Enzymes that cleave non-glycosidic ether bonds between lignins or derivatives thereof and saccharides

Abstract

The patent application relates to isolated polypeptides that specifically cleave non-glycosidic ether bonds between lignins or derivatives thereof and saccharides, and to cDNAs encoding the polypeptides. The patent application also relates to nucleic acid constructs, expression vectors and host cells comprising the cDNAs, as well as methods of producing and using the isolated polypeptides for treating pulp and biomass to increase soluble saccharide yield and enrich lignin fractions.

What is claimed is: 
     
         1 . An isolated polypeptide that specifically cleaves a non-glycosidic ether bond between a lignin or a derivative thereof and a saccharide, wherein said polypeptide is encoded by a cDNA that specifically cleaves a non-glycosidic ether bond between a lignin or a derivative thereof and a saccharide. 
     
     
         2 . The isolated polypeptide of  claim 1 , wherein said polypeptide is selected from the group consisting of:
 (a) a polypeptide having at least about 80% sequence identity to the mature polypeptide of SEQ ID NO:2;   (b) a polypeptide having at least about 90-95% sequence identity to the mature polypeptide of SEQ ID NO:2;   (c) a polypeptide encoded by a polynucleotide that hybridizes under medium to high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1 or (ii) the full length complement of (i);   (d) a polypeptide encoded by a polynucleotide having at least about 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:1;   (e) a polypeptide encoded by a polynucleotide having at least about 90-95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:1;   (f) a variant of the mature polypeptide of SEQ ID NO:2 comprising a substitution, deletion and/or insertion at one or several positions; and   (g) a fragment of the polypeptide of (a), (b), (c) (d) or (e) that specifically cleaves a non-glycosidic ether bond between a lignin or a derivative thereof and a saccharide.   
     
     
         3 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide comprises a catalytic domain that is selected from the group consisting of:
 (a) a catalytic domain having at least about 80% sequence identity to the amino acids of SEQ ID NO:4;   (b) a catalytic domain having at least about 90-95% sequence identity to the amino acids of SEQ ID NO:4;   (c) a catalytic domain encoded by a polynucleotide that hybridizes under medium to high stringency conditions with (i) the nucleotide sequence of SEQ ID NO:3 or (ii) the full length complement of (i);   (d) a catalytic domain encoded by a polynucleotide having at least about 80% sequence identity to the nucleotide sequence of SEQ ID NO:3; and   (e) a catalytic domain encoded by a polynucleotide having at least about 90-95% sequence identity to the nucleotide sequence of SEQ ID NO:3.   
     
     
         4 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide is mannan:lignin etherase or xylan:lignin etherase. 
     
     
         5 . The isolated polypeptide of  claim 1 , wherein cleavage of the non-glycosidic ether bond is between an aromatic carbon of the lignin or the derivative thereof and the saccharide. 
     
     
         6 . The isolated polypeptide of  claim 1 , wherein cleavage of the non-glycosidic ether bond is between a non-aromatic carbon of the lignin or the derivative thereof and the saccharide. 
     
     
         7 . The isolated polypeptide of  claim 6 , wherein said non-aromatic carbon of the lignin is an α-linked benzyl carbon or a β-linked benzyl carbon. 
     
     
         8 . The isolated polypeptide of  claim 1 , wherein said saccharide is selected from the group consisting of a monosaccharide, a disaccharide, an oligosaccharide, and a polysaccharide. 
     
     
         9 . The isolated polypeptide of  claim 8 , wherein said polysaccharide is hemicellulose. 
     
     
         10 . The isolated polypeptide of  claim 1 , wherein said polypeptide is selected from the group consisting of:
 (a) a polypeptide having at least about 85% sequence identity to the mature polypeptide of SEQ ID NO:50;   (b) a polypeptide having at least about 90-95% sequence identity to the mature polypeptide of SEQ ID NO:50;   (c) a polypeptide encoded by a polynucleotide that hybridizes under medium to high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO:49 or (ii) the full length complement of (i);   (d) a polypeptide encoded by a polynucleotide having at least about 85% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:49;   (e) a polypeptide encoded by a polynucleotide having at least about 90-95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:49;   (f) a variant of the mature polypeptide of SEQ ID NO:50 comprising a substitution, deletion and/or insertion at one or several positions; and   (g) a fragment of the polypeptide of (a), (b), (c) (d) or (e) that specifically cleaves a non-glycosidic ether bond between a lignin or a derivative thereof and a saccharide.