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，作者利用重叠延伸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28KD. 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℃。
ep8, a thermostabilized mutant of Penicillum expansum lipase obtained in a previous study, contained a single amino acid substitution. To further improve the thermostability of the lipase, the Lys of wild-type (lip07) and mutant (ep8) in 202 were separately substituted by Ala using the Overlap extension PCR technique. The mutant genes (lip07-K202A and ep8-K202A) were subcloned into pAO815, and then transformed into the Pichia pastoris GS115 for extracelluar expression, separately. 15% SDS-PAGE analysis indicated that the molecular mass of PEL-ep8-K202A and PEL-lip07-K202A are both about 28KD, which are just the same with the wild-type lipase. The comparison experiments showed that: 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.
邹有土,吴义真,施文芳,林琳. K202A突变对扩展青霉脂肪酶热稳定性的影响[J]. 中国生物工程杂志, 2007, 27(12): 52-56.
. Effect of K202A mutation in the thermostability of Penicillum expansum Lipase. China Biotechnology, 2007, 27(12): 52-56.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I12/52
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