用于降解邻苯二甲酸酯的红球菌来源酰胺酶RhoⅡ突变改造  

Mutational Modification of Amidohydrolase RhoⅡ Derived from Rhodococcus sp.for Degradation of Phthalate Esters

作　　者：黄慧芹 徐友强[1,2] 李微微 张成楠 李秀婷 HUANG Huiqin;XU Youqiang;LI Weiwei;ZHANG Chengnan;LI Xiuting(Beijing Advanced Innovation Center for Food Nutrition and Human Health,Beijing Technology and Business University,Beijing 100048,China;Beijing Engineering and Technology Research Center of Food Additives,Beijing 100048,China)

机构地区：[1]北京工商大学食品营养与人类健康高精尖创新中心,北京100048 [2]北京市食品添加剂工程技术研究中心,北京100048

出　　处：《食品科学技术学报》2024年第4期125-134,共10页Journal of Food Science and Technology

基　　金：国家自然科学基金资助项目(32072165)。

摘　　要：邻苯二甲酸酯(phthalate esters,PAEs)具有生理毒性,去除毒性的关键在于侧链酯键的完全水解。红球菌源酰胺酶RhoⅡ是作用于邻苯二甲酸单酯酯键的水解酶,但不能水解PAEs。采用计算机模拟和定点突变的方法,对RhoⅡ进行改造,以实现水解PAEs的目的。将RhoⅡ与邻苯二甲酸单丁酯(monobutyl phthalate,MBP)进行分子对接,结果显示,RhoⅡ以Lys200、Arg185的R基稳定MBP的羧基,MBP与RhoⅡ的两个单体均形成氢键相互作用。位点突变表明,Asp39、Lys127和Cys160构成了RhoⅡ的催化三联体,作为活性中心存在于酶的疏水凹槽内。此外,通过定点突变获得了具有水解PAEs的突变酶F44N,与原酶相比,其水解邻苯二甲酸二丁酯(dibutyl phthalate,DBP)、邻苯二甲酸二异丁酯(diisobutyl phthalate,DIBP)的能力显著提升,这是由于苯丙氨酸突变为天门冬酰胺后,明显削弱了酶对底物的空间位阻作用,使酶的底物结合空腔体积增大,DBP、DIBP能够与酶进行结合,实现酯键水解。结合突变前后对接结果推测,突变影响酶的底物结合空腔,导致突变酶不能有效催化邻苯二甲酸单酯。通过验证RhoⅡ的催化活性中心,突变关键位点,实现了RhoⅡ底物谱的改变,希望可以丰富催化水解PAEs的酶资源,促进相关水解酶更好的应用。Phthalate esters(PAEs)show physiological toxicity,and the key to reduce toxicity lies in the complete cleavage of side chain ester bonds.Amidohydrolase RhoⅡ is a hydrolytic enzyme acting on ester bond of monoalkyl phthalate esters,but cannot hydrolyze PAEs.RhoⅡ was modified by computer simulation and site-directed mutation to achieve the purpose of PAEs hydrolysis.Results of the molecular docking between RhoⅡ and monobutyl phthalate(MBP)showed that RhoⅡ stabilized the carboxyl group of MBP with R-groups of Lys200 and Arg185,and both monomers of MBP and RhoⅡ formed hydrogen bond interactions.The site-directed mutation indicated that Asp39,Lys127 and Cys160 formed the catalytic triplet of RhoⅡ,which as the active center positioned in the hydrophobic cavity of the enzymes.Additionally,the mutant enzyme F44N was obtained through site-directed mutation and could hydrolyze PAEs,which significantly improved the enzymatic hydrolysis efficiency toward dibutyl phthalate(DBP)and diisobutyl phthalate(DIBP)compared with the original enzyme.After phenylalanine mutated to asparagine,the steric hindrance effect of the enzyme on the substrate was significantly weakened,and the substrate binding cavity of the enzyme increased.DBP and DIBP could bind to the catalytic pocket of the enzyme to achieve ester bond cleavage.The catalytic mechanism was speculated based on molecular docking and mutation.The mutation might affect the substrate binding cavity,and the mutant enzyme could not effectively bind to the monoalkyl phthalate esters.This work performed sequence and structure analysis of RhoⅡ,and verified the catalyzed triplet.By finding the catalytic activity center of RhoⅡ and mutating the key site,the substrate specificity of RhoⅡ was changed.This study hoped that the enzyme resources related to enzymatic catalytic hydrolysis of PAEs could be expanded and promoted the application of related hydrolases.

关 键 词：酰胺水解酶 邻苯二甲酸单酯 分子对接 催化三联体 定点突变 

分 类 号：TS201.2[轻工技术与工程—食品科学] TS201.6[轻工技术与工程—食品科学与工程] Q814.9[生物学—生物工程]