扩展青霉脂肪酶K56R叠加突变对热稳定性的影响

K56R Double-Mutagenesis Effected Thermostability of Penicillium expansum Lipase

目的:扩展青霉脂肪酶随机突变体ep8是一株热稳定性比野生型有所提高的突变体。获得热稳定性提高的优良菌株。方法:在ep8的基础上利用重叠延伸PCR构建叠加突变重组质粒pPIC3.5K-ep8-K56R,将该质粒电转毕赤酵母(Pichia pastoris)GS115进行异源表达。结果:该叠加突变脂肪酶在毕赤酵母中获得了活性表达。15%SDS-PAGE结果分析表明突变脂肪酶PEL-ep8-K56R-GS分子量与野生型PEL-GS一致,约为28kDa。叠加突变脂肪酶在37℃时酶活为852U/mL、野生型为760u/mL、随机突变体为824u/mL,叠加突变体酶活相比野生型提高了21.1%,相比随机突变体提高了3.4%。热稳定性分析数据表明叠加突变脂肪酶Tm值为40.1℃、野生型为38.7℃、随机突变体为39.9℃,Tm值相比野生型提高了1.4℃,相比随机突变体提高了0.2℃。

Objective：A random mutant named ep8, which contained a single amino acid substitution, was obtained by using the lipase gene from Penicillium expansum （lip07） as an error-prone PCR template in previous study. The ep8 has higher thermostability than lip07. Method：Depend on it, a recombinant vetor pPIC3.5K-ep8-K56R which contain double mutant genes was constructed by overlap extension PCR using the cDNA of ep8 and two primers contained mutant K56R as the template. Result：The recombinant vetor was transformed into Pichia pastoris GS115 by electroporation for extracelluar expression. 15% SDS-PAGE analysis indicated that the molecular mass of double-mutant （PEL-ep8-K56R-GS） is 28kDa, the same as the wild-type （lip07） and random-mutant （ep8）. The yield of the double-mutant lipase 852U/mL, which is 21.1% higher than that of the wild-type lipase （760u/mL） and 3.4% higher than that of random-mutant lipase （824u/mL） at their optimum temperature. The Tm of double-mutant lipase is 40.1℃, 1.4℃ higher than that of wild-type lipase （38.7℃） and 0.2℃ higher than that of random-mutant lipase （39.9℃）.