D92P点突变对扩展青霉碱性脂肪酶最适作用温度的改善  

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Mutation of D92P

作　　者：陈木妹[1] 林琳[1] 

机构地区：[1]福建师范大学生物工程学院,福建福州350007

出　　处：《生物技术通讯》2005年第1期31-33,共3页Letters in Biotechnology

基　　金：国家自然科学基金项目(30270033);福建省自然科学基金重点项目(B0120001)

摘　　要：利用重叠延伸PCR法对扩展青霉碱性脂肪酶(PEL)基因进行体外定点突变,并构建了含突变基因的重组质粒pPIC3.5K-lip-D92P。将该质粒在毕赤酵母GS115菌株中表达。与野生型表达产物PEL-GS相比较,突变体表达产物PEL-D92P-GS最适作用温度为45℃,比野生型提高了5℃;其热稳定性与野生型相当;突变体在40℃下的表达量为109U/mL,约为野生型的29%。结果分析表明,Pro替代Asp92后,可能是由于Pro一级结构的特点,使酶结构更加稳定,在高温下更适于与底物结合。In order to improve the optimum temperature of Penicillium expansum lipase(PEL), Asp92 was decided to be the target to be replaced by overlap extension PCR technique. The recombinant plasmid pPIC3.5K-lip-D92P containing mutant site was expressed in Pichia pastoris GS115. The comparison experiments of PEL-D92P-GS with wild type PEL-GS showed that the optimum temperatue of PEL-D92P-GS was increased by 5℃. The thermostability of PEL-D92P-GS was similar to the wild-type. The enzymatic activity of the mutant was 109 U/mL at 40℃, which was 29% that of the wild type at the same conditions. It was supposed that the substitution of Pro for Asp at positon 92 introduced a pyrrolidine ring which make the protein structure more rigid, therefore the mutant D92P improvent the optimum temperature.

关 键 词：扩展青霉 碱性脂肪酶 最适作用温度 定点突变 重叠延伸PCR法 蛋白质工程 

分 类 号：Q556[生物学—生物化学]