青霉素G酰化酶基因Ser177的定点突变

Site-directed Mutagenesis at Ser177 of Penicillin G Acylase Gene

本研究用缺口双链法对大肠杆菌青霉素Ｇ酰化酶（ＰＧＡ）基因Ｓｅｒ１７７进行寡核苷酸定点突变。通过ＮＩＰＡＢ（２－硝基－５－苯乙酰胺苯甲酸）试纸法筛选和测序鉴定，获得突变体Ｃｙｓ１７７，Ｇｌｙ１７７，Ａｒｇ１７７和Ａｓｎ１７７。它们的ＰＧＡ活性均已丧失。酶蛋白电泳分析表明突变体蛋白在体内正常表达。推测ＰＧＡＳｅｒ１７７很可能位于酶底物结合中心，是酶活性所必需，不能被置换。

Site-directed mutagenesis was performed at Ser177 of Penicillin G Acylase(PGA) gene of E. coli by gap duplex method.Screened by 2-nitro-5-phenylacetaminobenzoic acid(NIPAB)test paper assay and confirmed by DNA sequencing,the mutants Cys177,Gly177,Arg177 and Asn177 were obtained.They have no activity of PGA,The mutant enzyme was expressed and detected by PAGE.It is suggested that Ser177 is at the substrate binding center of PGA and can not be replaced.