K55R与ep8叠加突变对扩展青霉脂肪酶热稳定性的改善

Improvement of the Thermostability of Penicillium expansum Lipase by Mutagenesis the Random Mutant Ep8 at K55R

利用重叠延伸PCR对扩展青霉脂肪酶(PEL)基因进行体外定点突变,构建了K55R与随机突变体ep8叠加突变的重组质粒pAO815-ep8-K55R。将该质粒电转化引入毕赤酵母(Pichia pastoris)GS115,进行异源表达。实验结果表明:该叠加突变体在毕赤酵母中获得了活性表达,得到表达产物脂肪酶PEL-ep8-K55R-GS。其表达量为508u/mL,分别约为野生型脂肪酶PEL-GS(627u/mL)的81%,随机突变脂肪酶PEL-ep8-GS(924u/mL)的55%;其比活力为2309.1u/mg,与随机突变脂肪酶PEL-ep8-GS和野生型脂肪酶PEL-GS的相仿。叠加突变脂肪酶PEL-ep8-K55R-GS的最适作用温度为37℃,与野生型脂肪酶PEL-GS和随机突变脂肪酶PEL-ep8-GS一致;其Tm值为41.0℃,比野生型脂肪酶PEL-GS提高了2.3℃,比随机突变脂肪酶PEL-ep8-GS提高了0.8℃。表明叠加突变脂肪酶PEL-ep8-K55R-GS的热稳定性有了进一步的提高。

In order to improve the thermostability of the Penicillium expansum Lipase （ PEL）, the lipase encoding genes was mutated by site-directed mutagenesis. A recombinant vector pAOg15-epg-K55R which contain 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 K55R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAOgl5-ep8- K55R can secret double-mutant lipase PEL-epg- K55R-GS into the medium when it was induced by Methanol. The yield of the double-mutant lipase is 508u/mL, which is 81% that of the wild type lipase PEL-GS（627u/mL） and 55 % that of random-mutant PEL-epg-GS（924u/mL）. The specific activity of double-mutant lipase is 2309.1 u/mg, which is similar to random-mutant lipase PEL-epS-GS and the wild type lipase PEL-GS. The optimum temperature of the double-mutant lipase is same with the wild type lipase PEL-GS and random-mutant lipase PEL- epS-GS. While the Tm of the double-mutant lipase is 41.0℃, 2.3℃ higher than the wild type lipase PEL-GS and 0.8% higher than the random-mutant lipase PEL-ep8-GS, indicating that the double-mutant lipase PEL-ep8-K55R-GS has higher thermostability.