Solubilized enzyme and uses thereof

Abstract

The present invention relates to mixtures comprising a polypeptide or a plurality of polypeptides having biomass-degrading activity that is solubilized from an inclusion body, and retaining biomass-degrading activity, and methods for producing and using the same. The invention described herein provides methods for increasing the yield of recombinant protein with biomass-degrading activity that can be isolated from host cells.

What is claimed is: 
     
         1 . A mixture comprising a plurality of polypeptides having biomass-degrading activity and a solubilizing agent, wherein the polypeptides have at least 8-10% of the biomass-degrading activity compared to a native polypeptide having biomass-degrading activity. 
     
     
         2 . The mixture of  claim 1 , further comprising one or more proteins associated with an inclusion body. 
     
     
         3 . The mixture of  claim 1 , wherein the mixture does not comprise one or more proteins associated with an inclusion body. 
     
     
         4 . The mixture of any of the preceding claims, further comprising cellular debris, one or more ribosomal component, one or more host protein, and/or host nucleic acid comprising DNA and/or RNA. 
     
     
         5 . The mixture of any of the preceding claims, wherein the biomass-degrading activity is cellobiase activity, ligninase activity, endoglucanase activity, cellobiohydrolase activity, or xylanase activity. 
     
     
         6 . The mixture of any of the preceding claims, wherein the polypeptide is partially unfolded, partially misfolded, or partially denatured. 
     
     
         7 . The mixture of  claim 1 , wherein the polypeptide comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1. 
     
     
         8 . The mixture of any of the preceding claims, wherein the polypeptide comprises a Cel3A enzyme from  T. reesei , or a functional variant or fragment thereof. 
     
     
         9 . The mixture of  claim 8 , wherein the Cel3A enzyme comprises the amino acid sequence SEQ ID NO: 1, or an amino acid sequence with at least 90% identity thereof. 
     
     
         10 . The mixture any of the preceding claims, wherein the polypeptide is encoded by a nucleic acid sequence comprising at least 90% identity to SEQ ID NO: 2 or SEQ ID NO: 3. 
     
     
         11 . The mixture of any of the preceding claims, wherein the polypeptide is aglycosylated. 
     
     
         12 . The mixture of any of  claims 1 - 3  or  7 , wherein the solubilizing agent comprises urea, and optionally, is present at a concentration between 0.2M-6M. 
     
     
         13 . The mixture of any of the preceding claims, further comprising at least one additional polypeptide having a biomass-degrading activity or a microorganism that produces one or more enzymes having a biomass-degrading activity. 
     
     
         14 . The mixture of  claim 13 , wherein the additional polypeptide is selected from a ligninase, an endoglucanase, a cellobiohydrolase, a cellobiase, and a xylanase, or any combination thereof. 
     
     
         15 . The mixture of  claim 13  or  14 , wherein the additional polypeptide is selected from:
 a. a polypeptide comprising an amino acid sequence with at least 90% identity to SEQ ID NO: 1; 
 b. a Cel3A enzyme from  T. reesei , or a functional variant or fragment thereof; or 
 c. a polypeptide encoded by a nucleic acid sequence comprising (e.g., consisting of) SEQ ID NO: 2 or SEQ ID NO: 3. 
 
     
     
         16 . The mixture of any of  claims 13 - 15 , wherein the additional polypeptide is aglycosylated. 
     
     
         17 . The mixture of any of  claims 13 - 15 , wherein the additional polypeptide is glycosylated. 
     
     
         18 . A mixture comprising a plurality of polypeptides having an amino acid sequence with at least 90% identity to SEQ ID NO: 1 and a solubilizing agent, wherein the plurality of polypeptides have at least 20%-40% of the activity of the native polypeptide comprising SEQ ID NO: 1. 
     
     
         19 . The mixture of  claim 18 , further comprising one or more proteins associated with an inclusion body. 
     
     
         20 . The mixture of  claim 18 , wherein the mixture does not comprise one or more proteins associated with an inclusion body. 
     
     
         21 . The mixture of any of  claims 18 - 20 , further comprising cellular debris, one or more ribosomal component, one or more host protein, and/or host nucleic acid comprising DNA and/or RNA. 
     
     
         22 . The mixture of any of  claims 18 - 21 , wherein the polypeptide is partially unfolded, partially misfolded, or partially denatured. 
     
