摘要
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℃).
出处
《生物技术》
CAS
CSCD
北大核心
2009年第2期23-26,共4页
Biotechnology
基金
福建省科技平台项目(2006H0085)
国家自然科学基金项目(30270033)
福建省自然科学基金项目(No.BOI20001
C0410009)资助
关键词
扩展青霉脂肪酶
叠加突变
热稳定性
Penicillium expansum lipase,double-mutagenesis,thermostability