定点突变提高青霉素G酰化酶的稳定性  

Enhancing Penicillin G Acylase Stability by Site-directed Mutagenesis

作　　者：杨晟[1] 黄鹤[1] 李士云[1] 叶玉珍[1] 万琳 张凤文[1] 袁中一[1] 

机构地区：[1]中国科学院上海生物化学研究所,上海200031

出　　处：《生物化学与生物物理学报》2000年第6期581-585,共5页

摘　　要：以大肠杆菌青霉素G酰化酶的晶体结构为模板 ,用软件PMODELING同源模建巨大芽孢杆菌青霉素G酰化酶的三维结构。在此基础上 ,将 β亚基 4 2 7位 (突变A)和 4 3 0位 (突变B)赖氨酸残基突变为丙氨酸 ,降低了该酶的等电点 ,增加了疏水性 ,从而提高其在酸性和有机溶剂环境中的稳定性。两个突变体与亲本相比 ,比活力和Km相近 ,最适pH减少了 0 .5个单位 ,突变B在 pH 5 .2的溶液中的稳定性明显提高。突变A和B在 15 %DMF中的半衰期分别比亲本酶提高了 60 %和 166%To improve the stability of penicillin G acylase(PGA ) from Bacillus megaterium, a three dimensional model of B.megaterium P GA was constructed based on crystal structure of penicillin G acylase from E.c oli using PMODELING program. The mutation of Lys at β427 and 430 to Ala was p redicted to enhance the stability of PGA in acidic or organic solvent environmen t. The results showed that 2 mutant PGA had similar specific activity and Km as the parent PGA. Their optimum pH dropped 0.5 pH units. The stability of Lys4 30Ala was enhanced obviously at pH 5.2 . The half lives of Lys427Ala and Lys430Ala were improved by 60 % and 166 %, respectively, in comparison with the parent PGA.

关 键 词：青霉素G酰化酶 定点突变 稳定性 细菌菌种 

分 类 号：Q556[生物学—生物化学]