Cloning, Codon Optimization And Expression Of Lipase Gene Penicillum Expansum

As an important industrial enzyme, lipase is widespread in nature. Many lipase genes have been cloned, and developed into highly effective expressed engineering strains. Penicillium expansum lipase (PEL), an alkaline lipase, showing a preference for triacylglycerols, has been widely used in industrialization. In this study, P.expansum CICC 40356 lipase gene based DNA sequence and cDNA sequence was cloned by PCR and RT-PCR, respectively. And then, codons used in lower frequencies in Pichia pastoris were optimized to achieve high expression level. Based on these, the property of recombinant lipase was preliminary characterized. Major ones are as follows.(1) The alkaline lipase genes were successfully amplified using PCR and RT-PCR methods. Analysis of nucleotide sequence revealed that the complete gene PEL possessed 1140 bp with six exons and five introns, the complete cDNA of 858 bp contained a open reading frame (ORF) encoding a protein of 286 amino acid residues, including a potential signal sequence of 20 amino acid residues. The primary sequence alignment through BLAST revealed 99 % identity to the reported PEL in the GenBank.(2) The heterologous protein expression levels are affected by many factors. Codon bias is regarded as the key factor affecting overexpression in heterologous host. In order to functionally express the recombinant PEL in high level in P. pastoris GS115, 10 amino codons of PEL and 9 amino acid codons of theα-signal peptide of the expression vector pPIC9K were optimized by overlap extension PCR. The native and codon-optimized PEL genes were subcloned into pPIC9K, pPIC9KM, and pPIC3.5K vectors, respectively. The constructed six expression vectors were pPIC9K-PEL1, pPIC9KM-PELM1, pPIC3.5K-PEL1, pPIC3.5K-PELM1 with PEL'own signal sequence, and pPIC9K-PEL2, pPIC9KM-PELM2 without PEL'own signal sequence. Then, they were transformed into P. pastoris GS115 after linearization by Sal I. Following MD and MM plates screening, PCR confirmation, resisting screening on G418-YPD medium plates, and functional screening on YPD-rhodamine B-olive oil medium plates, the recombinant transformants with multiple copy integrated genes were obtained. The best P. pastoris GS115 transformants were induced to express lipase with methanol, and the relative molecular weight of the secreted lipase was approximate 28 kDa determined by SDS-PAGE. After inducing for 100 hours in flasks, the secreted lipase present in the culture supernatant showed hydrolysis activity of 3.65 U/ml, 30.49 U/ml, 90.85 U/ml, 212.05 U/ml, 15.29 U/ml, 76.32 U/ml toward PNPP, respectively.(3) The recombinant lipase was also characterized. The results showed that the optimal temperature and pH of the lipase were 35℃and 9.5, respectively, and it was fairly stable in the range of pH 7.0-10.0. It had a maximal activity for medium chain ester (C₈), and showed relatively higher hydrolysis preference for medium chain esters (C₈-C₁₂). The enzyme activity was strongly stimulated in the presence of Ca²⁺ and Mg²⁺, while significantly inhibited by EDTA, and slightly inhibited by Fe²⁺, Zn²⁺, Cu²⁺.