Cloning And Expression Of Lipase Gene From Candida Rugosa

In recent years, the industrialized application of lipases has attracted an increasing attention, it is becoming a hot spot in biotechnological field to get more and more the versatile lipases and their gene resources. C. rugosa (formerly C. cylindracea) lipase (CRL) is an important industrial enzyme capable of catalyzing lipolytic, transesterification, esterification reaction, has been applied in many fields such as oil processing, food, pharmaceuticals, and refined chemical manufacture. The CRL family includes several lipase isozymes, and they have different properties from each other, some of them have been successfully expressed in heterologous hosts. The lipase exhibits a good prospect in industrialized application.In this paper, six lipase genes (lip1-lip5 and lipJ08) were amplified from the total genomic DNA of the C. rugosa ATCC 14830, among which the full sequence of lipJ08 was reported for the first time. Moreover, lipJ08 gene was functionally expressed in Pichia pastoris GS115 system after substituting 17 CTGser triple codons with TCTser universal codons. The main work was as follows:(1) By means of bioinformatics, we aligned nucleotide sequences of reported lipase isoforms from C. rugosa. Primers were designed based on the conservative nucleotide sequences, and six lipase genes (lip1-lip5 and lipJ08) of C.rugosa ATCC 14830 were cloned. The nucleotide sequencing revealed that the lipJ08 gene with an ORF of 1650 bp without any intron, was a novel gene of CRL isoform, encoding a protein of 534 amino acid residues, including a potential signal sequence of 15 amino acid residues. The deduced amino acid sequence of the lipase showed an overall identity of 66 % to lip1-lip5 from C.rugosa. The nucleotide sequences of the other 5 isoforms of lipase genes cloned from C. rugosa ATCC 14830 were identical to CRL isoforms, lip1-lip5, as previously reported, respectively.(2) The deviations from the universal genetic code for the asporogenic yeast C.rugosa, in which the universal codon for leucine, CUG, is used to code for serine, including the catalytic Ser-209, has hindered the use of recombinant CRL isoenzymes (isoforms). The conversion of most or all CTG codons is required for the expression of functional lipase proteins in heterologous system. To overcome the difficulty, in this work, 17 of the non-nuniversal serine codons (CTG) of lipJ08 were converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis, and the optimized gene was termed as MlipJ08.(3) The native and codon-optimized MlipJ08 genes were subcloned into pPIC9K vector, and expressed in P.pastoris GS115, respectively. SDS-PAGE analysis of LipJ08 produced by P. pastoris harboring the plasmid pPIC9K-lipJ08 or pPIC9K-MlipJ08 respectively showed a single protein band about 60 kDa. The assays of emzyme characteristics demonstrated that the hydrolysis active of MLIPJ08 toward olive oil from the supernatant was 4.65 U/mL, whereas the native lipase LIPJ08 has no hydrolysis activity after methanol induction for 120 hours in shake flasks. Furthermore, the observation of the MLIPJ08 hydrolyzing pNp esters with variety of carbon chain length showed that it exhibited a preference for both long-chain (C16 acyl group) and short-chain (C4 acyl group) pNp esters to medium-chain cholesteryl esters (C8, C10 and C12 acyl groups), and its optimal reaction temperature and pH were 37℃and pH 7.0, respectively.