【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。更多还原

【Abstract】 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.更多还原