Chrysosporium Cellulase and Methods of Use

Abstract

The subject invention relates to novel compositions of neutral and/or alkaline cellulase and methods for obtaining neutral and/or alkaline cellulase compositions from Chrysosporium cultures, in particular Chrysosporium lucknowense. This invention also provides mutants and methods of generating mutants of Chrysosporium capable of producing neutral and/or alkaline cellulase. This invention also relates to the genes encoding the enzymes comprising the neutral and/or alkaline cellulase composition. In addition, this invention provides methods of culturing Chrysosporium to produce neutral and/or alkaline cellulases. The neutral and/or alkaline cellulase compositions of the subject invention can be used in a variety of processes including stone washing of clothing, detergent processes, deinking, color brightening, depilling and biobleaching of paper and pulp and treatment of waste streams. The present invention also relates to the isolation and purification of cellulase enzymes, having glucanase and cellobiohydrolase activity, and useful for stonewashing applications.

1 . A composition having neutral and/or alkaline cellulase activity, obtained by a method which comprises growing a wild type or mutant fungus of the genus  Chrysosporium  in culture in a suitable medium, wherein the fungus is  Chrysosporium lucknowense, Chrysosporium pannorum, Chrysosporium keratinophilum, Chrysosporium lobatum, Chrysosporium merdarium, Chrysosporium queenslandicum,  or  Chrysosporium tropicum.    
     
     
         2 . A composition according to  claim 1  having optimal cellulose activity at a temperature from about 40° C. to about 60° C., at a pH from about 5.0 to about 12.0. 
     
     
         3 . A composition according to  claim 1  having at least 50% of the optimal cellulase activity, at a pH from about 6.0 to about 7.0, at a temperature from about 40° C. to about 60° C. 
     
     
         4 . A composition according to  claim 1  wherein said cellulase activity is assayed by any one of the CMCase, RBBCMCase, endoviscometric or filter paper activity assays. 
     
     
         5 . A substantially purified and isolated protein fraction, obtained from a composition according to  claim 1 , and having at least 50% of its maximal cellulase activity at a pH between about 6.0 and about 7.0 as measured by any one of the CMCase, RBBCMCase, endoviscometric or filter paper activity assays. 
     
     
         6 . An endoglucanase obtained from a fraction according to  claim 5 , having a molecular weight of about 25 kD and pI of about 4. 
     
     
         7 . An endoglucanase obtained from a fraction according to  claim 5 , having a molecular weight of about 70 kD and a pI of about 4. 
     
     
         8 . An endoglucanase obtained from a fraction according to  claim 5 , having, a molecular weight of about 60 kD and a pI of about 3. 
     
     
         9 . An endoglucanase obtained from a fraction according to  claim 5 , having a molecular weight of about 43 kD and a pI of about 3. 
     
     
         10 . A cellobiohydrolase obtained from a fraction according to  claim 5 , having a molecular weight of about 60 kD and a pI of about 4. 
     
     
         11 . A substantially purified and isolated neutral and/or alkaline cellulase enzyme, isolated from a protein fraction according to  claim 5 , and having a pI of between about 3 and about 5.5. 
     
     
         12 . A cellulase according to  claim 11  wherein said cellulase possesses either endoglucanase or cellobiohydrolase activity. 
     
     
         13 . A cellulase according to  claim 12  wherein said cellulase retains at least 50% of its maximal cellulase activity at a pH between about 6.0 and about 7.0. 
     
     
         14 . An endoglucanase obtained from a fraction according to  claim 5  and having a molecular weight of about 25 kD. 
     
     
         15 . An endoglucanase obtained from a fraction according to  claim 5  and having a molecular weight of about 70 kD. 
     
     
         16 . An endoglucanase obtained from a fraction according to  claim 5  and having a molecular weight of about 43 kD. 
     
     
         17 . A detergent composition containing one or more purified enzymes isolated from a protein fraction according to  claim 5 , and further comprising a surfactant. 
     
     
         18 . A fabric softening composition containing one or more purified enzymes obtained from the protein fraction according to  claim 5 . 
     
     
         19 . A composition for the enzymatic treatment of cellulosic fibers or cellulosic fabrics, comprising a cellulase whose amino acid sequence is encoded by a nucleic acid sequence from a wild-type or mutant fungus of the genus  Chrysosporium,  said composition having a pH between about 8.0 and about 12.0, wherein the fungus is selected from the group consisting of  Chrysosporium lucknowense, Chrysosporium pannorum, Chrysosporium pruinosum, Chrysosporium keratinophilum, Chrysosporium lobatum, Chrysosporium merdarium, Chrysosporium queenslandicum,  and  Chrysosporium tropicum.    
     
     
         20 . A composition according to  claim 19 , wherein the cellulase is isolated or obtained from a wild-type or mutant fungus of the genus  Chrysosporium.