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Cloning and Expression of Lipase Gene from Candida Rugosa

Author: JiangXueQing
Tutor: ZuoYunJun；XuLi
School: Huazhong University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Candida folds Lipase Clone lipJ08 Expression
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 91
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Abstract


In recent years, the industrialization of lipase increasingly wide application field for more lipase with different characteristics and their genetic resources, and to achieve large-scale production, a biotechnology research and development focus. Folds Candida (Candida rugosa, formerly Candida cylindracea) lipase (CRL) is a lipase varieties of the more important, can catalyze lipid hydrolysis, transesterification, ester synthesis reactions, are widely used in oil processing, food, pharmaceutical, cosmetic and other industrial areas, which contain multiple isozymes, specific nature, and can be implemented in a heterologous host a degree of expression, secretion, and has good prospects for development. This paper folds Candida (ATCC 14830) strain of departure, the total genomic DNA as template was amplified six lipase gene (lip1-lip5 and lipJ08). Among them, the gene lipJ08 reported for the first full-length sequence. At the same time, by replacing the serine 17 codon unconventional realized lipJ08 Pichia pastoris gene (GS115) in heterologous expression. Main tasks are as follows: (a) by means of bioinformatics, the folds have been cloned Candida lipase full-length gene sequence homology comparison, primers were designed based on conserved sequences in the genomic DNA level, from the folds of Candida yeast (ATCC 14830) strain was cloned six lipase gene (lip1-lip5 and lipJ08), which lip1-lip5 sequence folds reported Candida lipase isozymes same, lipJ08 gene folds Candida lipase new isozyme genes. Sequence analysis showed that, lipJ08 gene length of 1650 bp, encoding 549 amino acids including a 15 amino acid residues containing the signal peptide and mature lipase from the 534 amino acids. (2) As C.rugosa lipase gene exists in non-universal codon CTG, encoding Ser, while in Pichia pastoris is encoded Leu. Therefore, so as to Ser active center folds lipase Candida lipase obtained when heterologous expression is the expression product of no catalytic activity. By overlap extension PCR (Over-lap extension PCR) technique lipJ08 in 17 sites were mutated CTG transformation into Pichia encoded at Ser of TCT, the mutant gene named MlipJ08. (3) the lipJ08 and MlipJ08 were subcloned into the expression vector pPIC9K in successfully constructed expression plasmid pPIC9K-lipJ08 and pPIC9K-MlipJ08, expression vectors and transduced into electrical Pichia pastoris (GS115), the success obtained secretion expression. SDS-PAGE showed that the expression of LIPJ08 and MLIPJ08 the molecular size of approximately 60 kDa. Enzymatic properties demonstrated by the codon modified MLIPJ08 obtained recombinant lipase activity reached for the hydrolysis of olive oil 4.65 U / mL, while LIPJ08 vitality barely detectable; MLIPJ08 the optimum temperature is 37 ℃, the optimum pH was 7.0, the long carbon chain (C16) and short chain (C4) pNp ester hydrolysis activity is higher than the medium chain C8, C10, and C12 pNp esters.

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