Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/62052
標題: Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase
作者: Chang, S.W.
蕭介夫
Lee, G.C.
Shaw, J.F.
關鍵字: Candida rugosa lipase
codon optimization
isoforms
Pichia pastoris
multiple mutagenesis
alcohol oxidase
yeast
purification
cylindracea
usage
sequences
cloning
forms
期刊/報告no:: Journal of Agricultural and Food Chemistry, Volume 54, Issue 3, Page(s) 815-822.
摘要: An important industrial enzyme, Candida rugosa lipase (CRL) possesses several different isoforms encoded by the lip gene family (lip1-lip7), in which the recombinant LIP1 is the major form of the CRL multigene family. Previously, 19 of the nonuniversal serine codons (CTG) of the lip1 gene hav been successfully converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP1 in the yeast Pichia pastoris. To improve the expression efficiency of recombinant LIP1 in P. pastoris, a regional synthetic gene fragment of lip1 near the 5' end of a transcript has been constructed to match P. pastoris-pref erred codon usage for simple scale-up fermentation. The present results show that the production level (152 mg/L) of coLIP1 (codon-optimized LIP1) has an overall improvement of 4.6-fold relative to that (33 mg/L) of non-codon-optimized LIP1 with only half the cultivation time of P. pastoris. This finding demonstrates that the regional codon optimization the lip1 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP1 in the P. pastoris system. More distinct biochemical properties of the purified recombinant LIP1 for further industrial applications are also determined and discussed in detail.
URI: http://hdl.handle.net/11455/62052
ISSN: 0021-8561
文章連結: http://dx.doi.org/10.1021/jf052183k
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