Treatment of cellulosic material and enzymes useful thererin

Abstract

The present invention relates to the production of sugar hydrolysates from cellulosic material. The method may be used e.g. for producing fermentable sugars for the production of bioethanol from lignocellulosic material. Cellulolytic enzymes and their production by recombinant technology is described, as well as uses of the enzymes and enzyme preparations.

1 . A method for treating cellulosic material with cellobiohydrolase, endoglucanase and beta-glucosidase, whereby said cellobiohydrolase comprises an amino acid sequence having at least 80% identity to SEQ ID NOS: 2, 4, 6 or 8, or to an enzymatically active fragment thereof. 
     
     
         2 - 4 . (canceled) 
     
     
         5 . The method of  claim 1 , comprising using a cellobiohydrolase obtainable from  Thermoascus aurantiacus  or  Acremonium thermophilum  to which a cellulose binding domain, derived from  Trichoderma reesei  or Chaetomium termophilum, has been genetically attached, and the resulting fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NOS: 28 or 30. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the endoglucanase comprises an amino acid sequence having at least 80% identity to SEQ ID NOS: 10, 12, 14 or 16, or to an enzymatically active fragment thereof. 
     
     
         8 - 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the beta-glucosidase comprises an amino acid sequence having at least 80% identity to SEQ ID NOS: 22, 24 or 26, or to an enzymatically active fragment thereof. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the cellulosic material is lignocellulosic material. 
     
     
         13 . The method of  claim 1 , comprising treating lignocellulosic material with at least one further enzyme having an amino acid sequence having at least 80% identity to SEQ ID NOS: 18 or 20, or to an enzymatically active fragment thereof. 
     
     
         14 - 17 . (canceled) 
     
     
         18 . An enzyme preparation comprising cellobiohydrolase, endoglucanase and beta-glucosidase, wherein said cellobiohydrolase comprises an amino acid sequence having at least 80% identity to SEQ ID NOS: 2, 4, 6 or 8, or to an enzymatically active fragment thereof. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . The enzyme preparation of  claim 18 , wherein the enzymes are recombinant enzymes, comprising a cellobiohydrolase obtainable from  Thermoascus aurantiacus  to which a cellulose binding domain has been genetically attached. 
     
     
         22 . (canceled) 
     
     
         23 . The enzyme preparation of  claim 21 , wherein the attached cellulose binding domain is derived from  Trichoderma reesei  or  Chaetomium termophilum , and the resulting fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NOS: 28 or 30. 
     
     
         24 . The enzyme preparation of  claim 18 , wherein the endoglucanase comprises an amino acid sequence having at least 80% identity to SEQ ID NOS: 10, 12, 14 or 16, or to an enzymatically active fragment thereof. 
     
     
         25 . (canceled) 
     
     
         26 . The enzyme preparation of  claim 18 , wherein the beta-glucosidase comprises an amino acid sequence having at least 80% identity to SEQ ID NOS: 22, 24 or 26, or to an enzymatically active fragment thereof. 
     
     
         27 . (canceled) 
     
     
         28 . The enzyme preparation of  claim 18 , comprising at least one xylanase, which comprises an amino acid sequence having at least 80% identity to SEQ ID NOS: 18 or 20, or to an enzymatically active fragment thereof. 
     
     
         29 . (canceled) 
     
     
         30 . The enzyme preparation of  claim 18 , wherein at least one of the enzymes is encoded by a gene similar to that included in a microorganism having accession number DSM 16723, DSM 16728, DSM 16729, DSM 16727, DSM 17326, DSM 17324, DSM 17323, DSM 17729, DSM 16723, DSM 16726, DSM 16725, DSM 17325 or DSM 17667. 
     
     
         31 . (canceled) 
     
     
         32 . Use of an enzyme preparation according to  claim 18  for degrading cellulosic material. 
     
     
         33 . (canceled) 
     
     
         34 . Use of the method according to  claim 1  in a process for preparing ethanol from cellulosic material. 
     
