P197E与ep8叠加突变对扩展青霉脂肪酶热稳定性的影响

为提高脂肪酶的热稳定性,作者利用重叠延伸PCR对扩展青霉脂肪酶(PEL)基因进行了体外定点突变,构建了P197E(即将第197位的脯氨酸突变为谷氨酸)与随机突变体ep8叠加突变的重组质粒pPIC3.5K-ep8-P197E。将该质粒电转化至毕赤酵母Pichiapastoris GS115中,进行异源表达。与野生型酶和单点突变酶PEL-ep8的酶学性质比较,结果表明:叠加突变体PEL-ep8-P197E在40°C温育处理30min后,残余酶活分别比野生型PEL和随机突变体PEL-ep8提高了42.13%和37.3%。叠加突变体PEL-ep8-P197E的Tm值为41.51°C,比野生型酶PEL提高了2.81°C,比随机突变体脂肪酶PEL-ep8提高了2.25°C。通过对脂肪酶PEL的叠加突变,提高了该酶的热稳定性,并为结构与功能的进一步研究提供了材料。

Improving of the Thermostability of Penicillium expansum Lipase by Creating Mutation P197E

In order to improve the thermostability of Penicillium expansum lipase (PEL), a mutation P197E was created by site-directed mutagenesis. The mutation is generated by overlap extension PCR using the cDNA of a single site mutant lipase ep8 as the template and two special primers that corresponding to mutation P197E. Recombinant vector pPIC3.5K-ep8-P197E which contain double-mutant genes was constructed and electroporated into Pichia pasteris GS115. The recombinant transformant was selected and grow in the methanol containing media for expressing double-mutant lipase, PEL-ep8-P197E. Thermostability analysis reveals that the residual activity of the double-mutant are 42.13% and 37.3% greater than that of the wild type PEL and PEL-ep8 after incubated at 40°C for 30 min. The Tm of the double-mutant lipase is 41.51°C, 2.81°C higher than PEL and 2.25°C higher than PEL-ep8.