is the DNA molecular weight ladder.

[126] Figure 102 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is β-galactosidase polynucleotides of SEQ ID NO. 340 and the M line is the DNA molecular weight ladder.

[127] Figure 103 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is β-galactosidase polynucleotides of SEQ ID NO. 343 and the M line is the DNA molecular weight ladder.

[128] Figure 104 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is β-galactosidase polynucleotides of SEQ ID NO. 346 and the M line is the DNA molecular weight ladder.

[129] Figure 105 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is endo-1,4-β-galactanase polynucleotides from SEQ ID NO. 352 and the M line is the DNA molecular weight ladder.

[130] Figure 106 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is endo-1,4-β-galactanase polynucleotides from SEQ ID NO. 355 and the M line is the DNA molecular weight ladder.

[131] Figure 107 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is endo-1,4-β-galactanase polynucleotides from SEQ ID NO. 358 and the M line is the DNA molecular weight ladder.

[132] Figure 108 is agarose gel electrophoresis image showing result of PCR amplification in which lane 1 is endo-1,6-β-galactanase polynucleotides from SEQ ID NO. 361 and the M line is the DNA molecular weight ladder.

[133] Figure 109 is agarose gel electrophoresis image showing the result of PCR amplification in which lane 1 is endo-1,4-β-mannanase polynucleotides from SEQ ID NO. 364 and the M line is the DNA molecular weight ladder.

DETAILED DESCRIPTION OF THE INVENTION

[134] The definitions and/or methods provided herein define the present invention and guide those skilled in the art in practicing the present invention. Unless otherwise indicated, the terms are to be understood in accordance with conventional usage by those skilled in the relevant art. To the extent that any of the definitions and/or methods are found to be inconsistent with any of the definitions and/or methods provided in any patent or non-patent incorporated herein or in any reference found elsewhere, it is understood that said definition and/or method that was expressly provided/adopted in this order will be used here. The singular terms “a”, “an,” “the” and “the” include plural references unless the context clearly indicates otherwise. Likewise, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Therefore, "comprising A or B" means including A, or B, or A and B. It should be further understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given by nucleic acids or polypeptides are approximate, and are provided for description. While methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, illustrative materials and methods are now described.

[135] The present invention provides the nucleotide sequences of the M. phaseolina genes involved in the degradation of cellulose and/or hemicelluloses. Genes encode proteins with cellulolytic activity that are in use in an industry or of interest to an industry. Described here below are the genes that encode cellulolytic enzymes of invention, their identifications, characterizations, modifications and methods of use in various industrial processes.

[136]Nucleotide sequences of M. phaseolina genomic DNA were obtained by a whole-genome random shotgun DNA sequencing effort. Genomic DNA was prepared from a strain isolate of M. phaseolina ms6 that was isolated from the infected jute plant (Corchorus spp.). The generated nucleotide sequences were assembled to form contiguous sequences and structures by the Newbler assembler. Nucleotide sequences were initially annotated by software programs such as Augustus, Glimmer M (The Institute of Genome Research, Rockville, Md.) and Evidence Modeler (EVM), which can identify putative coding regions, introns, and splice junctions. . In addition, automated and manual curation of nucleotide sequences was performed to refine and establish accurate characterization of coding regions and other gene features.

[137] The genomic sequences of the invention that encode cellulose and/or hemicellulose degradation enzymes are identified primarily by comparing nucleotide sequences of genomic DNA from M. phaseolina and the nucleotide sequences of known enzyme genes from other micro- organisms. Prior to this invention, the nucleotide sequences of these M. phaseolina genes (involved in the degradation of cellulose and/or hemicelluloses), the reading frames, the positions of exons and introns, the structure of the enzymes, and their potential utility in various industries , including those involved in food and feed preparation, beverages, textiles, bioethanol and detergents, were not known.

[138]Over 14,000 M. phaseolina cDNAs have been partially or fully sequenced. Among these, one hundred thirty-four cDNAs encoding novel enzymes with putative roles in cellulose and/or hemicellulose degradation were discovered.

[139] Open reading frames (ORFs) are analyzed following complete or partial sequencing of cDNA library clones derived from M. phaseolina mRNA and are further analyzed using sequence analysis software and determining homology to known sequences in libraries data (public/private).

[140]In the context of the disclosure, a number of terms used throughout the specification have the indicated meaning unless expressly stated to have a different meaning.

[141] The term "cellulolytic activity", as used herein, is defined as a biological activity that hydrolyzes a cellulosic material. For purposes of the present invention, cellulolytic activity is determined by measuring the increase in hydrolysis of cellulosic material by a cellulolytic mixture under appropriate/effective conditions, e.g., an appropriate/effective amount (e.g., 1-10 mg) of protein cellulolytic properties/g cellulose on pre-treated corn stover (PCS) for an appropriate/effective number of days (such as 5-7 days) at an appropriate/effective temperature level (eg 50°C), which is then compared with a controlled hydrolysis without addition of cellulolytic protein.

[142] The term “PCS” or “Pretreated Corn Husk”, as used herein, is defined as a cellulosic material derived from corn husks by treatment with heat and dilute acid.

[143] The term "cellulose" is intended to include soluble and insoluble, amorphous and crystalline forms of cellulose.

[144] The term “hemicellulose” is intended to include glycans, mannans, xylans, arabinans, or polyglucuronic or polygalacturonic acid.

[145] The term “cellulosic material” is defined here as any material containing cellulose. Cellulose is generally found, for example, in the stems, leaves, hulls, bark and spikes of plants or leaves, branches and wood of trees. Cellulosic material can also be, but is not limited to, found in herbaceous material, agricultural waste, forestry waste, municipal solid waste, paper waste, and pulp and paper mill waste. It is understood here that the cellulose may be in the form of lignocellulose, which is a plant cell wall material containing lignin, cellulose and hemicellulose in a mixed condition.

[146] The term “gene”, as used herein (unless otherwise stated/inferred), is generally defined as the genomic sequences of the fungus M. phaseolina (or any of its strains), particularly the polynucleotide sequence encoding polypeptide from a series of enzymes involved in the degradation of cellulose and/or hemicellulose. The term may further include nucleic acid molecules comprising upstream, downstream, and/or intron nucleotide sequences.

[147] The term "open reading frame (ORF)" means a series of nucleotide triplets that encode amino acids without any terminating codons, and the triplet sequence is translatable into protein using the appropriate codon usage information for a particular organism.

[148] A "coding sequence" or "coding region" refers to a nucleic acid molecule having sequence information necessary to produce a gene product, such as an amino acid or polypeptide, when the sequence is expressed. The coding sequence may comprise untranslated sequences (including introns or 5' and 3' untranslated regions) within translated regions, or may lack such intermediate untranslated sequences (e.g., as in cDNA).

[149] The term "cDNA" is defined here as a DNA molecule that can be prepared by reverse transcription of a mature, processed mRNA molecule obtained from a eukaryotic cell. cDNA is devoid of intron sequences that are normally present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor mRNA that is processed through a series of steps before appearing as processed mature mRNA. These steps include the removal of intron sequences by a process called processing.

[150] As used herein, a "polynucleotide" is a sequence of nucleotides as a fragment of nucleic acid. A polynucleotide can be a single-stranded or double-stranded RNA or DNA polymer, which optionally contains synthetic, unnatural, or altered nucleotide bases. A polynucleotide in the form of a DNA polymer can be composed of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures and/or combinations thereof. An isolated polynucleotide of the present invention can be derived from, but not limited to, SEQ ID NOs. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 2 23, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 2 98, 301, 304, 307, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364 or any complement thereof.

[151] “Isolated” means altered “by the hand of man” from the natural state. If a substance or composition occurs in nature, it would be considered “isolated” if it were altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a plant or animal is not "isolated", but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is used herein.

[152] The term "recombinant", when used herein to refer to a polypeptide or protein, usually means that a polypeptide or protein is derived from recombinant expression systems (eg, microbial or mammalian). "Microbial" refers to recombinant polypeptides or proteins prepared in bacterial or fungal expression systems. Polypeptides or proteins expressed in most bacterial systems, eg Escherichia coli, will be free of glycosylation modifications; polypeptides or proteins expressed in fungi will be glycosylated.

[153] A "vector" generally refers to a replicon, such as a plasmid, phage, cosmid, yeast, or virus, or an artificial replication sequence (ARS) or an artificial chromosome for expressing a polypeptide from a nucleotide sequence. The term "vector" is also intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a “plasmid,” which refers to a loop of circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be spliced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors can be integrated into the genome of a host cell by introduction into the host cell and thereby replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retrovirus, adenovirus, and adeno-associated virus), which serve equivalent functions.

[154] The term "expression vector" is defined herein as a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the invention, which is operatively linked to additional nucleotides which provide for its expression.

[155] The term “expression construct” can comprise an assembly of genetic elements having a regulatory role in gene expression, for example, promoters or enhancers, or a coding sequence that is transcribed into RNA, mRNA and translated into protein, and which is operably linked to the appropriate promoter or transcriptional initiation and termination sequences.

[156] The term "operably linked" here denotes a configuration in which a control sequence is placed in an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs expression of the coding sequence of a polypeptide .

[157] The term "host cell" refers to a cell of any organism. Preferred host cells are derived from plants, bacteria, yeast, fungi, insects or other animals. It should be understood that the term "host cell" is intended to refer not only to the cell of the particular subject, but to the descendants of that cell. Due to the fact that certain modifications may occur in succeeding generations due to mutation or environmental influences, these descendants may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. The term "host cell", as used herein, includes any type of cell that is susceptible to transformation, transfection, transduction and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. Methods for introducing polynucleotide sequences into various types of host cells are well known in the art. Host cells or progeny of host cells transformed with the recombinant expression cassettes of the present invention are provided. The host cells can be plant cells. Preferably, the plant cells are jute cells.

[158] The term "nucleic acid construct" as used herein refers to a nucleic acid molecule, single-stranded or double-stranded, that is isolated from a naturally occurring gene or that is modified to contain segments of nucleic acids in a different way. that otherwise would not exist in nature. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the necessary control sequences for expression of a coding sequence of the present invention.

[159] The term "recombinant host cells" means cultured cells that comprise a recombinant transcriptional unit, and will express heterologous polypeptides or proteins and RNA encoded by the DNA segment or synthetic gene in the recombinant transcriptional unit. Cells can be prokaryotic or eukaryotic.

[160] "Polypeptide" as used herein, is a single linear chain of amino acids linked by peptide bonds and having a sequence greater than 100 amino acids in length.

[161] The term "promoter", as used herein, generally refers to a nucleic acid sequence that functions to direct the downstream transcription of a gene. The promoter will generally be appropriate for the host cell in which the target gene is being expressed. The promoter along with other transcriptional and translational regulatory nucleic acid sequences (also called "control sequences") is required to express a given gene. In general, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer or activator sequences.

[162] The term “in vitro” as used herein, refers to a biological reaction that takes place in an artificial environment outside of a living organism, which is typically carried out in a laboratory using components of an organism that has been isolated from its context. biological standard in order to allow a more detailed or convenient analysis to be performed.

[163] The term "% homology" is used interchangeably herein with the term "% identity" herein and typically refers to the level of nucleic acid or amino acid sequence identity between the nucleic acid sequence encoding any one of inventive polypeptides or amino acid sequence of the inventive polypeptide, when aligned using a sequence alignment program.

