E83V对扩展青霉脂肪酶最适作用温度的影响  

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-directed Mutagenesis

作　　者：陈国仁[1] 林琳[1] 

机构地区：[1]福建师范大学生物工程学院,福州350007

出　　处：《微生物学通报》2005年第1期85-89,共5页Microbiology China

基　　金：国家自然科学基金资助项目(No.30270033);福建省自然科学基金重点项目资助(No.B0120001)~~

摘　　要：利用重叠延伸PCR法对扩展青霉碱性脂肪酶(PEL)基因作了体外定点突变,获得了最适作用温度有所提高的突变体。含突变基因的重组质粒pPIC3.5K-lip-E83V在PichiapastorisGS115中表达。对突变体表达产物PEL-E83V-GS与野生型表达产物PELGS作了比较:前者最适作用温度为45℃,比野生型提高了5℃;其热稳定性基本不变;突变体在37℃下的表达量为188U/mL,约为野生型的80%。最适作用温度的提高可能是由于83位亲水性的Glu用疏水性的Val取代,增加了脂肪酶表面的疏水作用,使其在高温下更适于与底物的结合。In order to improve the optimum temperature of lipases, The Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique. The recombinant plasmid pPIC3.5K-lip-E83V containing matant gene was expressed in Pichia pastoris GS115. The comparison experiments of the mutant PEL-E83V-GS with the wild-type PEL-GS showed that: the optimum temperature of the mutant (45℃) was higher by 5℃ than that of the wild type. The thermostability of the mutant was similar to the wild type. The enzymatic activity of the mutant was 188 U/mL at 37 ℃, which was 80% that of the wild type at the same conditions. The hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid, Glu substituted with the hydrophobic amino acid, Val, and be responsible for the improvement of optimum temperature.

关 键 词：扩展青霉碱性脂肪酶 定点突变 最适作用温度 

分 类 号：Q814[生物学—生物工程]