     
         23 . The mixture any of  claims 18 - 22 , wherein the polypeptide is encoded by a nucleic acid sequence comprising at least 90% identity to SEQ ID NO: 2 or SEQ ID NO: 3. 
     
     
         24 . The mixture of any of  claims 18 - 23 , wherein the polypeptide is aglycosylated. 
     
     
         25 . The mixture of any of  claims 18 - 20 , wherein the solubilizing agent comprises urea, and optionally, is present at a concentration between 0.2M-6M. 
     
     
         26 . The mixture of any of  claims 18 - 25 , further comprising at least one additional polypeptide having a biomass-degrading activity or a microorganism that produces one or more enzymes having a biomass-degrading activity. 
     
     
         27 . The mixture of  claim 26 , wherein the additional polypeptide is selected from a ligninase, an endoglucanase, a cellobiohydrolase, a cellobiase, and a xylanase, or any combination thereof. 
     
     
         28 . The mixture of  claim 26  or  27 , wherein the additional polypeptide is selected from:
 a. a polypeptide comprising an amino acid sequence with at least 90% identity to SEQ ID NO: 1; 
 b. a Cel3A enzyme from  T. reesei , or a functional variant or fragment thereof; or 
 c. a polypeptide encoded by a nucleic acid sequence comprising (e.g., consisting of) SEQ ID NO: 2 or SEQ ID NO: 3. 
 
     
     
         29 . The mixture of any of  claims 26 - 28  wherein the additional polypeptide is aglycosylated. 
     
     
         30 . The mixture of any of  claims 26 - 28 , wherein the additional polypeptide is glycosylated. 
     
     
         31 . A method for producing a mixture of any of  claims 1 - 30  comprising contacting a cell expressing the polypeptide having biomass-degrading activity, or lysate thereof, with a solubilizing agent at a concentration suitable for solubilizing the polypeptide. 
     
     
         32 . The method of  claim 31 , further comprising lysing the cell to obtain a lysate, separating a soluble fraction from an insoluble fraction of the lysate, and resuspending the insoluble fraction in the solubilizing agent. 
     
     
         33 . The method of  claim 31  or  32 , wherein the solubilizing agent is urea, and optionally, wherein the concentration of the solubilizing agent is between 0.2M-6M. 
     
     
         34 . The method of any of  claims 31 - 33 , wherein the biomass-degrading activity is a cellobiase activity, a ligninase activity, an endoglucanase activity, a cellobiohydrolase, or a xylanase activity. 
     
     
         35 . The method of any of  claims 31 - 34 , wherein the polypeptide comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1. 
     
     
         36 . The method of any of  claims 31 - 35 , wherein the polypeptide comprises a Cel3A from  T. reesei , or a functional variant or fragment thereof. 
     
     
         37 . The method of any of  claims 31 - 36  wherein the polypeptide is aglycosylated. 
     
     
         38 . A method for producing a polypeptide having biomass-degrading activity comprising expressing the polypeptide in a cell and contacting the cell or a lysate thereof with a solubilizing agent at a concentration suitable for solubilizing the polypeptide. 
     
     
         39 . A method for producing a polypeptide having biomass-degrading activity comprising providing a cell that has been genetically modified to produce at least one polypeptide having biomass-degrading activity, wherein at least a portion of said polypeptide having biomass-degrading activity is found in inclusion bodies, and contacting the cell, or a lysate thereof containing the inclusion bodies, with a solubilizing agent at a concentration suitable for solubilizing the polypeptide. 
     
     
         40 . The method of  claim 38  or  39 , wherein the solubilizing agent comprises urea. 
     
     
         41 . The method of any of  claims 38 - 40 , wherein the concentration of the solubilizing agent is between 0.2M-6M. 
     
     
         42 . The method of any of  claims 38 - 41 , further comprising lysing the cell to obtain a lysate, separating a soluble fraction from an insoluble fraction of the lysate, and resuspending the insoluble fraction in the solubilizing agent. 
     
     
         43 . The method of any of  claims 38 - 42 , wherein the biomass-degrading activity is a cellobiase activity, a ligninase activity, an endoglucanase activity, a cellobiohydrolase activity, or a xylanase activity. 
     
