基于易错PCR定向进化扩展青霉 Penicillium expansum FS1884碱性脂肪酶

为改善扩展青霉FS1884碱性脂肪酶的活性及酶学性质,利用连续两轮易错PCR对扩展青霉FS1884脂肪酶基因PEL进行随机突变,在大肠杆菌JM109中构建突变文库。含突变脂肪酶基因的重组质粒电击转化巴斯德毕赤酵母GS115,经过YPOM板初筛和橄榄油检验板复筛,获得一株酶活性提高的脂肪酶突变体:PEL-ep25-GS。与野生型脂肪酶PEL-GS相比,在最适温度40℃、pH9.4时突变体的酶活力是野生型酶的1.3倍。测序结果表明:该突变体第253位氨基酸发生了突变,由赖氨酸变成蛋氨酸。

Directed Evolution of Alkaline Lipase from Penicillium Expansum FS1884 by Error-prone PCR

To improve the enzymes properties and increase its activity, two continuous rounds of error-prone PCR was introduced to Penicillium expansum FS1884 lipase (PEL) for random mutagenesis, which contributes to a library of mutated clones in E.coli JM109. The recombinant plasmid (pPIC3.5K-ep-PEL)harboring the mutated lipase genes were transformed to Pichia pastoris GS115, and screened by YPOM medium plate and olive oil plate, one mutant named as PEL-ep25-GS whose lipase activity has been increased was screened. The mutated lipase showed 1.3 times activity of that of the wild type under the reaction condition of pH9.4 and 40℃. Sequencing result indicated that the lysine in the site of 253 was changed into methionine (Lys253Met) in the mutated lipase when comparing to the wild type one.