K202A突变对扩展青霉脂肪酶热稳定性的影响  

Effect of K202A Mutation in the Thermostability of Penicillum expansum Lipase

作　　者：邹有土[1] 吴义真[1] 施文芳[1] 林琳[1] 

机构地区：[1]福建师范大学生命科学学院,福州350108

出　　处：《中国生物工程杂志》2007年第12期52-56,共5页China Biotechnology

基　　金：国家自然科学基金资助项目(30270033);福建省自然科学基金资助项目(B0120001;C0410009)

摘　　要：利用易错PCR定向进化扩展青霉脂肪酶(PEL),获得了一株热稳定性有所提高的随机突变体(ep8),ep8包含有一个氨基酸的改变。为进一步提高其热稳定性,利用重叠延伸PCR法,以ep8基因为模板,将第202位赖氨酸突变为丙氨酸(K202A),构建表达质粒pAO815-ep8-K202A。并将其引入毕赤酵母GS115构建叠加突变体(PEL-ep8-K202A)。同时以野生型lip07为模板构建单点突变体:PEL-lip07-K202A。15%SDS-PAGE结果分析表明突变体分子量与野生型一致,约为28kDa。表达产物热稳定性分析结果表明:野生型(PEL)的Tm值为39.03℃,而以野生型为模板进行定点突变得到的单点突变酶(PEL-lip07-K202A),其Tm却降低了2℃,为37.08℃。叠加突变酶(PEL-ep8-K202A)的Tm为41.66℃,比野生型酶提高2.63℃,比随机突变体ep8生产的酶(PEL-ep8)的Tm提高了1.21℃。Lipase gene from PeniciUium expansum （lip07） was cloned and over-expressed in Pichia pastoris, a random mutant named ep8, which contained a single amino acid substitution, was obtained by using the lip07 as an error-prone PCR template in previous study, ep8 shows higher thermostability than that of lip07, To further improve the thermostability of the lipase, the Lys of wild-type （lip07） and mutant （ep8） in 202 were substituted by Ala using the Overlap extension PCR technique respectively. The mutant genes （lip07-K202A and ep8-K202A） were subcloned into pAO815, and then transformed into the Pichia pastoris GSll5 for extracelluar expression, respectively. 15% SDS-PAGE analysis indicated that the molecular mass of PEL-ep8-K202A and PEL -lip07-K202A are both about 28kDa, which is same with the wild-type lipase. The Tm of PEL-ep8-K202A is 41.66℃ ,2.63℃ higher than that of the wild-type （39.03℃） and 1.21℃ higher than the random mutant（ PEL -ep8：40.45℃ ） ; the Tm of single mutant （PEL-lip07-K202A） is 37.08℃ ,2℃ lower than that of the wild-type lipase.

关 键 词：扩展青霉脂肪酶 定点突变热稳定性 

分 类 号：Q78[生物学—分子生物学]