Patent NZ-597118-A (NZ)
Title: A method for increased expression of a variant lipolytic enzyme
Inventor: MIASNIKOV ANDREI
IPC: A23L5/20
Filed: 2010-06-23
Abstract: 597118 Disclosed is a nucleic acid comprising a nucleotide sequence encoding a lipolytic enzyme with improved expression and/or functionality and/or activity compared with the wild-type enzyme and comprises at least one modification at a position which corresponds in the encoded amino acid sequence to the introduction of at least one glycosylation site in the amino acid sequence wherein each amino acid position corresponds to the position of the amino acid sequence of SEQ ID No.2, wherein when the nucleotide sequence encodes the fungal lipolytic enzyme shown as SEQ ID No. 22 or SEQ ID No. 23 the modification is not a substitution at position 63 and the deletion is not at position 311-312; wherein the nucleotide sequence has at least 90% identity with SEQ ID No.1, with SEQ ID No. 24, or with a nucleotide sequence shown in positions 23-106 of SEQ ID No. 24, or with a nucleotide sequence shown in positions 113-1063 of SEQ ID No. 24 or with a nucleotide sequence shown in positions 113-929 of SEQ ID No. 24. Further disclosed is a variant polypeptide with improved expression and/or functionality and/or activity compared with the wild-type enzyme which has hydrolytic activity towards an ester bond in a polar lipid and comprises an amino acid sequence which has at least 90% identity with amino acids 33-296 of SEQ ID No. 2 and which has been modified compared with the sequence shown in SEQ ID No.2 to introduce at least one glycosylation site in the amino acid sequence, wherein each amino acid position corresponds to the position of the amino acid sequence shown in SEQ ID No.2. Further disclosed are methods of preparing said variant lipolytic enzymes comprising expressing said nucleotides in a host organism. SEQ ID NO: 2 MLLLSLLSAVTLAVASPVALEEYANSLEDRAVGVTSTDFTNFKFYIQHGAAAYCNSGTAAGAKITCSNNGCPTIESNGVTVVASFTGSKTGIGGYVSTDSSRKEIVVAIRGSSNIRNWLTNLDFDQSDCSLVSGCGVHSGFQNAWAEISAQASAAVAKARKANPSFKVVATGHSLGGAVATLSAANLRAAGTPVDIYTYGAPRVGNAALSAFISNQAGGEFRVTHDKDPVPRLPPLIFGYRHTTPEYWLSGGGGDKVDYAISDVKVCEGAANLMCNGGTLGLDIDAHLHYFQATDACNAGGFSWRRYRSAKRESIDMRATMTDAQLEAKLNSYVAMDQEYVKTHQNRT SEQ ID NO: 1 ccgcggactggcatcatgcttcttctatccctcctctcggctgtcacccttgcggtggccagtcctgtagccctcgaagaatacgccaactctcttgaagacagagccgttggagtcacctcaacagacttcaccaacttcaagttctacatccagcatggcgccgcagcatactgcaactccgggaccgcagccggtgcaaagatcacctgttccaacaatggttgcccaacgattgagtccaacggcgtgactgtcgtggcatctttcactggctccaagactggcatcggcgggtacgtctcgacagatagctcccgtaaagaaatcgtcgtcgcgatccgtggtagcagcaacatccgcaactggcttacaaacctcgactttgaccagtccgactgcagtcttgtctctggctgtggtgtgcactctggcttccagaacgcctgggccgagatctcggcgcaagcaagcgctgctgtagcaaaagctcgcaaggcgaacccttccttcaaggtcgtcgccacaggccactccctcggcggcgctgtggccacactgagtgctgcaaaccttcgagctgctggtacacccgtcgacatctacacatatggtgctcctcgagtaggaaacgccgcgctctctgctttcatctcgaaccaggctggcggagaatttcgcgttacgcacgacaaggatcccgtgcctcgtcttccccctctgatcttcggataccgacacacaaccccagagtactggctgtctggcggcggcggcgacaaggttgactacgccatcagcgacgtcaaggtctgtgagggtgctgccaatctcatgtgcaacggtggaactctgggtctggatattgatgctcatctgcactacttccaggcgactgatgcttgcaacgctggtggcttctcttggagacgttataggagcgccaagcgtgagagcatcgacatgagggctaccatgacagacgcacagttggaggccaagctcaactcttatgttgccatggatcaggagtatgtcaagactcaccaaaaccgcacatgaggcgcgcc SEQ ID NO: 24 ttggagaatattccttgtcacgatgcttcttctaccactcctctcggccatcaccctcgcggtagccagtcctgtagctctcgacgactacgtcaactctcttgaggagcgagctgttggtgtcactacaaccgacttcagcaacttcaagttctacatccaacacggcgccgcagcttactgcaactctgaagccgcagctggttccaagatcacctgctccaacaatggctgtccaaccgttcagggcaacggagcgaccatcgtgacatctttcgttggctccaagacaggtatcggtggctacgtcgcgacagactctgcccgaaaggaaatcgtcgtctcgttccgcggaagcatcaatattcgaaactggcttaccaacctcgacttcggccaggaagactgcagtctcgtctctggatgcggtgtgcactctggcttccagcgagcctggaatgagatctcgtctcaagcaaccgctgctgttgcctccgcccgcaaggcgaacccttctttcaacgtcatttctacaggccactcccttggaggtgccgtggccgttcttgctgccgcaaacttgagagtcggtggaacacccgtcgatatttacacctacggctctccccgtgtcggaaacgcgcagctctcagccttcgtctcaaaccaggctggtggagagtaccgcgttacacacgctgatgaccctgtcccccgtctccctcctctgatcttcggatacaggcacacaactcctgagttctggctgtccggcggtggaggcgacaaggttgactacaccatcagcgatgtcaaggtctgtgagggtgctgccaaccttggatgcaacggtggaactcttggtttggatattgctgctcatctgcattacttccaggcgactgacgcctgtaacgctggtggcttctcttggcgacgatacagaagcgccgagagcgtcgacaagagggccaccatgactgatgccgagcttgagaagaagctgaactcttatgtccagatggataaggagtatgtgaagaataaccaggcccgctcttaacgagggtatgaggtttgatgggaaatgacatgattcatgaacgaaaccatagtacatatgatgcaaataggatataaaaacatatttcattcactagctttacacaa