Laccase variants

Abstract

The present invention relates to variants of a parent laccase. The present invention also relates to polynucleotides encoding the variant laccases and to nucleic acid constructs, vectors, and host cells comprising the polynucleotides, and methods of using the variant enzymes.

1 . A method for enzymatic oxidation of a lignin or a lignin containing material, comprising contacting the lignin or a lignin containing material with a variant of a parent laccase, comprising a substitution at one or more positions corresponding to positions M168, E198, Y199, Y200, S262, M266, H74, A120, G121, Y122, W123, H124, D212, N213, R214, D223, P224, S225, R226, V227, I228, D229, N230, K231, I232, T233 A236, D237, S238, F239, S43, S81, S82, S101, M33, M65, M87, M255, T8, T11, T56, T62, T64, T97, T99, T101, T114, T132, T136, T160, T162, T169, T177, T180, T184, T215, T219, A29P, K39P, D111P, K152P, TBP, A9P, I80P, A86P, M87P, I232P, M255P, V260P, T56P, H58P, K106D, K106E, K152D, K152E, D30E, H176R, G96S, G54S, G54T, L143S, L143T, K41N, R68N, E79N, S262N, G54N, E60N, T62N, G95N, T97N, T99N, D158N, E182N, I217N, and D278N of the mature polypeptide of SEQ ID NO: 2; wherein the variant has laccase activity and the parent laccase is:
 (a) a polypeptide comprising an amino acid sequence having at least 70% identity to the mature polypeptide of SEQ ID NO:2 or to amino acids 9 to 293 of SEQ ID NO: 2; or   (b) a polypeptide encoded by a polynucleotide that hybridizes under medium stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 1, or its complementary strand.   
     
     
         2 . The method of  claim 1 , wherein the method is a method for bleaching of lignin-containing material. 
     
     
         3 . The method of  claim 1 , wherein the method is a method for bleaching of pulp for paper production. 
     
     
         4 . The method of  claim 1 , wherein the method is lignin modification in manufacture of wood composites. 
     
     
         5 . The method of  claim 4 , wherein the wood composites can be selected from the group consisting of wood fiber material, chipboards, fibre boards or particle boards. 
     
     
         6 . The method of  claim 1 , wherein the lignin or a lignin containing material is contacted with the polypeptide at pH 8-12. 
     
     
         7 . The method of  claim 1 , wherein the parent laccase is (a) a polypeptide comprising an amino acid sequence having at least 75% identity, preferably at least 80% identity, more preferably at least 85% identity, most preferably at least 90% identity, and even most preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2 or to amino acids 9 to 293 of SEQ ID NO: 2; or (b) a polypeptide encoded by a polynucleotide that hybridizes under medium-high stringency conditions, and preferably high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 1, or its complementary strand. 
     
     
         8 . The method of  claim 1 , wherein the parent laccase comprises or consists of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:8. 
     
     
         9 . The method of  claim 1 , wherein the variant comprises one or more substitutions selected from the group consisting of G54S, E133A, M168A, M168G, E198S, Y199W, I217A, I217L, P224A, S225N, M255L, S262E, S262Q, E262K, M266A, and M266W. 
     
     
         10 . The method of  claim 1 , wherein the variant comprises one or more substitutions selected from the group consisting of M168G+M266W; M168G+M266A; M168A+E198S+S262E; M168A+E198S+S262Q; M168A+E198S+S262K; M168A+E198S+S262E+Y199W; M168G+S225N; and M168G+Y199W+M266W. 
     
     
         11 . The method of  claim 1 , wherein the variant comprises an amino acid sequence having at least 70% identity, preferably at least 75% identity, more preferably at least 80% identity, even more preferably at least 85% identity, most preferably at least 90% identity, and even most preferably at least 95% identity to the amino acid sequence of the parent laccase. 
     
     
         12 . The method of  claim 1 , wherein the variant has one or more improved properties compared to the parent laccase, wherein the improved properties are selected from the group consisting of thermal activity, thermostability, pH activity, and oxidative stability.