[164] For example, as used here, 80% homology means the same as 80% sequence identity determined by a defined algorithm, and consequently a homologue of a given sequence has more than 80% sequence identity along the way. of a given string length. Exemplary levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity for a given sequence, e.g., the coding sequence for any of the inventive polypeptides as described here.

[165] Exemplary computer programs that can be used to determine the identity between two sequences include, but are not limited to, the BLAST suite of programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly accessible at www.ncbi.nlm.nih.gov/BLAST.

[166] Sequence searches are typically performed using the BLASTN program when evaluating a given nucleic acid sequence against nucleic acid sequences in GenBank DNA Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in GenBank Protein Sequences and other public databases.

[167] A preferential alignment of selected sequences in order to determine the "% identity" between two or more sequences is performed using, for example, the CLUSTAL-W program.

[168] The term "primer" as used herein, is an oligonucleotide capable of binding to a target nucleic acid sequence and initiating nucleic acid synthesis. An amplification oligonucleotide as defined herein will preferably be from 10 to 50, more preferably from 15 to 25 nucleotides in length. While the amplification oligonucleotides of the present invention can be chemically synthesized, these oligonucleotides are non-naturally occurring nucleic acids.

[169] The abbreviations used throughout the specification to refer to nucleic acids comprising nucleotide sequences are the conventional one-letter abbreviations. Thus, when included in a nucleic acid, naturally occurring coding nucleotides are abbreviated as follows: adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Also, unless otherwise specified, nucleic acid sequences shown here are in the 5' ^ 3' direction.

[170] As used herein, the term “complementary” and derivatives thereof are used in reference to the pairing of nucleic acids by the well-known rules that A pairs with T or U and C pairs with G. Complement can be “partial” or “ complete". In partial complement, only some of the nucleic acid bases are combined according to base pairing rules; while in full or full complement, all bases are matched according to the matching rule. The degree of complement between strands of nucleic acid can have significant effects on the efficiency and strength of hybridization between strands of nucleic acid well known in the art. The efficiency and strength of said hybridization is dependent on the detection method.

[171] The DNA sequences of the invention were generated by sequencing reactions and may contain small errors that may exist as misidentified nucleotides, insertions and deletions. However, these small errors, if present, should not disturb the identification of the sequences as an M. phaseolina gene that encodes an enzyme of industrial interest and are specifically included in the scope of the invention.

[172] Included in the present invention are genomic nucleotide sequences and gene coding sequences that encode M. phaseolina enzymes of industrial interest. Accordingly, in one embodiment, SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362 and 365 are provided, wherein each of which it identifies a nucleotide sequence of the open reading frame (ORF) of an identified gene. In another embodiment, genomic sequences of genes identified by SEQ ID NOs. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 2 23, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 2 98, 301, 304, 307, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364 are provided.

[173] As used herein, "gene" also refers to (i) a gene comprising at least one of the nucleotide sequences and/or fragments thereof that are set forth in SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 2 32, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 3 07, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364; (ii) any nucleotide sequence or fragment thereof encoding the amino acid sequence which is set forth in SEQ ID NOs. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 2 25, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 3 00, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363 and 366; (iii) any nucleotide sequence that hybridizes to the complement of the nucleotide sequences set forth in SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364 under conditions of medium restriction, e.g. Filter-bound DNA in an appropriate/effective amount of 6x sodium chloride/sodium citrate (SSC) at an appropriate/effective temperature level (such as 45°C) followed by one or more washes in an appropriate/effective level of SDS , such as 0.2xSSC/0.1% SDS at an appropriate/effective temperature level (such as 50 to 65°C) or under highly stringent conditions, for example, hybridization to filter-bound nucleic acid in an appropriate amount/ effective 6x SSC at an appropriate/effective temperature level (such as 45°C) followed by one or more washes at an appropriate/effective level of SDS (such as 0.1XSSC/0.2% SDS) at an appropriate level /effective temperature (such as 68°C) or other hybridization conditions that are apparent to those skilled in the art (see, for example, Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current Protocols in Molecular Biology, 1994; Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York). Preferably, the polynucleotides that hybridize to the complements of the DNA sequences disclosed herein encode gene products, for example, those gene products that are functionally equivalent to a gene product encoded by one of the enzyme genes or fragments thereof.

[174] As described above, gene sequences include not only degenerate nucleotide sequences encoding the amino acid sequences SEQ ID NOs. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 2 25, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 3 00, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363 and 366, but also degenerate nucleotide sequences which when translated in organisms other than M. phaseolina, would produce a polypeptide comprising one of the amino acid sequences of SEQ ID NOs. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 2 25, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 3 00, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363 and 366 or a fragment thereof. One skilled in the art would know how to select the appropriate codons or modify the nucleotide sequences of SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 2 32, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 3 07, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364, when using the gene sequences in M. phaseolina or in other organisms. For example, in Candida albicans, the CTG codon encodes a serine residue instead of a leucine residue.

[175] The nucleotide sequences of the invention can be used as genetic markers and/or sequence markers to aid in the development of a genetic, physical or sequence map of the M. phaseolina genome. The nucleotide sequences and corresponding gene products of the invention can also be used to detect the presence of M. phaseolina. Hybridization and antibody-based methods known in the art can be used to determine the presence and concentration of the nucleotide sequences and corresponding gene products of the invention.

[176]Nucleotide sequences can also be used to identify inhibitors of enzymes that may have therapeutic effects, given the fact that enzymes can play a role in invading a host during an infection.

[177] In another embodiment, in addition to the M. phaseolina nucleotide sequences described above, homologs or orthologs of the genes of the invention, as may be present in M. phaseolina and other fungal species, are also included. Particularly preferred are homologues or orthologs in filamentous fungi. These enzyme genes can be identified and isolated by known molecular biological techniques.

[178] The term "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota (Hawksworth DL, Kirk PM, Sutton BC, Pegler DN. Ainsworth and Bisby's Dictionary of Fungi (8th Ed.). 1995; CAB International , Wallingford, United Kingdom. 616 p) and yeast. Representative groups of Ascomycota include, for example, Neurospora, Penicillium, Aspergillus. Representative groups of Basidiomycota include mushrooms, rusts and cereal rusts. Representative groups of Chytridiomycota include Allomyces, Blastocladiella, Coelomomyces. Representative groups of Zygomycota include, for example, Rhizopus and Mucor.

[179] The term “filamentous fungi” includes all filamentous forms of fungi. Filamentous fungi are characterized by a vegetative mycelium composed of chitin, cellulose, glycan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by elongation of hyphae and carbon catabolism is obligate aerobic.

[180] Therefore, the present invention provides fungal nucleotide sequences that are hybridizable to gene polynucleotides. In one embodiment, the present invention includes an isolated nucleic acid comprising and/or consisting of a nucleotide sequence that is at least 50% identical to a nucleotide sequence selected from the group comprising and/or consisting of: SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 2 32, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 3 07, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364.

[181] In another embodiment, the present invention includes an isolated nucleic acid comprising a fungal nucleotide sequence that hybridizes under conditions of medium stringency to a second nucleic acid comprising and/or consisting of a nucleotide sequence selected from the group comprising and/or or consisting of SEQ ID NO. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 14 9, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 2 32, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 3 07, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364.

[182] In yet another embodiment, the present invention includes an isolated nucleic acid comprising a fungal nucleotide sequence encoding an amino acid sequence polypeptide that is at least 50% identical to an amino acid sequence selected from the group comprising and/or consisting of SEQ ID NOs. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 2 25, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 3 00, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363 and 366.

[183] The nucleotide sequences of the invention further include fungal nucleotide sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more nucleotide sequence identity to the nucleotide sequences set forth in SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364.

[184] To isolate homologous genes, the M. phaseolina gene sequence described above can be labeled and used to screen a cDNA library constructed from mRNA obtained from the organism of interest, including, but not limited to, M. phaseoline. Therefore, nucleic acid probes, preferably detectably labeled, comprising any of the nucleotide sequences of SEQ ID NO. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 2 32, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 3 07, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364 are included. Hybridization conditions should be less stringent when the cDNA library was derived from an organism other than the type of organism from which the tagged sequence was derived. cDNA screening can also identify clones derived from alternatively recombinant transcripts in the same species. Alternatively, the labeled probe can be used to screen a genomic library derived from the organism of interest, again using appropriately stringent conditions. Low stringency conditions will be well known to those skilled in the art, and will predictably vary depending on the specific organisms from which the library and tagged sequences are derived. (Details in Sambrook J, Russell DW. Molecular Cloning, A Laboratory Manual, Third edition, 2001, Cold Spring Harbor Press, N.Y.; and Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current Protocols in Molecular Biology, 1994; Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[185] In addition, a homologous gene sequence can be isolated by performing a polymerase chain reaction (PCR) using two combined degenerate oligonucleotide primers designed based on amino acid sequences within the gene of interest. The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from the organism of interest. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of an enzyme homologous gene sequence.

[186] The PCR fragment can then be used to isolate a full-length cDNA clone by a variety of methods known to those skilled in the art. Alternatively, the labeled fragment can be used to screen a genomic library.

[187] In another embodiment of the invention, M. phaseolina gene sequences can be used in the development of modified or new enzymes that have particularly desirable chemical and/or physical characteristics. Because of the apparent kinship in amino acid sequences between M. phaseolina enzymes and other filamentous fungi, the structure of an enzyme from another fungus can be used to predict the structure of the M. phaseolina enzyme, and aid in rational modification of the enzyme. of M. phaseolina for useful and superior properties. The sequences provided by the present invention can also be used as raw materials for the rational modification or design of new enzymes with characteristics that allow the enzymes to perform better in demanding processes.

[188]Gen nucleotide sequences can be altered by random and site-directed mutagenesis techniques or directed molecular evolution techniques, such as, but not limited to, methods described in (Arnold FH. Protein engineering for unusual environments. Curr. Opinion Biotechnol. 1993;4:450-455), oligonucleotide-directed mutagenesis (Reidhaar-Olson JF, Sauer RT. Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science, 1988; 241:5357), chemical mutagenesis (Eckert KA, Drinkwater NR. recA-dependent and recA-independent N-ethyl-N-nitrosourea mutagenesis at a plasmid-encoded herpes simplex virus thymidine kinase gene in Escherichia coli. Mutat Res. 1987;178:1-10), mutagenesis site-directed (Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA, 1985;82:488-492; Oliphant A, Nussbaum AL, Struhl K. Cloning of random-sequence oligodeoxynucleotides. Gene 1986;44 177-183), error-prone PCR (Cadwell RC, Joyce GF. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992;2:28-33), cassette mutagenesis (Stauss Hj, Davies H, Sadovnikova E, Chain B, Horowitz N, Sinclair C. Induction of cytotoxic T lymphocytes with peptides in vitro: identification of candidate T-cell epitopes in human papilloma virus PNAS 1992;89(17):7871-7875 ) DNA scrambling methods are described in Stemmer WP. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. PNAS 1994;91(22):10747-10751 and in US Pat. U.S. Nos. 5,605,793; 6,117,679; and 6,132,970 and the methods described in U.S. Pat. U.S. Nos. 5,939,250, 5,965,408, 6,171,820. Mutations in the nucleotide sequence can be determined by sequencing the gene in the clones.

