Carbohydrate-Binding Modules of a New Family

Abstract

The present invention relates to non-catalytic carbohydrate-binding modules (CBM) belonging to a new family of CBM's. A CBM of the invention was found attached to a glycosyl hydrolase family 61 (GH61) polypeptide and was shown to have little homology with known CBM's indicating that it is the first known member of a new family of CBM's. The present invention further relates to CBM's preferably exhibiting binding affinity for cellulose; to a method of producing such CBM's; and to methods for using such CBM's in the textile, detergent and cellulose fiber processing industries, for purification of polypeptides, immobilisation of active enzymes, baking, manufacturing of biofuel, modification of plant cell walls.

What is claimed is: 
     
         1 . An isolated polynucleotide molecule encoding an enzyme hybrid comprising a carbohydrate-binding module and a catalytic domain exhibiting enzyme activity, wherein the carbohydrate-binding module is heterologous to the catalytic domain exhibiting enzyme activity and wherein the carbohydrate-binding module has at least 90% identity with the sequence of amino acids 34-174 of SEQ ID NO: 2. 
     
     
         2 . The isolated polynucleotide molecule of  claim 1 , wherein the catalytic domain exhibits endo-beta-1,4-glucanase activity. 
     
     
         3 . The isolated polynucleotide molecule of  claim 1 , wherein the catalytic domain exhibits enzyme activity is an enxyme selected from the group consisting of an acetyl xylan esterase, alpha-L-arabinofuranosidase, amylase, arabinose, beta-glucosidase, beta-mannosidase, beta-1.3-1.4-glucanase, cellulose, cellulose 1.4-cellobiosidase, Cellobiohydrolase, cinnamoyl esterase, endo-1.4-beta-xylanase, endo-1.4-galactanase, pectinase, exo-polygalacturonase, feruloyl esterase, glucan 1.3-beta-glucosidase, licheninase, lipase, mannan endo-1.4-mannosidase, oxidoreductase, pectin methylesterase, pectin acetylesterase, phenol-oxidizing oxidase, polygalacturonase, protease, thamnogalacturonase, thamnogalacturonan acetyl esterase, rhamnogalacturonan hydrolase, rhamnogalacturonan lyase, rhamnogalacturonan methyl esterase, xylanase, xyloglucanase, and xylan methyl esterase. 
     
     
         4 . The isolated polynucleotide molecule of  claim 1 , wherein the carbohydrate-binding module has at least 95% identity with the sequence of amino acids 34-174 of SEQ ID NO: 2. 
     
     
         5 . The isolated polynucleotide molecule of  claim 1 , wherein the carbohydrate-binding module has at least 97% identity with the sequence of amino acids 34-174 of SEQ ID NO: 2. 
     
     
         6 . The isolated polynucleotide molecule of  claim 1 , wherein the carbohydrate-binding module is a fragment of the sequence of amino acids 34-174 of SEQ ID NO: 2, which retains carbohydrate-binding module activity. 
     
     
         7 . The isolated polynucleotide molecule of  claim 1 , wherein the carbohydrate-binding module comprises the sequence of amino acids 34-174 of SEQ ID NO: 2. 
     
     
         8 . The isolated polynucleotide molecule of  claim 7 , which comprises a nucleotide sequence as shown in SEQ ID NO:1 from nucleotide 109 to nucleotide 531. 
     
     
         9 . The isolated polynucleotide molecule of  claim 7 , wherein the carbohydrate-binding module is encoded by a DNA sequence obtained from  Pseudoplectania nigrella  CBS 444.97. 
     
     
         10 . A polynucleotide construct comprising the polynucleotide molecule of  claim 1 . 
     
     
         11 . An expression vector comprising the following operably linked elements: a transcription promoter, the polynucleotide molecule of  claim 1 , and a transcription terminator. 
     
     
         12 . An isolated recombinant host cell comprising the expression vector of  claim 11 . 
     
     
         13 . The recombinant host cell of  claim 12 , which is a eukaryotic cell. 
     
     
         14 . The recombinant host cell of  claim 13 , wherein the eukaryotic cell is a fungal cell. 
     
     
         15 . The recombinant host cell of  claim 14 , wherein the cell belongs to a strain of  Aspergillus.    
     
     
         16 . The recombinant host cell of  claim 15 , wherein the cell is a strain of  Aspergillus oryzae.    
     
     
         17 . A method of producing a polypeptide having carbohydrate-binding module activity comprising culturing the recombinant host cell of  claim 12 , and recovering the polypeptide. 
     
     
         18 . An isolated polynucleotide molecule encoding a polypeptide having carbohydrate-binding module activity which polynucleotide molecule hybridizes to a denatured double-stranded DNA probe under low stringency conditions, wherein the probe is selected from the group consisting of DNA probes comprising the sequence shown in positions 109-531 of SEQ ID NO:1 and DNA probes comprising a subsequence of positions 109-531 of SEQ ID NO:1 having a length of at least about 300 base pairs.