定点突变引入离子键提高植酸酶YiAPPA热稳定性的研究  

Research on Improving the Thermostability of Phytase YiAPPA by Introducing Ionic Bonds through Site-directed Mutagenesis

作　　者：曾静[1] 郭建军[1] 王通 袁林[1] ZENG Jing;GUO Jianjun;WANG Tong;YUAN Lin(Institute of Microbiology,Jiangxi Academy of Sciences,Nanchang 330096,China)

机构地区：[1]江西省科学院微生物研究所,江西南昌330096

出　　处：《食品工业科技》2024年第24期157-166,共10页Science and Technology of Food Industry

基　　金：国家自然科学基金地区科学基金项目(32160579);江西省主要学科学术和技术带头人培养计划项目(20212BCJ23033,S2021GDQN2403);江西省科学院基础研究项目(2022YJC2006)。

摘　　要：本研究旨在通过定点突变引入离子键来提高植酸酶YiAPPA的热稳定性。对比YiAPPA与热稳定性优良的植酸酶r PhyXT52的分子结构,采用定点突变技术向YiAPPA中引入与rPhyXT52热稳定性相关的分子表面离子键,构建离子键突变体。通过热稳定性筛选,获得热稳定性显著提高的离子键突变体T209K/S220E/N237D。突变体T209K/S220E/N237D的酶学特性研究结果表明:突变体T209K/S220E/N237D于37℃、pH4.5的绝对酶活为3982.06 U/mg,与YiAPPA基本一致;T209K/S220E/N237D的最适反应温度、最适反应pH、pH稳定性以及蛋白酶抗性也与YiAPPA基本一致;与YiAPPA相比,T209K/S220E/N237D于80℃半衰期由14.81 min延长至24.72 min,半失活温度T_(50)^(30)由55.12℃提升至64.05℃,T_(m)值由48.36℃提升至55.04℃。分子动力学模拟显示,T209K/S220E/N237D中引入了新的离子键,提高了酶分子中构成这些离子键的氨基酸残基所在的结构单元的稳定性,从而提高了酶的热稳定性。本研究结果表明,向YiAPPA中引入离子键可有效提高其热稳定性,使其更适用于食品加工领域。本研究也可为植酸酶以及其他类型酶的热稳定性改造提供理论依据。To improve the thermostability of the phytase YiAPPA by introducing ionic bonds through site-directed mutagenesis.By comparing the molecular structure of YiAPPA and phytase rPhyXT52 with excellent thermal stability,site-directed mutagenesis was used to introduce molecular surface ionic bonds related to the thermostability of rPhyXT52 into YiAPPA to construct mutants.The mutant T209K/S220E/N237D with significantly improved thermo stability were obtained through screening.The mutant T209K/S220E/N237D had an specific activity of 3982.06 U/mg at 37℃ and pH4.5,which was essentially consistent with the respective values for YiAPPA.The optimal reaction temperature,optimal reaction pH,pH stability,and protease resistance of T209K/S220E/N237D were also basically the same as those of YiAPPA.However,compared with that of YiAPPA,the half-life of T209K/S220E/N237D at 80℃ was extended from 14.81 to 24.72 min,the half-inactivation temperature T_(50)^(30) was increased from 55.12 to 64.05℃,and the T_(m) value was increased from 48.36℃to 55.04℃.Molecular dynamics simulations showed that the introduction of new ionic bonds in T209K/S220E/N237D improved the stability of the structural units in the enzyme molecule in which the amino acid residues constituting these ionic bonds were located,thereby improving the thermostability of the enzyme.Results showed that the introduction of ionic bonds into YiAPPA could effectively improve its thermostability and make it more applicable for food processing.The results would be also provide a theoretical basis for the improved thermostability of phytase and other enzymes.

关 键 词：植酸酶 定点突变 离子键 热稳定性 分子动力学模拟 

分 类 号：Q814.4[生物学—生物工程]