Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme

Shu Wei Chang, Guan Chiun Lee, Jei Fu Shaw*

*此作品的通信作者

研究成果: 雜誌貢獻期刊論文同行評審

35 引文 斯高帕斯(Scopus)

摘要

Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip1 to lip7). In this study, an additional N-terminal peptide in front of the lip3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip3 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 pGAPZαC vector. The results show that the production yield (0.687 unit/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip3 and of 52-fold (0.47 unit/mL) of codon-optimized lip3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip3 (lip3), 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 lip3 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.

原文英語
頁(從 - 到)5831-5838
頁數8
期刊Journal of Agricultural and Food Chemistry
54
發行號16
DOIs
出版狀態已發佈 - 2006 8月 9
對外發佈

ASJC Scopus subject areas

  • 一般化學
  • 一般農業與生物科學

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