[189] In one embodiment, the 699 bp long polynucleotide illustrated in SEQ ID NO. 2 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a 232 amino acid polypeptide, as in SEQ ID NO. 3, with a predicted molecular weight of about 24 kD. Bioinformatics analysis of SEQ ID NO. 2 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[190] In another embodiment, the 696 bp long polynucleotide illustrated in SEQ ID NO. 5 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a polypeptide of 231 amino acids, as in SEQ ID NO. 6, with a calculated molecular weight of about 24 kD. Bioinformatics analysis of SEQ ID NO. 5 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[191] In another embodiment, the 786 bp long polynucleotide illustrated in SEQ ID NO. 8 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a polypeptide of 261 amino acids, as in SEQ ID NO. 9, with a calculated molecular weight of about 28 kD. Bioinformatics analysis of SEQ ID NO. 8 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[192] In another embodiment, the 873 bp long polynucleotide illustrated in SEQ ID NO. 11 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a polypeptide of 290 amino acids, as in SEQ ID NO. 12, with a calculated molecular weight of about 32 kD. Bioinformatics analysis of SEQ ID NO. 11 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[193] In another embodiment, the 732 bp long polynucleotide illustrated in SEQ ID NO. 14 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a 243 amino acid polypeptide, as in SEQ ID NO. 15, with a calculated molecular weight of about 25 kD. Bioinformatics analysis of SEQ ID NO. 14 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[194] In another embodiment, the 834 bp long polynucleotide illustrated in SEQ ID NO. 17 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a polypeptide of 277 amino acids, as in SEQ ID NO. 18, with a calculated molecular weight of about 29 kD. Bioinformatics analysis of SEQ ID NO. 17 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[195] In another embodiment, the 1431 bp long polynucleotide illustrated in SEQ ID NO. 20 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a 476 amino acid polypeptide, as in SEQ ID NO. 21, with a calculated molecular weight of about 51 kD. Bioinformatics analysis of SEQ ID NO. 20 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[196] In another embodiment, the 762 bp long polynucleotide illustrated in SEQ ID NO. 23 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a polypeptide of 253 amino acids, as in SEQ ID NO. 24, with a calculated molecular weight of about 27 kD. Bioinformatics analysis of SEQ ID NO. 23 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[197] In another embodiment, the 1113 bp long polynucleotide illustrated in SEQ ID NO. 26 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a polypeptide of 370 amino acids, as in SEQ ID NO. 27, with a calculated molecular weight of about 38 kD. Bioinformatics analysis of SEQ ID NO. 26 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[198] In another embodiment, the 678 bp long polynucleotide illustrated in SEQ ID NO. 29 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a 225 amino acid polypeptide, as in SEQ ID NO. 30, with a calculated molecular weight of about 24 kD. Bioinformatics analysis of SEQ ID NO. 29 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[199] In another embodiment, the 669 bp long polynucleotide illustrated in SEQ ID NO. 32 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a 222 amino acid polypeptide, as in SEQ ID NO. 33, with a calculated molecular weight of about 24 kD. Bioinformatics analysis of SEQ ID NO. 32 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[200] In another embodiment, the 963 bp long polynucleotide illustrated in SEQ ID NO. 35 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a 320 amino acid polypeptide, as in SEQ ID NO. 36, with a calculated molecular weight of about 35 kD. Bioinformatics analysis of SEQ ID NO. 35 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[201] In another embodiment, the 1479 bp long polynucleotide illustrated in SEQ ID NO. 38 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a 492 amino acid polypeptide, as in SEQ ID NO. 39, with a calculated molecular weight of about 58 kD. Bioinformatics analysis of SEQ ID NO. 38 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[202] In another embodiment, the 1743 bp long polynucleotide illustrated in SEQ ID NO. 41 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a polypeptide of 580 amino acids, as in SEQ ID NO. 42, with a calculated molecular weight of about 64 kD. Bioinformatics analysis of SEQ ID NO. 41 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[203] In another embodiment, the 1083 bp long polynucleotide illustrated in SEQ ID NO. 44 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a 360 amino acid polypeptide, as in SEQ ID NO. 45, with a calculated molecular weight of about 39 kD. Bioinformatics analysis of SEQ ID NO. 44 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[204] In another embodiment, the 1395 bp long polynucleotide illustrated in SEQ ID NO. 47 is the full-length cDNA clone encoding the β-1,4-endoglycanase protein having an open reading frame encoding a polypeptide of a 464 amino acid polypeptide, as in SEQ ID NO. 48, with a calculated molecular weight of about 47.68 kD. Bioinformatics analysis of SEQ ID NO. 47 reveals that this sequence produces β-1,4-endoglycanase protein that specifically cleaves the internal bonds of the cellulose chain.

[205] In another embodiment, the 1368 bp long polynucleotide illustrated in SEQ ID NO. 50 is the full-length cDNA clone encoding the cellobiohydrolase protein having an open reading frame encoding a polypeptide of a polypeptide of 455 amino acids, as in SEQ ID NO. 51, with a calculated molecular weight of about 48 kD. Bioinformatics analysis of SEQ ID NO. 50 reveals that this sequence produces cellobiohydrolase protein that attacks cellulose from the reducing or non-reducing ends of the cellulose polymer and produces a glucose dimer, cellobiose.

[206] In another embodiment, the 1392 bp long polynucleotide illustrated in SEQ ID NO. 53 is the full-length cDNA clone encoding the cellobiohydrolase protein having an open reading frame encoding a polypeptide of a polypeptide of 463 amino acids, as in SEQ ID NO. 54, with a calculated molecular weight of about 50 kD. Bioinformatics analysis of SEQ ID NO. 53 reveals that this sequence produces cellobiohydrolase protein that attacks cellulose from the reducing or non-reducing ends of the cellulose polymer and produces a glucose dimer, cellobiose.

[207] In another embodiment, the 528 bp long polynucleotide illustrated in SEQ ID NO. 56 is the full-length cDNA clone encoding the cellobiohydrolase protein having an open reading frame encoding a polypeptide of a polypeptide of 175 amino acids, as in SEQ ID NO. 57, with a calculated molecular weight of about 19 kD. Bioinformatics analysis of SEQ ID NO. 56 reveals that this sequence produces cellobiohydrolase protein that attacks cellulose from the reducing or non-reducing ends of the cellulose polymer and produces a glucose dimer, cellobiose.

[208] In another embodiment, the 2448 bp long polynucleotide illustrated in SEQ ID NO. 59 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a polypeptide of a polypeptide of 815 amino acids, as in SEQ ID NO. 60, with a calculated molecular weight of about 87 kD. Bioinformatics analysis of SEQ ID NO. 59 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[209] In another embodiment, the 2511 bp long polynucleotide illustrated in SEQ ID NO. 62 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a polypeptide of a polypeptide of 836 amino acids, as in SEQ ID NO. 63, with a calculated molecular weight of about 90 kD. Bioinformatics analysis of SEQ ID NO. 62 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[210] In another embodiment, the 2202 bp long polynucleotide illustrated in SEQ ID NO. 65 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a polypeptide of a polypeptide of 733 amino acids, as in SEQ ID NO. 66, with a calculated molecular weight of about 77 kD. Bioinformatics analysis of SEQ ID NO. 65 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[211] In another embodiment, the 2712 bp long polynucleotide illustrated in SEQ ID NO. 68 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 903 amino acid polypeptide, as in SEQ ID NO. 69, with a calculated molecular weight of about 96 kD. Bioinformatics analysis of SEQ ID NO. 68 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[212] In another embodiment, the 3249 bp long polynucleotide illustrated in SEQ ID NO. 71 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 1082 amino acid polypeptide, as in SEQ ID NO. 72, with a calculated molecular weight of about 119 kD. Bioinformatics analysis of SEQ ID NO. 71 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[213] In another embodiment, the 2616 bp long polynucleotide illustrated in SEQ ID NO. 74 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding an 871 amino acid polypeptide, as in SEQ ID NO. 75, with a calculated molecular weight of about 93 kD. Bioinformatics analysis of SEQ ID NO. 74 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[214] In another embodiment, the 2418 bp long polynucleotide illustrated in SEQ ID NO. 77 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding an 805 amino acid polypeptide, as in SEQ ID NO. 78, with a calculated molecular weight of about 84 kD. Bioinformatics analysis of SEQ ID NO. 77 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[215] In another embodiment, the 2346 bp long polynucleotide illustrated in SEQ ID NO. 80 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 781 amino acid polypeptide, as in SEQ ID NO. 81, with a calculated molecular weight of about 84 kD. Bioinformatics analysis of SEQ ID NO. 80 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[216] In another embodiment, the 2499 bp long polynucleotide illustrated in SEQ ID NO. 83 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding an 832 amino acid polypeptide, as in SEQ ID NO. 84, with a calculated molecular weight of about 91 kD. Bioinformatics analysis of SEQ ID NO. 83 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[217] In another embodiment, the 2337 bp long polynucleotide illustrated in SEQ ID NO. 86 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 778 amino acid polypeptide, as in SEQ ID NO. 87, with a calculated molecular weight of about 84 kD. Bioinformatics analysis of SEQ ID NO. 86 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[218] In another embodiment, the 2361 bp long polynucleotide illustrated in SEQ ID NO. 89 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 786 amino acid polypeptide, as in SEQ ID NO. 90, with a calculated molecular weight of about 85 kD. Bioinformatics analysis of SEQ ID NO. 89 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[219] In another embodiment, the 1905 bp long polynucleotide illustrated in SEQ ID NO. 92 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 634 amino acid polypeptide, as in SEQ ID NO. 93, with a calculated molecular weight of about 70 kD. Bioinformatics analysis of SEQ ID NO. 92 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[220] In another embodiment, the 1731 bp long polynucleotide illustrated in SEQ ID NO. 95 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 576 amino acid polypeptide, as in SEQ ID NO. 96, with a calculated molecular weight of about 65 kD. Bioinformatics analysis of SEQ ID NO. 95 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[221] In another embodiment, the 1443 bp long polynucleotide illustrated in SEQ ID NO. 98 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 480 amino acid polypeptide, as in SEQ ID NO. 99, with a calculated molecular weight of about 55 kD. Bioinformatics analysis of SEQ ID NO. 98 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[222] In another embodiment, the 1617 bp long polynucleotide illustrated in SEQ ID NO. 101 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 538 amino acid polypeptide, as in SEQ ID NO. 102, with a calculated molecular weight of about 61 kD. Bioinformatics analysis of SEQ ID NO. 101 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[223] In another embodiment, the 1863 bp long polynucleotide illustrated in SEQ ID NO. 104 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 620 amino acid polypeptide, as in SEQ ID NO. 105, with a calculated molecular weight of about 69 kD. Bioinformatics analysis of SEQ ID NO. 104 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[224] In another embodiment, the 1629 bp long polynucleotide illustrated in SEQ ID NO. 107 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 542 amino acid polypeptide, as in SEQ ID NO. 108, with a calculated molecular weight of about 61 kD. Bioinformatics analysis of SEQ ID NO. 107 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[225] In another embodiment, the 654 bp long polynucleotide illustrated in SEQ ID NO. 110 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 217 amino acid polypeptide, as in SEQ ID NO. 111, with a calculated molecular weight of about 25 kD. Bioinformatics analysis of SEQ ID NO. 110 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[226] In another embodiment, the 2406 bp long polynucleotide illustrated in SEQ ID NO. 113 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding an 801 amino acid polypeptide, as in SEQ ID NO. 114, with a calculated molecular mass of about 77 kD. Bioinformatics analysis of SEQ ID NO. 113 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[227] In another embodiment, the 2214 bp long polynucleotide illustrated in SEQ ID NO. 116 is the full-length cDNA clone encoding the β-glycosidase protein having an open reading frame encoding a 737 amino acid polypeptide, as in SEQ ID NO. 117, with a calculated molecular mass of about 78 kD. Bioinformatics analysis of SEQ ID NO. 116 reveals that this sequence produces β-glucosidase protein that hydrolyses cellobiose and, in some cases, cellooligosaccharides into glucose.