     
         44 . The method of any of  claim 38  or  39 , wherein the polypeptide comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1. 
     
     
         45 . The method of any of  claims 38 - 44 , wherein the polypeptide comprises a Cel3A from  T. reesei , or a functional variant or fragment thereof. 
     
     
         46 . The method of any of  claims 38 - 45 , wherein the cell is a prokaryotic or bacterial cell, e.g.,  E. coli  cell, origami  E. coli  cell. 
     
     
         47 . The method of any of  claims 38 - 46 , wherein the polypeptide is aglycosylated. 
     
     
         48 . A method of producing a product from a biomass comprising contacting a biomass with the mixture of any of  claims 1 - 30 , and, optionally, a microorganism that produces one or more biomass-degrading enzyme and/or an enzyme mixture comprising biomass-degrading enzymes, under conditions suitable for the production of the product. 
     
     
         49 . The method of  claim 48 , further comprising treating the biomass with an electron beam prior to contacting the biomass with the mixture. 
     
     
         50 . The method of  claim 48  or  49 , wherein the product is a sugar product. 
     
     
         51 . The method of  claim 50 , wherein the sugar product is glucose and/or xylose. 
     
     
         52 . The method of any of  claims 48 - 51 , further comprising isolating the product. 
     
     
         53 . The method of  claim 52 , wherein the isolating of the product comprises precipitation, crystallization, chromatography, centrifugation, and/or extraction. 
     
     
         54 . The method of any of  claims 48 - 53 , wherein the enzyme mixture comprises at least two of the enzymes selected from B2AF03, CIP1, CIP2, Cel1a, Cel3a, Cel5a, Cel6a, Cel7a, Cel7b, Cel12a, Cel45a, Cel74a, paMan5a, paMan26a, and Swollenin. 
     
     
         55 . The method of any of  claims 48 - 54 , wherein the biomass comprises one or more of an agricultural product or waste, a paper product or waste, a forestry product, or a general waste, or any combination thereof, wherein:
 a) an agricultural product or waste comprises sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, canola straw, wheat straw, barley straw, oat straw, rice straw, corn cobs, corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean stover, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a combination thereof;   b) a paper product or waste comprises paper, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, cardstock, cardboard, paperboard, or paper pulp, or a combination thereof;   c) a forestry product comprises aspen wood, particle board, wood chips, or sawdust, or a combination thereof; and   d) a general waste comprises manure, sewage, or offal, or a combination thereof.   
     
     
         56 . The method of any of  claims 48 - 55 , further comprises a step of treating the biomass prior to introducing the microorganism or the enzyme mixture to reduce the recalcitrance of the biomass, wherein the treating comprises bombardment with electrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, or freeze grinding. 
     
     
         57 . The method of any of  claims 48 - 56 , wherein the microorganism that produces a biomass-degrading enzyme is from species in the genera selected from  Bacillus, Coprinus, Myceliophthora, Cephalosporium, Scytalidium, Penicillium, Aspergillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, Chrysosporium  or  Trichoderma.    
     
     
         58 . The method of any of  claims 48 - 57 , wherein the microorganism that produces a biomass-degrading enzyme is selected from  Aspergillus, Humicola insolens  ( Scytalidium thermophilum )  Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris, Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, Acremonium furatum, Chrysosporium lucknowense, Trichoderma viride, Trichoderma reesei , or  Trichoderma koningii.    
     
     
         59 . The method of any of  claims 48 - 58 , wherein the microorganism has been induced to produce biomass-degrading enzymes by combining the microorganism with an induction biomass sample under conditions suitable for increasing production of biomass-degrading enzymes compared to an uninduced microorganism. 
     
     
         60 . The method of any of  claims 48 - 59 , wherein the induction biomass sample comprises one or more of an agricultural product or waste, a paper product or waste, a forestry product, or a general waste, or any combination thereof, wherein:
 a) an agricultural product or waste comprises sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, canola straw, wheat straw, barley straw, oat straw, rice straw, corn cobs, corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean stover, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a combination thereof;   b) a paper product or waste comprises paper, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, cardstock, cardboard, paperboard, or paper pulp, or a combination thereof;   c) a forestry product comprises aspen wood, particle board, wood chips, or sawdust, or a combination thereof; and   d) a general waste comprises manure, sewage, or offal, or a combination thereof.