     
         35 . A polypeptide comprising a fragment having cellulolytic activity and being selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence having at least 66% identity to SEQ ID NO:4, 79% identity to SEQ ID NO:6, 78% identity to SEQ ID NO:12, 68% identity to SEQ ID NO: 14, 72% identity to SEQ ID NO: 16, 68% identity to SEQ ID NO:20, 74% identity to SEQ ID NOS:22 or 24, or 78% identity to SEQ ID NO:26;   b) a variant of a) comprising a fragment having cellulolytic activity; and   c) a fragment of a) or b) having cellulolytic activity.   
     
     
         36 . (canceled) 
     
     
         37 . An isolated polynucleotide selected from the group consisting of:
 a) a nucleotide sequence of SEQ ID NOS: 3, 5, 11, 13, 15, 19, 21, 23 or 25, or a sequence encoding a polypeptide of  claim 35 ;   b) a complementary strand of a)   c) a fragment of a) or b) comprising at least 20 nucleotides; and   d) a sequence that is degenerate as a result of the genetic code to any one of the sequences as defined in a), b) or c).   
     
     
         38 - 39 . (canceled) 
     
     
         40 . A vector, which comprises as a heterologous sequence a polynucleotide of  claim 37 . 
     
     
         41 . The vector of  claim 40 , which is capable of expressing a polypeptide comprising a fragment having cellulolytic activity and being selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence having at least 66% identity to SEQ ID NO:4, 79% identity to SEQ ID NO:6, 78% identity to SEQ ID NO:12, 68% identity to SEQ ID NO:14, 72% identity to SEQ ID NO:16, 68% identity to SEQ ID NO:20, 74% identity to SEQ ID NO:22 or 24, or 78% identity to SEQ ID NO:26;   b) a variant of a) comprising a fragment having cellulolytic activity; and   c) a fragment of a) or b) having cellulolytic activity.   
     
     
         42 . A host cell comprising the vector of  claim 40 . 
     
     
         43 - 44 . (canceled) 
     
     
         45 . An  Escherichia coli  strain having accession number DSM 16728, DSM 16729, DSM 17324, DSM 17323, DSM 17729, DSM 16726, DSM 16725, DSM 17325 or DSM 17667. 
     
     
         46 . An enzyme preparation comprising a polypeptide of  claim 35 . 
     
     
         47 - 49 . (canceled) 
     
     
         50 . Use of a polypeptide according to  claim 35  or an enzyme preparation comprising a polypeptide of  claim 35  in an industry selected from the group consisting of fuel industry, textile industry, detergent industry, pulp and paper industry, food industry, feed industry and beverage industry. 
     
     
         51 . (canceled) 
     
     
         52 . The use according to  claim 50 , wherein the enzyme preparation is spent culture medium. 
     
     
         53 . A method for preparing a polypeptide comprising a fragment having cellulolytic activity and being selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence having at least 66% identity to SEQ ID NO:4, 79% identity to SEQ ID NO:6, 78% identity to SEQ ID NO:12, 68% identity to SEQ ID NO:14, 72% identity to SEQ ID NO:16, 68% identity to SEQ ID NO:20, 74% identity to SEQ ID NO:22 or 24, or 78% identity to SEQ ID NO:26;   b) a variant of a) comprising a fragment having cellulolytic activity; and   c) a fragment of a) or b) having cellulolytic activity,   said method comprising transforming a host cell with a vector encoding said polypeptide, and culturing said host cell under conditions enabling expression of said polypeptide, and optionally recovering and purifying the polypeptide produced.   
     
     
         54 . A method of treating cellulosic material with a spent culture medium of at least one microorganism capable of producing a polypeptide comprising a fragment having cellulolytic activity and being selected from the group consisting of:
 a) a polypeptide comprising an amino acid sequence having at least 66% identity to SEQ ID NO:4, 79% identity to SEQ ID NO:6, 78% identity to SEQ ID NO:12, 68% identity to SEQ ID NO:14, 72% identity to SEQ ID NO:16, 68% identity to SEQ ID NO:20, 74% identity to SEQ ID NO:22 or 24, or 78% identity to SEQ ID NO:26;   b) a variant of a) comprising a fragment having cellulolytic activity; and   c) a fragment of a) or b) having cellulolytic activity,   
       said method comprising reacting the cellulosic material with the spent culture medium to obtain hydrolysed cellulosic material.