[228] In another embodiment, the 2664 bp long polynucleotide illustrated in SEQ ID NO. 119 is the full-length cDNA clone encoding the α-glycosidase protein having an open reading frame encoding an 887 amino acid polypeptide, as in SEQ ID NO. 120 with a calculated molecular weight of about 97 kD. Bioinformatics analysis of SEQ ID NO. 119 reveals that this sequence produces α-glucosidase protein that hydrolyzes terminal non-reducing α-D-glucose residues (1->4)-linked to release α-D-glucose.

[229] In another embodiment, the 2172 bp long polynucleotide illustrated in SEQ ID NO. 122 is the full-length cDNA clone encoding the α-glycosidase protein having an open reading frame encoding a 723 amino acid polypeptide, as in SEQ ID NO. 123 with a calculated molecular mass of about 80 kD. Bioinformatics analysis of SEQ ID NO. 122 reveals that this sequence produces α-glucosidase protein that hydrolyzes terminal non-reducing α-D-glucose residues (1->4)-linked to release α-D-glucose.

[230] In another embodiment, the 2226 bp long polynucleotide illustrated in SEQ ID NO. 125 is the full-length cDNA clone encoding the α-glycosidase protein having an open reading frame encoding a 741 amino acid polypeptide, as in SEQ ID NO. 126 with a calculated molecular mass of about 83 kD. Bioinformatics analysis of SEQ ID NO. 125 reveals that this sequence produces α-glucosidase protein that hydrolyzes terminal non-reducing α-D-glucose residues (1->4)-linked to release α-D-glucose.

[231] In another embodiment, the 2967 bp long polynucleotide illustrated in SEQ ID NO. 128 is the full-length cDNA clone encoding the α-glycosidase protein having an open reading frame encoding a 988 amino acid polypeptide, as in SEQ ID NO. 129 with a calculated molecular mass of about 112 kD. Bioinformatics analysis of SEQ ID NO. 128 reveals that this sequence produces α-glucosidase protein that hydrolyzes terminal non-reducing α-D-glucose residues (1->4)-linked to release α-D-glucose.

[232] In another embodiment, the 3024 bp long polynucleotide illustrated in SEQ ID NO. 131 is the full-length cDNA clone encoding the α-glycosidase protein having an open reading frame encoding a 1007 amino acid polypeptide, as in SEQ ID NO. 132 with a calculated molecular mass of about 110 kD. Bioinformatics analysis of SEQ ID NO. 131 reveals that this sequence produces α-glucosidase protein that hydrolyzes terminal non-reducing α-D-glucose residues (1->4)-linked to release α-D-glucose.

[233] In another embodiment, the 2544 bp long polynucleotide illustrated in SEQ ID NO. 134 is the full-length cDNA clone encoding the α-glycosidase protein having an open reading frame encoding an 847 amino acid polypeptide, as in SEQ ID NO. 135 with a calculated molecular mass of about 94 kD. Bioinformatics analysis of SEQ ID NO. 134 reveals that this sequence produces α-glucosidase protein that hydrolyzes terminal non-reducing α-D-glucose residues (1->4)-linked to release α-D-glucose.

[234] In another embodiment, the 1359 bp long polynucleotide illustrated in SEQ ID NO. 137 is the full-length cDNA clone encoding the α-glycosidase protein having an open reading frame encoding a 452 amino acid polypeptide, as in SEQ ID NO. 138 with a calculated molecular weight of about 50 kD. Bioinformatics analysis of SEQ ID NO. 137 reveals that this sequence produces α-glucosidase protein that hydrolyzes terminal non-reducing α-D-glucose residues (1->4)-linked to release α-D-glucose.

[235] In another embodiment, the 1734 bp long polynucleotide illustrated in SEQ ID NO. 140 is the full-length cDNA clone encoding the exo-1,3-β-glycanase protein having an open reading frame encoding a 577 amino acid polypeptide, as in SEQ ID NO. 141 with a calculated molecular mass of about 63 kD. Bioinformatics analysis of SEQ ID NO. 140 reveals this sequence produces exo-1,3-β-glucanase protein that successively hydrolyzes the β-D-glucose units of the non-reducing ends of (1->3)-β-D-glycans, releasing α-glucose.

[236] In another embodiment, the 1341 bp long polynucleotide illustrated in SEQ ID NO. 143 is the full-length cDNA clone encoding the exo-1,3-β-glycanase protein having an open reading frame encoding a 446 amino acid polypeptide, as in SEQ ID NO. 144 with a calculated molecular mass of about 49 kD. Bioinformatics analysis of SEQ ID NO. 143 reveals this sequence produces exo-1,3-β-glucanase protein that successively hydrolyzes the β-D-glucose units of the non-reducing ends of (1->3)-β-D-glycans, releasing α-glucose.

[237] In another embodiment, the 1251 bp long polynucleotide illustrated in SEQ ID NO. 146 is the full-length cDNA clone encoding the exo-1,3-β-glycanase protein having an open reading frame encoding a 416 amino acid polypeptide, as in SEQ ID NO. 147 with a calculated molecular mass of about 46 kD. Bioinformatics analysis of SEQ ID NO. 146 reveals this sequence produces exo-1,3-β-glucanase protein that successively hydrolyzes the β-D-glucose units of the non-reducing ends of (1->3)-β-D-glycans, releasing α-glucose.

[238] In another embodiment, the 885 bp long polynucleotide illustrated in SEQ ID NO. 149 is the full-length cDNA clone encoding the glycan-1,3-β-glycanase protein having an open reading frame encoding a 294 amino acid polypeptide, as in SEQ ID NO. 150 with a calculated molecular weight of about 32 kD. Bioinformatics analysis of SEQ ID NO. 149 reveals this sequence produces glycan-1,3-β-glucanase protein that successively hydrolyzes the β-D-glucose units of the non-reducing ends of (1->3)-β-D-glycans, releasing α-glucose.

[239] In another embodiment, the 921 bp long polynucleotide illustrated in SEQ ID NO. 152 is the full-length cDNA clone encoding the glycan-1,3-β-glycanase protein having an open reading frame encoding a 306 amino acid polypeptide, as in SEQ ID NO. 153 with a calculated molecular mass of about 33 kD. Bioinformatics analysis of SEQ ID NO. 152 reveals this sequence produces glycan-1,3-β-glucanase protein that successively hydrolyzes the β-D-glucose units of the non-reducing ends of (1->3)-β-D-glycans, releasing α-glucose.

[240] In another embodiment, the 1242 bp long polynucleotide illustrated in SEQ ID NO. 155 is the full-length cDNA clone encoding the exo-1,3-β-eta-glycanase protein having an open reading frame encoding a 413 amino acid polypeptide, as in SEQ ID NO. 156 with a calculated molecular weight of about 46 kD. Bioinformatics analysis of SEQ ID NO. 155 reveals this sequence produces exo-1,3-β-glucanase protein that successively hydrolyzes the β-D-glucose units of the non-reducing ends of (1->3)-β-D-glycans, releasing α-glucose.

[241] In another embodiment, the 2757 bp long polynucleotide illustrated in SEQ ID NO. 158 is the full-length cDNA clone encoding the exo-1,3-β-glycanase protein having an open reading frame encoding a 918 amino acid polypeptide, as in SEQ ID NO. 159 with a calculated molecular mass of about 105 kD. Bioinformatics analysis of SEQ ID NO. 158 reveals this sequence produces exo-1,3-β-glucanase protein that successively hydrolyzes the β-D-glucose units of the non-reducing ends of (1->3)-β-D-glycans, releasing α-glucose.

[242] In another embodiment, the 2334 bp long polynucleotide illustrated in SEQ ID NO. 161 is the full-length cDNA clone encoding the exo-1,3-β-glycanase protein having an open reading frame encoding a 777 amino acid polypeptide, as in SEQ ID NO. 162 with a calculated molecular mass of about 86 kD. Bioinformatics analysis of SEQ ID NO. 161 reveals this sequence produces exo-1,3-β-glucanase protein that successively hydrolyzes the β-D-glucose units of the non-reducing ends of (1->3)-β-D-glycans, releasing α-glucose.

[243] In another embodiment, the 2529 bp long polynucleotide illustrated in SEQ ID NO. 164 is the full-length cDNA clone encoding the exo-1,3-β-glycanase protein having an open reading frame encoding an 842 amino acid polypeptide, as in SEQ ID NO. 165 with a calculated molecular mass of about 91 kD. Bioinformatics analysis of SEQ ID NO. 164 reveals this sequence produces exo-1,3-β-glucanase protein that successively hydrolyzes the β-D-glucose units of the non-reducing ends of (1->3)-β-D-glycans, releasing α-glucose.

[244] In another embodiment, the 3363 bp long polynucleotide illustrated in SEQ ID NO. 167 is the full-length cDNA clone encoding the α-1,4-glycan lyase protein having an open reading frame encoding a polypeptide of 1120 amino acids, as in SEQ ID NO. 168 with a calculated molecular mass of about 127 kD. Bioinformatics analysis of SEQ ID NO. 167 reveals that this sequence produces α-1,4-glycan lyase protein that catalyzes the sequential degradation of (1->4)-α-D-glycans from the non-reducing end with the release of 1,5-anhydro-D-fructose and D-glucose.

[245] In another embodiment, the 3345 bp long polynucleotide illustrated in SEQ ID NO. 170 is the full-length cDNA clone encoding the α-1,4-glycan lyase protein having an open reading frame encoding a polypeptide of 1114 amino acids, as in SEQ ID NO. 171 with a calculated molecular mass of about 128 kD. Bioinformatics analysis of SEQ ID NO. 170 reveals that this sequence produces α-1,4-glycan lyase protein that catalyzes the sequential degradation of (1->4)-α-D-glycans from the non-reducing end with the release of 1,5-anhydro-D-fructose and D-glucose.

[246] In another embodiment, the 2025 bp long polynucleotide illustrated in SEQ ID NO. 173 is the full-length cDNA clone encoding the α-xylosidase protein having an open reading frame encoding a 674 amino acid polypeptide, as in SEQ ID NO. 174 with a calculated molecular mass of about 76 kD. Bioinformatics analysis of SEQ ID NO. 173 reveals that this sequence produces α-xylosidase protein that catalyzes the hydrolysis of xyloglycan side chains and removes unsubstituted D-xylose residues attached to the glucose located at the non-reducing end. This enzyme is involved in the degradation of xyloglycan oligosaccharides.

[247] In another embodiment, the 2316 bp long polynucleotide illustrated in SEQ ID NO. 176 is the full-length cDNA clone encoding the α-xylosidase protein having an open reading frame encoding a 771 amino acid polypeptide, as in SEQ ID NO. 177 with a calculated molecular mass of about 86 kD. Bioinformatics analysis of SEQ ID NO. 176 reveals that this sequence produces α-xylosidase protein that catalyzes the hydrolysis of xyloglycan side chains and removes unsubstituted D-xylose residues attached to the glucose located at the non-reducing end. This enzyme is involved in the degradation of xyloglycan oligosaccharides.

