利用定点诱变提高膨胀青霉脂肪酶的热稳定性

Q4 Agricultural and Biological Sciences

应用与环境生物学报 Pub Date : 2013-12-13 DOI:10.3724/SP.J.1145.2013.00043

Cai Shaoli, Zou Youtu, Hu Jianzhong, Lin Lin

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引用次数: 0

摘要

为了提高膨胀青霉菌脂肪酶(PEL)的热稳定性，采用定点诱变的方法对脂肪酶基因进行了突变。以随机突变型脂肪酶ep8(单位点突变)cDNA为模板，采用重叠延伸PCR技术构建了含有双突变基因的重组质粒pAO815-ep8-K115R，并利用两种特殊引物构建了另一个突变位点K115R。通过电孔法将重组载体转化为毕赤酵母GS115，重组突变体GS-pAO815-ep8-K115R在甲醇诱导下可将双突变体脂肪酶PEL-ep8K115R-GS分泌到培养基中。热稳定性分析表明，双突变型脂肪酶PEL-ep8-K115R-GS在40℃孵育30 min后的剩余活性分别比野生型和随机突变型脂肪酶PEL-ep8-GS高54%和27%。双突变型脂肪酶PEL-ep8-K115RGS的温度为42.2℃，比野生型脂肪酶PEL-GS高3.5℃，比随机突变型脂肪酶PEL-ep8-GS高2.0℃。

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Improvement of Thermostability of Penicillium expansum Lipase by Site-directed Mutagenesis

In order to improve the thermostability of Penicillium expansum lipase(PEL),the lipase gene was mutated by sitedirected mutagenesis.A recombinant plasmid pAO815-ep8-K115R which contains double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8(a single site mutant) as the template and two special primers were used to generate another mutation site K115R.The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8-K115R can secret the double-mutant lipase PEL-ep8K115R-GS into the medium when it was induced by methanol.Thermostability analysis revealed that the residual activity of the double-mutant lipase PEL-ep8-K115R-GS after incubated at 40 ℃ for 30 min was 54% and 27% higher than that of the wild type lipase PEL-GS and the random-mutant lipase PEL-ep8-GS respectively.Tm of the double-mutant lipase PEL-ep8-K115RGS was 42.2 ℃,3.5 ℃ higher than that of the wild type lipase PEL-GS,and 2.0 ℃ higher than that of the random-mutant lipase PEL-ep8-GS.

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来源期刊

应用与环境生物学报 Agricultural and Biological Sciences-Agronomy and Crop Science

CiteScore

1.20

自引率

0.00%

发文量

4458

期刊介绍： The “Chinese Journal of Applied and Environmental Biology” is a national scientific and technological journal published both at home and abroad. It was started in 1995 and became bimonthly in 1999, and now it is a core journal for applied and environmental biology in China. It aims to introduce and exchange the new achievements, new technologies, new methods and new progresses made from the research fields of biology and its related disciplines both in China and abroad; enhance scientific and technological research serving for the development and construction of national economy, and improvement of people’s living standards.