Lipolytic enzyme genes

Abstract

The inventors have isolated novel genes with a high homology to the T. lanuginosus lipase gene and are thus well suited for use in gene shuffling. Accordingly, the invention provides a method of generating genetic diversity into lipolytic enzymes by family shuffling of two or more homologous genes which encode lipolytic enzymes. The DNA shuffling technique is used to create a library of shuffled genes, and this is expressed in a suitable expression system and the expressed proteins are screened for lipolytic enzyme activity. The expressed proteins may further be screened to identify lipolytic enzymes with improved properties. The invention also provides a polynucleotide comprising a nucleotide sequence encoding a lipolytic enzyme and a lipolytic enzyme (a polypeptide with lipolytic enzyme activity).

1 . A method of producing a lipolytic enzyme which comprises: 
 a) shuffling at least two polynucleotides which comprise: 
 i) a polynucleotide encoding a polypeptide which has lipolytic enzyme activity and has an amino acid sequence having at least 90% identity with the mature peptide of SEQ ID NO: 2, and  
 ii) a polynucleotide encoding a polypeptide which has lipolytic enzyme activity and has an amino acid sequence having 55-90% identity with the mature peptide of SEQ ID NO: 2  
   b) expressing the shuffled polynucleotides to form recombinant polypeptides,    c) screening the polypeptides to select a polypeptide having lipolytic enzyme activity, and    d) producing the selected polypeptide.    
     
     
         2 . The method of  claim 1  wherein the amino acid sequence encoded by polynucleotide (ii) has at least 90% identity to the mature part of SEQ ID NO: 4, 6, 8 or 10.  
     
     
         3 . The method of  claim 1  or  2  wherein the polynucleotides comprise a polynucleotide having a nucleotide sequence having at least 90% identity to the mature part of SEQ ID NO: 1, 3, 5, 7 or 9.  
     
     
         4 . A polynucleotide which comprises a nucleotide sequence which encodes a lipolytic enzyme and which: 
 a) is a DNA sequence cloned into a plasmid present in  Escherichia coli  deposit number DSM 14047, 14048,14049, or 14051, or    b) is the DNA sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9 or can be derived therefrom by substitution, deletion, and/or insertion of one or more nucleotides, or    c) has at least 90% identity with the DNA sequence encoding a mature peptide shown in SEQ ID NO: 3, at least 80% identity with the DNA sequence encoding a mature peptide shown in SEQ ID NO: 5, at least 65% identity with the DNA sequence encoding a mature peptide shown in SEQ ID NO: 7 or at least 60% identity with the DNA sequence encoding a mature peptide shown in SEQ ID NO: 9, or    d) is an allelic variant of the DNA sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9; or    e) hybridizes under high stringency conditions with a complementary strand of the nucleic acid sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9, or a subsequence thereof having at least 100 nucleotides.    
     
     
         5 . The polynucleotide of  claim 4  which further comprises one or more control sequences which are operably linked to said nucleotide sequence and capable of directing the expression of the lipolytic enzyme in a suitable expression host.  
     
     
         6 . A recombinant expression vector comprising the polynucleotide of  claim 5 , a promoter, and transcriptional and translational stop signals.  
     
     
         7 . A recombinant host cell transformed with the polynucleotide of  claim 5  or the vector of  claim 6 .  
     
     
         8 . A method for producing a polypeptide having lipolytic enzyme activity comprising cultivating the host cell of  claim 7  under conditions conducive to production of the polypeptide, and recovering the polypeptide.  
     
     
         9 . A polypeptide which has lipolytic enzyme activity and which: 
 f) is encoded by a DNA sequence cloned into a plasmid present in  Escherichia coli  deposit number DSM 14047 or 14049, or    g) has an amino acid sequence which is the mature peptide of SEQ ID NO: 6 or 10, or can be derived therefrom by substitution, deletion, and/or insertion of one or more amino acids, or    h) has an amino acid sequence which has at least 80% identity with the mature peptide of SEQ ID NO: 6 or at least 60% identity with the mature peptide of SEQ ID NO: 10, or    i) is immunologically reactive with an antibody raised against the mature peptide of SEQ ID NO: 6 or 10 in purified form, or    j) is an allelic variant of the mature peptide of SEQ ID NO: 6 or 10; or    k) is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with a complementary strand of the nucleic acid sequence encoding a mature peptide shown in SEQ ID NO: 5 or 9, or a subsequence thereof having at least 100 nucleotides.    
     