[248] In another embodiment, the 1236 bp long polynucleotide illustrated in SEQ ID NO. 179 is the full length cDNA clone encoding the unsaturated d-4,5-β-glucuronyl hydrolase protein having an open reading frame encoding a 411 amino acid polypeptide, as in SEQ ID NO. 180 with a calculated molecular weight of about 45 kD. Bioinformatics analysis of SEQ ID NO. 179 reveals that this sequence produces unsaturated d-4,5-β-glucuronyl hydrolase protein that catalyzes the hydrolytic release of unsaturated glucuronic acids from oligosaccharides produced by polysaccharide lyase reactions.

[249] In another embodiment, the 1143 bp long polynucleotide illustrated in SEQ ID NO. 182 is the full-length cDNA clone encoding the unsaturated d-4,5-β-glucuronyl hydrolase protein having an open reading frame encoding a 380 amino acid polypeptide, as in SEQ ID NO. 183 with a calculated molecular mass of about 44 kD. Bioinformatics analysis of SEQ ID NO. 182 reveals that this sequence produces unsaturated d-4,5-β-glucuronyl hydrolase protein that catalyzes the hydrolytic release of unsaturated glucuronic acids from oligosaccharides produced by polysaccharide lyase reactions.

[250] In another embodiment, the 1248 bp long polynucleotide illustrated in SEQ ID NO. 185 is the full-length cDNA clone encoding the unsaturated d-4,5-β-glucuronyl hydrolase protein having an open reading frame encoding a 415 amino acid polypeptide, as in SEQ ID NO. 186 with a calculated molecular mass of about 47 kD. Bioinformatics analysis of SEQ ID NO. 185 reveals that this sequence produces unsaturated d-4,5-β-glucuronyl hydrolase protein that catalyzes the hydrolytic release of unsaturated glucuronic acids from oligosaccharides produced by polysaccharide lyase reactions.

[251] In another embodiment, the 1158 bp long polynucleotide illustrated in SEQ ID NO. 188 is the full-length cDNA clone encoding the unsaturated d-4,5-β-glucuronyl hydrolase protein having an open reading frame encoding a 385 amino acid polypeptide, as in SEQ ID NO. 189 with a calculated molecular mass of about 43 kD. Bioinformatics analysis of SEQ ID NO. 188 reveals that this sequence produces unsaturated d-4,5-β-glucuronyl hydrolase protein that catalyzes the hydrolytic release of unsaturated glucuronic acids from oligosaccharides produced by polysaccharide lyase reactions.

[252] In another embodiment, the 1113 bp long polynucleotide illustrated in SEQ ID NO. 191 is the full-length cDNA clone encoding the unsaturated d-4,5-β-glucuronyl hydrolase protein having an open reading frame encoding a 370 amino acid polypeptide, as in SEQ ID NO. 192 with a calculated molecular mass of about 39 kD. Bioinformatics analysis of SEQ ID NO. 191 reveals that this sequence produces unsaturated d-4,5-β-glucuronyl hydrolase protein that catalyzes the hydrolytic release of unsaturated glucuronic acids from oligosaccharides produced by polysaccharide lyase reactions.

[253] In another embodiment, the 1683 bp long polynucleotide illustrated in SEQ ID NO. 194 is the full length cDNA clone encoding the 1,4-α-glucosidase glycan protein having an open reading frame encoding a 560 amino acid polypeptide, as in SEQ ID NO. 195 with a calculated molecular mass of about 61 kD. Bioinformatics analysis of SEQ ID NO. 194 reveals that this sequence produces glycan protein 1,4-α-glucosidase that hydrolyses successively terminal α-D-glucose residues (1->4)-linked from the non-reducing ends of the chains with release of β-D-glucose.

[254] In another embodiment, the 1917 bp long polynucleotide illustrated in SEQ ID NO. 197 is the full-length cDNA clone encoding the 1,4-α-glucosidase glycan protein having an open reading frame encoding a 638 amino acid polypeptide, as in SEQ ID NO. 198 with a calculated molecular mass of about 68 kD. Bioinformatics analysis of SEQ ID NO. 197 reveals that this sequence produces glycan protein 1,4-α-glucosidase that hydrolyses successively terminal α-D-glucose residues (1->4)-linked from the non-reducing ends of the chains with release of β-D-glucose.

[255] In another embodiment, the 2670 bp long polynucleotide illustrated in SEQ ID NO. 200 is the full-length cDNA clone encoding the α-1,2-mannosidase protein having an open reading frame encoding an 889 amino acid polypeptide, as in SEQ ID NO. 201 with a calculated molecular mass of about 95 kD. Bioinformatics analysis of SEQ ID NO. 200 reveals that this sequence produces α-1,2-mannosidase protein that removes α-1,2-linked mannose residues from Man(9)(GlcNAc)(2) by hydrolysis.

[256] In another embodiment, the 2361 bp long polynucleotide illustrated in SEQ ID NO. 203 is the full-length cDNA clone encoding the α-1,2-mannosidase protein having an open reading frame encoding a 786 amino acid polypeptide, as in SEQ ID NO. 204 with a calculated molecular weight of about 85 kD. Bioinformatics analysis of SEQ ID NO. 203 reveals that this sequence produces α-1,2-mannosidase protein that removes α-1,2-linked mannose residues from Man(9)(GlcNAc)(2) by hydrolysis.

[257] In another embodiment, the 2382 bp long polynucleotide illustrated in SEQ ID NO. 206 is the full-length cDNA clone encoding the α-1,2-mannosidase protein having an open reading frame encoding a 793 amino acid polypeptide, as in SEQ ID NO. 207 with a calculated molecular mass of about 88 kD. Bioinformatics analysis of SEQ ID NO. 206 reveals that this sequence produces α-1,2-mannosidase protein that removes α-1,2-linked mannose residues from Man(9)(GlcNAc)(2) by hydrolysis.

[258] In another embodiment, the 2415 bp long polynucleotide illustrated in SEQ ID NO. 209 is the full-length cDNA clone encoding the α-1,2-mannosidase protein having an open reading frame encoding an 804 amino acid polypeptide, as in SEQ ID NO. 210 with a calculated molecular mass of about 88 kD. Bioinformatics analysis of SEQ ID NO. 209 reveals that this sequence produces α-1,2-mannosidase protein that removes α-1,2-linked mannose residues from Man(9)(GlcNAc)(2) by hydrolysis.

[259] In another embodiment, the 2289 bp long polynucleotide illustrated in SEQ ID NO. 212 is the full-length cDNA clone encoding the α-1,2-mannosidase protein having an open reading frame encoding a 762 amino acid polypeptide, as in SEQ ID NO. 213 with a calculated molecular mass of about 83 kD. Bioinformatics analysis of SEQ ID NO. 212 reveals that this sequence produces α-1,2-mannosidase protein that removes α-1,2-linked mannose residues from Man(9)(GlcNAc)(2) by hydrolysis.

[260] In another embodiment, the 2484 bp long polynucleotide illustrated in SEQ ID NO. 215 is the full-length cDNA clone encoding the α-1,2-mannosidase protein having an open reading frame encoding an 827 amino acid polypeptide, as in SEQ ID NO. 216 with a calculated molecular mass of about 92 kD. Bioinformatics analysis of SEQ ID NO. 215 reveals that this sequence produces α-1,2-mannosidase protein that removes α-1,2-linked mannose residues from Man(9)(GlcNAc)(2) by hydrolysis.

[261] In another embodiment, the 1485 bp long polynucleotide illustrated in SEQ ID NO. 218 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 494 amino acid polypeptide, as in SEQ ID NO. 219 with a calculated molecular mass of about 52 kD. Bioinformatics analysis of SEQ ID NO. 218 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[262] In another embodiment, the 1533 bp long polynucleotide illustrated in SEQ ID NO. 221 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 510 amino acid polypeptide, as in SEQ ID NO. 222 with a calculated molecular mass of about 56 kD. Bioinformatics analysis of SEQ ID NO. 221 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[263] In another embodiment, the 1344 bp long polynucleotide illustrated in SEQ ID NO. 224 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 447 amino acid polypeptide, as in SEQ ID NO. 225 with a calculated molecular weight of about 49 kD. Bioinformatics analysis of SEQ ID NO. 224 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[264] In another embodiment, the 1365 bp long polynucleotide illustrated in SEQ ID NO. 227 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 454 amino acid polypeptide, as in SEQ ID NO. 228 with a calculated molecular mass of about 49 kD. Bioinformatics analysis of SEQ ID NO. 227 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[265] In another embodiment, the 765 bp long polynucleotide illustrated in SEQ ID NO. 230 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 254 amino acid polypeptide, as in SEQ ID NO. 231 with a calculated molecular mass of about 29 kD. Bioinformatics analysis of SEQ ID NO. 230 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[266] In another embodiment, the 1485 bp long polynucleotide illustrated in SEQ ID NO. 233 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 494 amino acid polypeptide, as in SEQ ID NO. 234 with a calculated molecular weight of about 55 kD. Bioinformatics analysis of SEQ ID NO. 233 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[267] In another embodiment, the 1503 bp long polynucleotide illustrated in SEQ ID NO. 236 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 500 amino acid polypeptide, as in SEQ ID NO. 237 with a calculated molecular mass of about 54 kD. Bioinformatics analysis of SEQ ID NO. 236 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[268] In another embodiment, the 810 bp long polynucleotide illustrated in SEQ ID NO. 239 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 269 amino acid polypeptide, as in SEQ ID NO. 240 with a calculated molecular weight of about 29 kD. Bioinformatics analysis of SEQ ID NO. 239 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[269] In another embodiment, the 783 bp long polynucleotide illustrated in SEQ ID NO. 242 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 260 amino acid polypeptide, as in SEQ ID NO. 243 with a calculated molecular weight of about 29 kD. Bioinformatics analysis of SEQ ID NO. 242 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[270] In another embodiment, the 1251 bp long polynucleotide illustrated in SEQ ID NO. 245 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 416 amino acid polypeptide, as in SEQ ID NO. 246 with a calculated molecular weight of about 45 kD. Bioinformatics analysis of SEQ ID NO. 245 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[271] In another embodiment, the 1350 bp long polynucleotide illustrated in SEQ ID NO. 248 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 449 amino acid polypeptide, as in SEQ ID NO. 249 with a calculated molecular weight of about 50 kD. Bioinformatics analysis of SEQ ID NO. 248 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[272] In another embodiment, the 1485 bp long polynucleotide illustrated in SEQ ID NO. 251 is the full-length cDNA clone encoding the α-1,3-glycanase protein having an open reading frame encoding a 494 amino acid polypeptide, as in SEQ ID NO. 252 with a calculated molecular weight of about 55 kD. Bioinformatics analysis of SEQ ID NO. 251 reveals that this sequence produces α-1,3-glucanase protein that degrades α-1,3-glycans and also has the ability to remove dental plaque. Therefore, this enzyme could be applied as active ingredients in mouthwash, toothpaste, tooth gel or chewing gum to prevent the accumulation of glucose biopolymers and could also be useful in all other forms of preventive oral hygiene.

