定点突变提高毕赤酵母产葡萄糖氧化酶的氧化稳定性  

Enhancing Oxidative Stability of Glucose Oxidase from Aspergillus niger by Site-Directed Mutagenesis

作　　者：闻一凡 顾磊[1,2] 张娟[1,2] 堵国成[1,2] 

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2016年第12期1260-1267,共8页Journal of Food Science and Biotechnology

基　　金：国家863计划项目(2011AA100905);国家自然科学基金项目(31470160)

摘　　要：通过定点突变的方法,将来源于黑曲霉Aspergillus niger的葡萄糖氧化酶的甲硫氨酸替换为亮氨酸,以期提高葡萄糖氧化酶的耐氧化性。野生型的葡萄糖氧化酶和突变酶M305L、M524L、M556L和M582L的基因分别在巴斯德毕赤酵母中分泌表达,对突变酶与野生型葡萄糖氧化酶纯化后进行性质比较。结果表明：突变酶的耐氧化性与野生型葡萄糖氧化酶相比均有不同程度的提高,其中,突变酶M556L在100 mmol/L和500 mmol/L过氧化氢存在下的氧化稳定性是野生型葡萄糖氧化酶的近2倍,且催化效率（kcat/Km）提高至野生型葡萄糖氧化酶的2.08倍。定点突变后,突变酶的最适反应温度为35℃,与野生型葡萄糖氧化酶基本保持一致,同时,突变酶M524L的p H稳定范围由4.0~6.0扩展到3.0~7.0。Aiming to improve the catalytic activity and oxidative stability of glucose oxidase from Aspergillus niger. This work conducted site-directed mutagenesis based on an analysis of the protein structure. Four methionines（M582L,M524L,M556L and M305L） were selected as the mutation sites and individually replaced with leucine. The native GOD and the mutated GOD were separately expressed in P. pastoris GS115. In the presence of H2O2（10 mM,20 mM,50 mM,100 mM,and 500 mM） at 35 ℃ for 2 h,the oxidative stability of the mutants increased compared to the wild-type under most of the circumstances. Meanwhile,M556L showed a higher（2 folds than the wild-type） oxidative stability of all in the presence of both 100 mM and 500 mM H2O2 for 2 h. Compared to the wild-type enzyme,the kcat/Km value of M556L increased（2.08 folds than the wild-type）. The thermol stability of the mutants remained unchanged while the stable pH range of M524L was extended from 4.0-6.0 to 3.0-7.0 compared to the wild-type. It is a fact that a more stable enzyme with higher oxidative stability is an ideal choice in most of the industrial application of GOD.

关 键 词：葡萄糖氧化酶 重组 定点突变 黑曲霉 巴斯德毕赤酵母 

分 类 号：Q936[生物学—微生物学]