     
         10 . The polypeptide of  claim 9  which is native to a strain of Talaromyces or Thermomyces, particularly  Thermomyces ibadanensis  or  Talaromyces byssochlamydoides.    
     
     
         11 . A nucleic acid sequence comprising a nucleic acid sequence which encodes the polypeptide of  claim 9  or  10 .  
     
     
         12 . A process for hydrolyzing the fatty acyl group in a lysophospholipid, comprising treating the lysophospholipid with a polypeptide which has lysophospholipase activity and which: 
 a) is encoded by a DNA sequence cloned into a plasmid present in  Escherichia coli  deposit number DSM 14047, 14048, 14049 or 14051, or    b) has an amino acid sequence which is the mature peptide of SEQ ID NO: 4, 6, 8 or 10, or can be derived therefrom by substitution, deletion, and/or insertion of one or more amino acids, or    c) has an amino acid sequence which has at least 55% identity with the mature peptide of SEQ ID NO: 4, 6, 8 or 10, or    d) is immunologically reactive with an antibody raised against the mature peptide of SEQ ID NO: 4, 6, 8 or 10 in purified form, or    e) is an allelic variant of the mature peptide of SEQ ID NO: 4, 6, 8 or 10; or    f) is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with a complementary strand of the nucleic acid sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9, or a subsequence thereof having at least 100 nucleotides.    
     
     
         13 . A process according to  claim 12  for improving the filterability of an aqueous solution or slurry of carbohydrate origin which contains lysophospholipid.  
     
     
         14 . The process of the preceding claim wherein the solution or slurry contains a starch hydrolysate, particularly a wheat starch hydrolysate.  
     
     
         15 . A detergent composition comprising a surfactant and a polypeptide which has lipolytic enzyme activity and which: 
 a) is encoded by a DNA sequence cloned into a plasmid present in  Escherichia coli  deposit number DSM 14047, 14048, 14049 or 14051, or    b) has an amino acid sequence which is the mature peptide of SEQ ID NO: 4, 6, 8 or 10, or can be derived therefrom by substitution, deletion, and/or insertion of one or more amino acids, or    c) has an amino acid sequence which has at least 55% identity with the mature peptide of SEQ ID NO: 4, 6, 8 or 10, or    d) is immunologically reactive with an antibody raised against the mature peptide of SEQ ID NO: 4, 6, 8 or 10 in purified form, or    e) is an allelic variant of the mature peptide of SEQ ID NO: 4, 6, 8 or 10; or    f) is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with a complementary strand of the nucleic acid sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9, or a subsequence thereof having at least 100 nucleotides.    
     
     
         16 . A flour composition comprising flour and a polypeptide which has lipolytic enzyme activity and which: 
 a) is encoded by a DNA sequence cloned into a plasmid present in  Escherichia coli  deposit number DSM 14047, 14048, 14049 or 14051, or    b) has an amino acid sequence which is the mature peptide of SEQ ID NO: 4, 6, 8 or 10, or can be derived therefrom by substitution, deletion, and/or insertion of one or more amino acids, or    c) has an amino acid sequence which has at least 55% identity with the mature peptide of SEQ ID NO: 4, 6, 8 or 10, or    d) is immunologically reactive with an antibody raised against the mature peptide of SEQ ID NO: 4, 6, 8 or 10 in purified form, or    e) is an allelic variant of the mature peptide of SEQ ID NO: 4, 6, 8 or 10; or    f) is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with a complementary strand of the nucleic acid sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9, or a subsequence thereof having at least 100 nucleotides.    
     
     
         17 . A process for producing a dough or a baked product made from dough, comprising adding to the dough a polypeptide which has lipolytic enzyme activity and which: 
 g) is encoded by a DNA sequence cloned into a plasmid present in  Escherichia coli  deposit number DSM 14047, 14048, 14049 or 14051, or    h) has an amino acid sequence which is the mature peptide of SEQ ID NO: 4, 6, 8 or 10, or can be derived therefrom by substitution, deletion, and/or insertion of one or more amino acids, or    i) has an amino acid sequence which has at least 55% identity with the mature peptide of SEQ ID NO: 4, 6, 8 or 10, or    j) is immunologically reactive with an antibody raised against the mature peptide of SEQ ID NO: 4, 6, 8 or 10 in purified form, or    k) is an allelic variant of the mature peptide of SEQ ID NO: 4, 6, 8 or 10; or    l) is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with a complementary strand of the nucleic acid sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9, or a subsequence thereof having at least 100 nucleotides.