Cell-wall degrading enzyme variants

Abstract

The present invention relates to variants of a cell-wall degrading enzyme having a beta-helix structure, which variant has at least one substituent in a position determined by identifying all residues potentially belonging to a stack; characterizing the stack as interior or exterior; characterizing the stack as polar, hydrophobic or aromatic/heteroaromatic based on the dominating characteristics of the parent or wild-type enzyme stack residues and/or its orientation relative to the beta-helix (interior or exterior); optimizing all stack positions of a stack either to hydrophobic aliphatic amino acids, hydrophobic aromatic or polar amino acids by allowing mutations within one or all positions to amino acids belonging to one of these groups; measuring thermostability of the variants by DSC or an application-related assay such as a Pad-Steam application test; and selecting the stabilized variants. Variants of a wild-type parent pectate lyase (EC 4.2.2.2) having the conserved amino acid residues D111, D141 or E141, D145, K165, R194 and R199 when aligned with the pectate lyase comprising the amino acid sequence of SEQ ID NO: 2 are preferred.

1 - 27 . (canceled)  
     
     
         28 . A variant of a parent pectate lyase (EC 4.2.2.2), which variant is encoded by a polynucleotide hybridizing to the polynucleotide sequence of SEQ ID NO:1 under medium stringency conditions.  
     
     
         29 . An enzyme preparation comprising the variant of  claim 28 .  
     
     
         30 . The preparation of  claim 29 , which further comprises one or more enzymes selected from the group consisting of proteases, cellulases (endoglucanases), beta-glucanases, hemicellulases, lipases, peroxidases, laccases, alpha-amylases, glucoamylases, cutinases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, arabinosidases, mannanases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases, other pectate lyases, pectin methylesterases, cellobiohydrolases, transglutaminases; or mixtures thereof.  
     
     
         31 . A detergent composition comprising the variant of  claim 28  and a surfactant.  
     
     
         32 . A method for improving the properties of cellulosic fibers, yarn, woven or non-woven fabric in which method the fibers, yarn or fabric is treated with an effective amount of a variant of  claim 28 .  
     
     
         33 . The method of  claim 32 , wherein the variant is used in a scouring process step.  
     
     
         34 . A method for degradation or modification of plant material in which method the plant material is treated with an effective amount of a variant of  claim 28 .  
     
     
         35 . The method of  claim 34 , wherein the plant material is recycled waste paper, mechanical paper-making pulps or fibers subjected to a retting process.  
     
     
         36 . An isolated polynucleotide molecule encoding a variant of  claim 28 .  
     
     
         37 . An expression vector comprising the following operably linked elements: 
 (a) a transcription promoter, (b) the polynucleotide molecule of  claim 36 , and (c) degenerate nucleotide sequences of (a) or (b); and a transcription terminator.    
     
     
         38 . A host cell into which has been introduced an expression vector of  claim 37 , wherein said cell expresses the polypeptide encoded by the DNA segment.  
     
     
         39 . A method of producing a variant, comprising 
 (a) culturing a host cell of  claim 38  under conditions conducive for expression of the polypeptide; and    (b) recovering the variant.