[273] In another embodiment, the 2349 bp long polynucleotide illustrated in SEQ ID NO. 254 is the full-length cDNA clone encoding the α-L-fucosidase protein having an open reading frame encoding a 782 amino acid polypeptide, as in SEQ ID NO. 255 with a calculated molecular weight of about 86 kD. Bioinformatics analysis of SEQ ID NO. 254 reveals that this sequence produces α-L-fucosidase protein which is responsible for the hydrolysis of α-1,6-linked fucose coupled to the N-acetylglucosamine reducing end of the carbohydrate fractions of glycoproteins. Therefore, this α-L-fucosidase is involved in the degradation of fucosylated xyloglycans.

[274] In another embodiment, the 2208 bp long polynucleotide illustrated in SEQ ID NO. 257 is the full-length cDNA clone encoding the α-L-fucosidase protein having an open reading frame encoding a 735 amino acid polypeptide, as in SEQ ID NO. 258 with a calculated molecular mass of about 81 kD. Bioinformatics analysis of SEQ ID NO. 257 reveals that this sequence produces α-L-fucosidase protein which is responsible for the hydrolysis of α-1,6-linked fucose coupled to the N-acetylglucosamine reducing end of the carbohydrate fractions of glycoproteins. Therefore, this α-L-fucosidase is involved in the degradation of fucosylated xyloglycans.

[275] In another embodiment, the 1491 bp long polynucleotide illustrated in SEQ ID NO. 260 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 496 amino acid polypeptide, as in SEQ ID NO. 261 with a calculated molecular mass of about 56 kD. Bioinformatics analysis of SEQ ID NO. 260 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[276] In another embodiment, the 1548 bp long polynucleotide illustrated in SEQ ID NO. 263 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 515 amino acid polypeptide, as in SEQ ID NO. 264 with a calculated molecular mass of about 56 kD. Bioinformatics analysis of SEQ ID NO. 263 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[277] In another embodiment, the 1560 bp long polynucleotide illustrated in SEQ ID NO. 266 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 519 amino acid polypeptide, as in SEQ ID NO. 267 with a calculated molecular mass of about 56 kD. Bioinformatics analysis of SEQ ID NO. 266 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[278] In another embodiment, the 2403 bp long polynucleotide illustrated in SEQ ID NO. 269 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding an 800 amino acid polypeptide, as in SEQ ID NO. 270 with a calculated molecular mass of about 87 kD. Bioinformatics analysis of SEQ ID NO. 269 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[279] In another embodiment, the 2916 bp long polynucleotide illustrated in SEQ ID NO. 272 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 971 amino acid polypeptide, as in SEQ ID NO. 273 with a calculated molecular mass of about 104 kD. Bioinformatics analysis of SEQ ID NO. 272 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[280] In another embodiment, the 978 bp long polynucleotide illustrated in SEQ ID NO. 275 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 325 amino acid polypeptide, as in SEQ ID NO. 276 with a calculated molecular mass of about 36 kD. Bioinformatics analysis of SEQ ID NO. 275 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[281] In another embodiment, the 1731 bp long polynucleotide illustrated in SEQ ID NO. 278 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 576 amino acid polypeptide, as in SEQ ID NO. 279 with a calculated molecular mass of about 63 kD. Bioinformatics analysis of SEQ ID NO. 278 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[282] In another embodiment, the 1737 bp long polynucleotide illustrated in SEQ ID NO. 281 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 578 amino acid polypeptide, as in SEQ ID NO. 282 with a calculated molecular mass of about 63 kD. Bioinformatics analysis of SEQ ID NO. 281 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[283] In another embodiment, the 1731 bp long polynucleotide illustrated in SEQ ID NO. 284 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 576 amino acid polypeptide, as in SEQ ID NO. 285 with a calculated molecular mass of about 64 kD. Bioinformatics analysis of SEQ ID NO. 284 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[284] In another embodiment, the 1764 bp long polynucleotide illustrated in SEQ ID NO. 287 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 587 amino acid polypeptide, as in SEQ ID NO. 288 with a calculated molecular mass of about 64 kD. Bioinformatics analysis of SEQ ID NO. 287 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[285] In another embodiment, the 1623 bp long polynucleotide illustrated in SEQ ID NO. 290 is the full-length cDNA clone encoding the β-1,4-xylosidase protein having an open reading frame encoding a 540 amino acid polypeptide, as in SEQ ID NO. 291 with a calculated molecular mass of about 60 kD. Bioinformatics analysis of SEQ ID NO. 290 reveals that this sequence produces β-1,4-xylosidase protein that hydrolyses (1->4)-β-D-xylans to successively remove D-xylose residues from non-reducing ends.

[286] In another embodiment, the 960 bp long polynucleotide illustrated in SEQ ID NO. 293 is the full-length cDNA clone encoding the endo-1,5-α-L-arabinosidase protein having an open reading frame encoding a 319 amino acid polypeptide, as in SEQ ID NO. 294 with a calculated molecular weight of about 35 kD. Bioinformatics analysis of SEQ ID NO. 293 discloses that this sequence produces endo-1,5-α-L-arabinosidase protein that catalyzes the hydrolysis of the α-1,5-linked L-arabinofuranoside structure in (1->5)-arabinans.

[287] In another embodiment, the 1056 bp long polynucleotide illustrated in SEQ ID NO. 296 is the full-length cDNA clone encoding the endo-1,5-α-L-arabinosidase protein having an open reading frame encoding a 351 amino acid polypeptide, as in SEQ ID NO. 297 with a calculated molecular mass of about 38 kD. Bioinformatics analysis of SEQ ID NO. 296 discloses that this sequence produces endo-1,5-α-L-arabinosidase protein that catalyzes the hydrolysis of the α-1,5-linked L-arabinofuranoside structure in (1->5)-arabinans.

[288] In another embodiment, the 957 bp long polynucleotide illustrated in SEQ ID NO. 299 is the full-length cDNA clone encoding the endo-1,5-α-L-arabinosidase protein having an open reading frame encoding a 318 amino acid polypeptide, as in SEQ ID NO. 300 with a calculated molecular weight of about 34 kD. Bioinformatics analysis of SEQ ID NO. 299 discloses that this sequence produces endo-1,5-α-L-arabinosidase protein that catalyzes the hydrolysis of the α-1,5-linked L-arabinofuranoside structure in (1->5)-arabinans.

[289] In another embodiment, the 1110 bp long polynucleotide illustrated in SEQ ID NO. 302 is the full-length cDNA clone encoding the endo-1,5-α-L-arabinosidase protein having an open reading frame encoding a 369 amino acid polypeptide, as in SEQ ID NO. 303 with a calculated molecular weight of about 40 kD. Bioinformatics analysis of SEQ ID NO. 302 discloses that this sequence produces endo-1,5-α-L-arabinosidase protein that catalyzes the hydrolysis of the α-1,5-linked L-arabinofuranoside structure in (1->5)-arabinans.

[290] In another embodiment, the 972 bp long polynucleotide illustrated in SEQ ID NO. 305 is the full-length cDNA clone encoding the endo-1,4-β-xylanase protein having an open reading frame encoding a 323 amino acid polypeptide, as in SEQ ID NO. 306 with a calculated molecular weight of about 34 kD. Bioinformatics analysis of SEQ ID NO. 305 reveals that this sequence produces endo-1,4-β-xylanase protein that catalyzes the endo-hydrolysis of (1->4)-β-D-xylosidic bonds in xylans and produces xylose. This enzyme is essential for the depolymerization of hemicelluloses and has a potential industrial application, such as in biobleaching, papermaking and in the food and animal feed industries. Other potential applications include the conversion of xylan in agricultural and food industry waste to xylose, and in the production of raw materials for fuels and chemicals.

[291] In another embodiment, the 987 bp long polynucleotide illustrated in SEQ ID NO. 308 is the full-length cDNA clone encoding the endo-1,4-β-xylanase protein having an open reading frame encoding a 328 amino acid polypeptide, as in SEQ ID NO. 309 with a calculated molecular weight of about 35 kD. Bioinformatics analysis of SEQ ID NO. 308 reveals that this sequence produces endo-1,4-β-xylanase protein that catalyzes the endo-hydrolysis of (1->4)-β-D-xylosidic bonds in xylans and produces xylose. This enzyme is essential for the depolymerization of hemicelluloses and has a potential industrial application, such as in biobleaching, papermaking and in the food and animal feed industries. Other potential applications include the conversion of xylan in agricultural and food industry waste to xylose, and in the production of raw materials for fuels and chemicals.

[292] In another embodiment, the 1353 bp long polynucleotide illustrated in SEQ ID NO. 311 is the full-length cDNA clone encoding the endo-1,4-β-xylanase protein having an open reading frame encoding a 450 amino acid polypeptide, as in SEQ ID NO. 312 with a calculated molecular mass of about 48 kD. Bioinformatics analysis of SEQ ID NO. 311 reveals that this sequence produces endo-1,4-β-xylanase protein that catalyzes the endo-hydrolysis of (1->4)-β-D-xylosidic bonds in xylans and produces xylose. This enzyme is essential for the depolymerization of hemicelluloses and has a potential industrial application, such as in biobleaching, papermaking and in the food and animal feed industries. Other potential applications include the conversion of xylan in agricultural and food industry waste to xylose, and in the production of raw materials for fuels and chemicals.

[293] In another embodiment, the 1518 bp long polynucleotide illustrated in SEQ ID NO. 314 is the full-length cDNA clone encoding the endo-1,4-β-xylanase protein having an open reading frame encoding a 505 amino acid polypeptide, as in SEQ ID NO. 315 with a calculated molecular weight of about 54 kD. Bioinformatics analysis of SEQ ID NO. 314 reveals that this sequence produces endo-1,4-β-xylanase protein that catalyzes the endo-hydrolysis of (1->4)-β-D-xylosidic bonds in xylans and produces xylose. This enzyme is essential for the depolymerization of hemicelluloses and has a potential industrial application, such as in biobleaching, papermaking and in the food and animal feed industries. Other potential applications include the conversion of xylan in agricultural and food industry waste to xylose, and in the production of raw materials for fuels and chemicals.

[294] In another embodiment, the 1149 bp long polynucleotide illustrated in SEQ ID NO. 317 is the full-length cDNA clone encoding the α-arabinofuranosidase protein having an open reading frame encoding a 382 amino acid polypeptide, as in SEQ ID NO. 318 with a calculated molecular weight of about 43 kD. Bioinformatics analysis of SEQ ID NO. 317 discloses that this sequence produces the α-arabinofuranosidase protein that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues to α-L-arabinosides.

[295] In another embodiment, the 1116 bp long polynucleotide illustrated in SEQ ID NO. 320 is the full length cDNA clone encoding the α-arabinofuranosidase protein having an open reading frame encoding a 371 amino acid polypeptide, as in SEQ ID NO. 321 with a calculated molecular weight of about 41 kD. Bioinformatics analysis of SEQ ID NO. 320 reveals that this sequence produces the α-arabinofuranosidase protein that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues into α-L-arabinosides.

[296] In another embodiment, the 990 bp long polynucleotide illustrated in SEQ ID NO. 323 is the full-length cDNA clone encoding the α-arabinofuranosidase protein having an open reading frame encoding a 329 amino acid polypeptide, as in SEQ ID NO. 324 with a calculated molecular weight of about 36 kD. Bioinformatics analysis of SEQ ID NO. 323 discloses that this sequence produces the α-arabinofuranosidase protein that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues into α-L-arabinosides.

