摘要
目的:构建并筛选高效表达洛伐他汀酰基转移酶(Lov D)的毕赤酵母重组菌株。方法:将突变的Lov D基因克隆到毕赤酵母胞外表达质粒p PIC9K和胞内表达质粒p AO815中,将重组表达质粒电转入毕赤酵母GS115中,得到毕赤酵母重组菌株,通过摇瓶发酵筛选高酶活力的重组菌株;在此基础上,研究重组菌在5L发酵罐中的高密度发酵,并将所得酶液进行辛伐他汀催化反应。结果:p PIC9K-Lov D胞外表达重组菌的酶活是p AO815-Lov D胞内表达重组菌的3倍。筛选到酶活高的p PIC9K-Lov D-3菌株进行5L发酵罐放大实验,经过96 h的甲醇诱导表达,酶活可达609.3 U/L;发酵所得酶液冻干后进行酶功效实验,反应45 h后,其底物转化率可达96%以上。结论:构建的毕赤酵母胞外表达菌株可高效表达洛伐他汀酰基转移酶,培养液上清杂蛋白较少,有利于后续分离和纯化,为洛伐他汀酰基转移酶的工业化生产奠定了基础。
Objective：To express lovastatin acytransferase（LovD） in Pichia pastoris efficiently.Methods：The LovD gene was cloned into the vector pPIC9 K and pAO815 respectively,and transformed into P.pastoris GS115 to construct recombinant P.pastoris stains.Clones with high enzyme activity were screened by shaking flask cultivation.Based on the results of shaking flask cultivation,fermentation was implemented in a 5L bioreactor and the enzyme of fermentation was used to catalyze simvastation synthesis.Results：Enzyme activity of secrtory strains was two-fold higher than that of intracellular recombinant strains in shaking flask fermentation.In the 5L bioreactor,the enzyme activity of selected clone GS115/pPIC9K-LovD-3 achieved 609.3 U/L after 96 h methanol induction.The conversion of substrate was up to 96%after 45 h of reaction in the simvastation synthesis when the freeze-dried enzyme of fermentation was added.Conclusion：This recombinant P.pastoris stain not only can efficiently express lovastatin acyltransferase,but also can benefit subsequent separation and purification because of its less expression of uninterested proteins,which would offer a solid base for large-scale production of LovD.
出处
《生物技术通讯》
CAS
2015年第6期767-771,共5页
Letters in Biotechnology
