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|標題:||Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme||作者:||Chang, S.W.
|關鍵字:||Candida rugosa lipase;codon optimization;isoforms;N-terminal peptide;Pichia pastoris;codon optimization;multiple mutagenesis;alcohol oxidase;expression;gene;purification;cylindracea;fermentation;isoenzymes;sequences||Project:||Journal of Agricultural and Food Chemistry||期刊/報告no：:||Journal of Agricultural and Food Chemistry, Volume 54, Issue 16, Page(s) 5831-5838.||摘要:||
Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip 1 to lip 7). In this study, an additional N-terminal peptide in front of the lip 3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip 3 gene were converted into universal serine codons (TCT) by means of an overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP3 in the yeast Pichia pastoris. The regional synthetic DNA fragment (339 bp) is first recombined by primer assembly with 20 overlapping nucleotides, followed by specific overlap extension PCR with outside primers containing restriction enzyme sites for directional cloning into the pGAPZ alpha C vector. The results show that the production yield (0.687 unit/mL) of N-fused lip 3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip 3 and of 52-fold (0.47 unit/mL) of codon-optimized lip 3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip 3 (lip 3), with the cultivation time set at 5 days. This finding demonstrates that the reservation of the N terminus and the regional codon optimization of the lip 3 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The purified recombinant LIP3 shows distinct biochemical properties compared with other isoforms.
|Appears in Collections:||食品暨應用生物科技學系|
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