[297] In another embodiment, the 1002 bp long polynucleotide illustrated in SEQ ID NO. 326 is the full length cDNA clone encoding the α-arabinofuranosidase protein having an open reading frame encoding a 333 amino acid polypeptide, as in SEQ ID NO. 327 with a calculated molecular weight of about 36 kD. Bioinformatics analysis of SEQ ID NO. 326 discloses that this sequence produces the α-arabinofuranosidase protein that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues into α-L-arabinosides.

[298] In another embodiment, the 1527 bp long polynucleotide illustrated in SEQ ID NO. 329 is the full-length cDNA clone encoding the α-arabinofuranosidase protein having an open reading frame encoding a 508 amino acid polypeptide, as in SEQ ID NO. 330 with a calculated molecular weight of about 57 kD. Bioinformatics analysis of SEQ ID NO. 329 discloses that this sequence produces the protein α-arabinofuranosidase which catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues into α-L-arabinosides.

[299] In another embodiment, the 2145 bp long polynucleotide illustrated in SEQ ID NO. 332 is the full-length cDNA clone encoding the α-arabinofuranosidase protein having an open reading frame encoding a 714 amino acid polypeptide, as in SEQ ID NO. 333 with a calculated molecular weight of about 80 kD. Bioinformatics analysis of SEQ ID NO. 332 discloses that this sequence produces the α-arabinofuranosidase protein that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues to α-L-arabinosides.

[300] In another embodiment, the 2994 bp length of polynucleotide illustrated in SEQ ID NO. 335 is the full-length cDNA clone encoding the β-galactosidase protein having an open reading frame encoding a 997 amino acid polypeptide, as in SEQ ID NO. 336 with a calculated molecular weight of about 108 kD. Bioinformatics analysis of SEQ ID NO. 335 reveals that this sequence produces β-galactosidase protein that catalyzes the hydrolysis of β-D-galactosides and α-L-arabinosides. β-galactosidase has the catalytic property of hydrolyzing lactose into glucose and galactose. Then, this enzyme is used to prepare milk and fermented milk products. It is used to prevent crystallization of lactose, to improve sweetness, to increase solubility of dairy product in dairy industries. Furthermore, it is used to produce low-lactose-containing food products for people with low lactose tolerance. Therefore, the use of β-galactosidase is one of the most promising enzyme applications for the food industry.

[301] In another embodiment, the 1932 bp long polynucleotide illustrated in SEQ ID NO. 338 is the full-length cDNA clone encoding the β-galactosidase protein having an open reading frame encoding a 643 amino acid polypeptide, as in SEQ ID NO. 339 with a calculated molecular mass of about 71 kD. Bioinformatics analysis of SEQ ID NO. 338 reveals that this sequence produces β-galactosidase protein that catalyzes the hydrolysis of β-D-galactosides and α-L-arabinosides. β-galactosidase has the catalytic property of hydrolyzing lactose into glucose and galactose. Then, this enzyme is used to prepare milk and fermented milk products. It is used to prevent crystallization of lactose, to improve sweetness, to increase solubility of dairy product in dairy industries. Furthermore, it is used to produce low-lactose-containing food products for people with low lactose tolerance. Therefore, the use of β-galactosidase is one of the most promising enzyme applications for the food industry.

[302] In another embodiment, the 3135 bp long polynucleotide illustrated in SEQ ID NO. 341 is the full-length cDNA clone encoding the β-galactosidase protein having an open reading frame encoding a polypeptide of 1044 amino acids, as in SEQ ID NO. 342 with a calculated molecular mass of about 118 kD. Bioinformatics analysis of SEQ ID NO. 341 reveals that this sequence produces β-galactosidase protein that catalyzes the hydrolysis of β-D-galactosides and α-L-arabinosides. β-galactosidase has the catalytic property of hydrolyzing lactose into glucose and galactose. Then, this enzyme is used to prepare milk and fermented milk products. It is used to prevent crystallization of lactose, to improve sweetness, to increase solubility of dairy product in dairy industries. Furthermore, it is used to produce low-lactose-containing food products for people with low lactose tolerance. Therefore, the use of β-galactosidase is one of the most promising enzyme applications for the food industry.

[303] In another embodiment, the 2070 bp long polynucleotide illustrated in SEQ ID NO. 344 is the full-length cDNA clone encoding the β-galactosidase protein having an open reading frame encoding a 689 amino acid polypeptide, as in SEQ ID NO. 345 with a calculated molecular weight of about 78 kD. Bioinformatics analysis of SEQ ID NO. 344 reveals that this sequence produces β-galactosidase protein that catalyzes the hydrolysis of β-D-galactosides and α-L-arabinosides. β-galactosidase has the catalytic property of hydrolyzing lactose into glucose and galactose. Then, this enzyme is used to prepare milk and fermented milk products. It is used to prevent crystallization of lactose, to improve sweetness, to increase solubility of dairy product in dairy industries. Furthermore, it is used to produce low-lactose-containing food products for people with low lactose tolerance. Therefore, the use of β-galactosidase is one of the most promising enzyme applications for the food industry.

[304] In another embodiment, the 2994 bp length of polynucleotide illustrated in SEQ ID NO. 347 is the full-length cDNA clone encoding the β-galactosidase protein having an open reading frame encoding a 997 amino acid polypeptide, as in SEQ ID NO. 348 with a calculated molecular mass of about 108 kD. Bioinformatics analysis of SEQ ID NO. 347 reveals that this sequence produces β-galactosidase protein that catalyzes the hydrolysis of β-D-galactosides and α-L-arabinosides. β-galactosidase has the catalytic property of hydrolyzing lactose into glucose and galactose. Then, this enzyme is used to prepare milk and fermented milk products. It is used to prevent crystallization of lactose, to improve sweetness, to increase solubility of dairy product in dairy industries. Furthermore, it is used to produce low-lactose-containing food products for people with low lactose tolerance. Therefore, the use of β-galactosidase is one of the most promising enzyme applications for the food industry.

[305] In another embodiment, the 2985 bp long polynucleotide illustrated in SEQ ID NO. 350 is the full-length cDNA clone encoding the β-galactosidase protein having an open reading frame encoding a 994 amino acid polypeptide, as in SEQ ID NO. 351 with a calculated molecular mass of about 109 kD. Bioinformatics analysis of SEQ ID NO. 350 reveals that this sequence produces β-galactosidase protein that catalyzes the hydrolysis of β-D-galactosides and α-L-arabinosides. β-galactosidase has the catalytic property of hydrolyzing lactose into glucose and galactose. Then, this enzyme is used to prepare milk and fermented milk products. It is used to prevent crystallization of lactose, to improve sweetness, to increase solubility of dairy product in dairy industries. Furthermore, it is used to produce low-lactose-containing food products for people with low lactose tolerance. Therefore, the use of β-galactosidase is one of the most promising enzyme applications for the food industry.

[306] In another embodiment, the 1068 bp long polynucleotide illustrated in SEQ ID NO. 353 is the full-length cDNA clone encoding the endo-1,4-β-galactanase protein having an open reading frame encoding a 355 amino acid polypeptide, as in SEQ ID NO. 354 with a calculated molecular weight of about 38 kD. Bioinformatics analysis of SEQ ID NO. 353 reveals that this sequence produces endo-1,4-β-galactanase protein that performs or causes the endo-hydrolysis of (1->4)-β-D-galactosidic bonds present in arabinogalactans.

[307] In another embodiment, the 1065 bp long polynucleotide illustrated in SEQ ID NO. 356 is the full-length cDNA clone encoding the endo-1,4-β-galactanase protein having an open reading frame encoding a 354 amino acid polypeptide, as in SEQ ID NO. 357 with a calculated molecular mass of about 38 kD. Bioinformatics analysis of SEQ ID NO. 356 reveals that this sequence produces endo-1,4-β-galactanase protein that performs or causes the endo-hydrolysis of (1->4)-β-D-galactosidic bonds present in arabinogalactans.

[308] In another embodiment, the 1146 bp long polynucleotide illustrated in SEQ ID NO. 359 is the full-length cDNA clone encoding the endo-1,4-β-galactanase protein having an open reading frame encoding a 381 amino acid polypeptide, as in SEQ ID NO. 360 with a calculated molecular weight of about 42 kD. Bioinformatics analysis of SEQ ID NO. 359 reveals that this sequence produces endo-1,4-β-galactanase protein that performs or causes the endo-hydrolysis of (1->4)-β-D-galactosidic bonds present in arabinogalactans.

[309] In another embodiment, the 1281 bp long polynucleotide illustrated in SEQ ID NO. 362 is the full-length cDNA clone encoding the endo-1,6-β-galactanase protein having an open reading frame encoding a 426 amino acid polypeptide, as in SEQ ID NO. 363 with a calculated molecular mass of about 48 kD. Bioinformatics analysis of SEQ ID NO. 362 reveals that this sequence produces endo-1,6-β-galactanase protein that catalyzes the random hydrolysis of (1->6) bonds in (1->6)-β-D-glycans.

[310] In another embodiment, the 1110 bp long polynucleotide illustrated in SEQ ID NO. 365 is the full-length cDNA clone encoding the endo-1,4-β-mannanase protein having an open reading frame encoding a 369 amino acid polypeptide, as in SEQ ID NO. 366 with a calculated molecular weight of about 40 kD. Bioinformatics analysis of SEQ ID NO. 365 reveals that this sequence produces endo-1,4-β-mannanase protein that catalyzes the random hydrolysis of (1->4)-β-D-mannosid bonds into mannans, galactomannans and glycomannans.

[311] The sequences provided by the present invention can also be used as raw materials for the rational modification or design of new enzymes with characteristics that allow the enzymes to perform better in demanding processes.

[312] Summary of the invention is given in Tables 1. Table 1. Invention in summary.

[313] The present invention also relates to (a) nucleic acid vectors comprising a nucleotide sequence comprising any of the foregoing gene sequences and/or complements thereof; (b) expression constructs comprising a nucleotide sequence comprising any of the foregoing coding sequences of genes operably linked to a regulatory element that directs expression of the coding sequences; and (c) recombinant host cells comprising any of the foregoing gene sequences, including coding regions operably linked with a regulatory element that directs expression of the coding sequences in the host cells.

[314] Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known. The various sequences may be coupled in accordance with known techniques such as restriction, joining of complementary restriction sites and ligation, blunt end by overhang stuffing and blunt ligation or the like. Polylinkers and adapters can be employed, where appropriate, and introduced or removed by known techniques to allow ease of assembly of DNA vectors and expression constructs. A large number of vectors are available for cloning and genetic manipulation. Normally, cloning can be performed in E. coli.

[315] In another embodiment of the invention, vectors comprising an enzyme gene sequence of the invention may further comprise replication functions that allow the transfer, maintenance and propagation of the vectors in one or more species of host cells, including, but not limited to a, E. coli cells, filamentous fungal cells, yeast cells, and Bacillus cells. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated along with the chromosome into which it has been integrated. The choice of vector will usually depend on the compatibility of the vector with the host cell into which the vector will be introduced.

