定点突变对扩展青霉脂肪酶热稳定性的改善  

Improvement of Thermostability of Penicillium expansum Lipase by Site-directed Mutagenesis

作　　者：蔡少丽[1,2,3] 邹有土[1,2,3] 黄建忠[1,2,3] 林琳[1,2,3] 

机构地区：[1]福建师范大学工业微生物教育部工程研究中心,福州350108 [2]福建师范大学生命科学学院,福州350108 [3]福建省现代发酵技术工程研究中心,福州350108

出　　处：《应用与环境生物学报》2013年第1期43-47,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(30270 033);福建省自然科学基金项目(B0120001;C0410009);福建工业微生物研发平台建设项目(2006H0085)资助~~

摘　　要：利用重叠延伸PCR方法对扩展青霉脂肪酶基因进行体外定点突变,构建了K115R与随机突变体ep8双突变的重组质粒pAO815-ep8-K115R.将该质粒电转化到毕赤酵母GS115中进行异源表达,获得双突变体脂肪酶PEL-ep8-K115R-GS.与野生型脂肪酶PEL-GS和随机突变体脂肪酶PEL-ep8-GS进行比较,发现双突变体脂肪酶PEL-ep8-K115R-GS在40℃温浴30 min后残余酶活为89%,比野生型脂肪酶PEL-GS和随机突变体脂肪酶PEL-ep8-GS分别提高54%和27%;双突变体脂肪酶PEL-ep8-K115R-GS的Tm值为42.2℃,比野生型脂肪酶PEL-GS提高3.5℃,比随机突变脂肪酶PEL-ep8-GS提高2.0℃.研究结果表明对扩展青霉脂肪酶的分子改造能提高其热稳定性.In order to improve the thermostability ofPenicillium expansum lipase （PEL）, the lipase gene was mutated by site-directed mutagenesis. A recombinant plasmid pAO815-ep8-K115R which contains double mutant genes was constructed by overlap extension PCR using the eDNA of a random-mutant lipase ep8 （a single site mutant） as the template and two special primers were used to generate another mutation site Kll5R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8-K115R can secret the double-mutant lipase PEL-ep8- Kll5R-GS into the medium when it was induced by methanol. Thermostability analysis revealed that the residual activity of the double-mutant lipase PEL-epS-K115R-GS after incubated at 40 ~C for 30 rain was 54% and 27% higher than that of the wild type lipase PEL-GS and the random-mutant lipase PEL-ep8-GS respectively. Tm of the double-mutant lipase PEL-ep8-K115R- GS was 42.2 ℃, 3.5℃ higher than that of the wild type lipase PEL-GS, and 2.0 ℃ higher than that of the random-mutant lipase PEL-ep8-GS. Fig 5, Ref20

关 键 词：扩展青霉 脂肪酶 定点突变 毕赤酵母 热稳定性 

分 类 号：TQ925[轻工技术与工程—发酵工程]