[316] The expression construct of the invention comprises a promoter, a nucleotide sequence encoding a gene sequence of the invention, a transcription termination sequence and selectable marker (optional). Any method known in the art to introduce such an expression construct into a host cell can be used. Fungal cells can be transformed by a process involving protoplast formation, protoplast transformation and cell wall regeneration in a manner known per se. Appropriate procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton MM, Hames JE, Timberlake WE. Transformation of Aspergillus nidulans by using a trpC plasmid. PNAS, 1984; 81(5):1470-1474. Appropriate methods for transforming Fusarium species are described by Malardier L, Daboussi MJ, Julien J, Roussel F, Scazzocchio C, Brygoo Y. Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum. Gene 1989; 78:147-156, and WO 96/00787. Yeast can be transformed using procedures described by Becker DM, Guarente L. High-efficiency transformation of yeast by electroporation. In: Abelson JN and Simon MI (eds), Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1991;194:182-187, Academic Press, Inc., New York; Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. Journal of Bacteriology, 1983;153: 163-168 and Hinnen A, Hicks JB, Fink GR. Transformation of yeast. PNAS, 1978; 75(4):1929-1933.

[317] For industrial applications, the enzymes of the present invention are produced by a fungal cell. Preferably, the expression host cell is a filamentous fungus cell that has been used in large-scale industrial fermentation. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed foreign protein. Preferably, an expression host is selected which is capable of efficient secretion of its endogenous proteins. A host cell can also be chosen for deficiencies in extracellular protease activities, as the secreted enzyme can be degraded in culture medium.

[318] The present invention also relates to methods for producing a polypeptide of the present invention, comprising and/or consisting of: (i) culturing a cell, which in its wild-type form is capable of producing the polypeptide, under conditions which contribute to the production of the polypeptide; and (ii) recovering the polypeptide. In a preferred aspect, the cell is M. phaseolina.

[319] Another embodiment of the invention also relates to methods for producing a polypeptide of the present invention, comprising and/or consisting of: (i) cultivating a host cell under conditions that contribute to the production of the polypeptide, in which the host cell comprises a nucleotide sequence or the complement of such sequences from any one or any combination of SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 2 32, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 3 07, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 or 364, wherein the nucleotide sequence encodes a polypeptide comprising and /or consists of any one of the mature polypeptides of SEQ ID NOs. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 2 25, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 3 00, and (ii) recovering the polypeptide .

[320] In another embodiment of the present invention, expression host cells or transformants are cultured in appropriate nutrient media for growth and protein expression using methods known in the art. For example, the cell can be cultured by shake-flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters, performed in an appropriate medium and under conditions allowing the polypeptide to be expressed and/or isolated. Cultivation can take place in suitable nutrient media comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see In: More Gene Manipulations in Fungi. Bennett J W, Lasure L., (eds. 1991; Academic Press, San Diego, CA). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.

[321] Polypeptides can be detected using methods known in the art that are specific for the polypeptides. These detection methods may include the use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. An enzyme assay can be used to determine polypeptide activity.

[322] The produced polypeptide can be recovered using methods known in the art. The polypeptide can be recovered in various methods from the nutrient medium by conventional procedures, including, but not limited to, filtration, centrifugation, extraction, spray drying, evaporation and precipitation or a combination thereof.

[323] The polypeptides of the present invention can be purified by a variety of procedures that are known in the art including, but not limited to, chromatography method (such as ion exchange, affinity, hydrophobic, chromatofocusing and size exclusion), electrophoretic procedures (such as isoelectric focusing), differential solubility (such as ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure polypeptides (see details in Protein Purification, Principles, High Resolution Methods and Applications. Janson JC, Rydén L. ( eds(). 1989; VCH Publishers Inc., New York).

[324] The present invention also relates to gene products (eg, RNA or proteins) which are encoded by gene sequences set forth in SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 2 24, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 2 99, The Enzyme Gene Products of the invention also include RNA or proteins that are encoded by the genomic sequences of the genes as set forth in SEQ ID NOs. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 2 23, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 2 98, 301, 304, 307, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361 and 364. amino acid sequence selected from the group comprising and/or consisting of SEQ ID NOs. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 2 25, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 3 00, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363 and 366.

[325] The enzymes of the present invention have at least one of the activities of an enzyme selected from the group comprising and/or consisting of β-1,4-endoglycanase, cellobiohydrolase, β-glucosidase, α-glucosidase, Exo-1,3 -β-glucanase, α-glycan lyase, α-xylosidase, d-4,5-unsaturated b-glucuronyl hydrolase, amyloglucosidase, α-1,2-mannosidase, α-1,3-glucanase, α-fucosidase, xylan 1 ,4-β-Xylosidase, endo-1,5-α-arabinosidase, Endo-1,4-β-xylanase, α-arabinofuranosidase, β-galactosidase, Endo-1,4-β-galactanase, Endo-1,6 -β-glucanase and endo-β-1,4-mannanase. The enzyme gene products of the invention can be readily produced, for example, by synthetic techniques or by the methods of recombinant DNA technology using techniques that are well known (see, Creighton TE. Proteins: Structures and Molecular Principles, 1983; W. H. Freeman and Co., N.Y.)

[326] In another embodiment, the methods and compositions of the invention include proteins and polypeptides that represent functionally equivalent gene products. Such functionally equivalent gene products include, but are not limited to, natural variants of the polypeptides having an amino acid sequence set forth in SEQ ID NOs. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 2 25, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 3 00, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363 and 366.

[327] These equivalent gene products may contain, for example, deletions, additions or substitutions of amino acid residues within the amino acid sequences encoded by the enzyme gene sequences described above, but which result in a silent change, thus producing a product functionally equivalent. Amino acid substitutions can be prepared based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

[328] Further modifications of the gene product coding sequences described above can be prepared to generate polypeptides that are more suitable, for example, for expression, scaling, etc. in a chosen host cell. For example, cysteine residues can be deleted or substituted with another amino acid in order to eliminate disulfide bonds.

[329] Another embodiment of the present invention further includes enzymes of the present invention in solid form. Enzymes in solid form or enzyme granules can be used, for example, in solid detergent and animal feed. Methods for preparing solid forms of enzymes are well known in the art, such as, but not limited to, pelletizing (spray cooling on a waxy material), extrusion, agglomeration or granulation (dilution with inert material and binders). Solid enzyme compositions comprising a solid form of an enzyme of the invention, in the form of mixed powders, tablets and the like, are contemplated.

[330] The present disclosure includes what appears in the appended claims, as well as what is described above. While this invention is described in its preferred form, with a degree of particularity, it is understood that the present disclosure of the preferred form is by way of example only, and that numerous changes in construction details and the combination and arrangement of parts may be invoked without departing from the scope and spirit of the invention and claims.

[331]Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

EXAMPLE

[332] The following example is intended to further illustrate the invention, without any intent for the invention to be limited to the specific embodiments described herein.

[333] Example 1 Isolation of Genomic DNA from M. phaseolina

[334] Genomic DNA was isolated from M. phaseolina strain ms6 using procedures described by Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical Streptomyces Genetics, 2000; John Innes Foundation, Norwich, UK, pp. 162-208. Briefly, mycelia were scraped from a stock Petri dish using an inoculation loop and inoculated into a liquid potato-dextrose medium in an Erlenmeyer flask. The Erlenmeyer was incubated without agitation for about 72 hours at 30°C. Mycelia grow on the surface at the air-medium interface. The mycelia were collected using a sterile toothpick and placed between sterile paper towels and washed a few times with saline solution (sodium phosphate buffer pH 7.0). The mycelia were squeezed to remove excess liquid and the collected mycelia were air-dried for 30 minutes. Semi-dried mycelia were placed in liquid nitrogen and ground to fine powder and finally isolated DNA following the protocol described in Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical Streptomyces Genetics, 2000; John Innes Foundation, Norwich, UK, pp. 162-208.

[335] Example 2 Primer Design and Synthesis

[336] The primers used in the study were designed from hand-cured transcriptome and the “gene templates” predicted from genomic sequences of M. phaseolina ms6 strain, choosing the sequences manually with complete ORFs or using databases where genes similar ones have been successfully isolated from other plants. Comparative bioinformatics analyzes of the nucleotide sequences obtained from the transcriptome were performed using the NCBI BLAST, BLASTP, RPS-BLAST, BLASTX and PSI-BLAST to identify homologues of related genes and for proper gene identification. Nucleotide sequence alignments were performed using clustalW version 1.82 whenever multiple sequences were found from the “gene mix”. The alignment was then edited. Gene-specific primers (forward and reverse) were selected manually or through the Primer 3 plus tool and the primers were custom synthesized.

[337] All oligonucleotides used in this study were synthesized and purified by HPLC by the supplier and purchased from Integrated DNA Technologies (IDT). Stock solutions of 100 pmol were prepared in autoclaved ddH2O and stored at about -20°C, in aliquots for use. Oligonucleotide sequences used as primers for PCR

[338] Example 3 Amplification, Cloning and Sequencing of Cellulases and Hemicellulases from M. phaseolina ms6

[339] Total RNA was isolated from three-day-old mycelium grown in liquid medium as described by Chomczynski P and Sacchi N, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987, 162: 156-159). RNA quality or integrity was verified by agarose gel electrophoresis and was quantified using Thermo Scientific Nano Drop 2000 according to standard procedure. The first strand of cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) following the manufacturer's instructions. The gene was amplified from cDNA by PCR using gene-specific primers. The PCR reaction (50μL) contained 1μL of cDNA, 20 pmoles of each primer, 5μL of 10x PCR Buffer, 5μL of 2.5 mM dNTP mix, and 1.0 unit of PfuTaq DNA polymerase. PCR was performed in a thermocycler (Applied Biosystems) using the following conditions: initial denaturation for 5 min at 95°C followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 5961°C for 30 sec and extension at 72° C for 1 to 2.0 min depending on the length of the target gene, with a final extension at 72°C for 7 min. The PCR product was analyzed on the 1% agarose gel using 1X TAE buffer and the amplicon was eluted from the gel using the QIAGEN Gel Extraction Kit following the manufacturer's instructions. The purified PCR product was ligated into pCR®8/GW/TOPO® TA cloning kit (Invitrogen) and transformed into competent E. coli cells (Invitrogen). Plasmids were isolated from putative colonies using the QIAprip Spin Miniprep Kit (QIAGEN) following the manufacturer's instructions. The presence of the insert was verified using gene-specific primers and positive plasmids were subjected to sequencing.

[340] Example 4 Sequence analysis

[341]Nucleotide sequence and amino acid sequence were analyzed by BLASTN and BLASTP programs, respectively. Reported sequences from other plants were aligned with ClustalW. Phylogenetic analysis was performed using Neighbor Joining (NJ).

INCORPORATION AS A REFERENCE

[342] All US patents, US published patent applications, and published PCT applications cited herein are incorporated herein by reference.

EQUIVALENTS

[343] While various embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision a variety of other means and/or structures to perform the functions and/or obtain the results and/or one or more of the advantages herein. described, and each such variation and/or modification is considered to be within the scope of the present invention. Those skilled in the art will recognize, or be able to discover using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. It should therefore be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereof; the invention may be practiced otherwise than as specifically described and claimed.

Claims (3)

1. Recombinant nucleic acid molecule, characterized in that it consists of a complementary DNA sequence (cDNA) set out in SEQ ID No. 2, wherein said nucleic acid molecule encodes a β-1,4-endoglucanase. 2. Recombinant expression construct, characterized in that it comprises the recombinant nucleic acid molecule as defined in claim 1. 3. Genetically engineered prokaryotic host cell, characterized in that it comprises the recombinant nucleic acid molecule as defined in claim 1.