定点突变提高环氧化物水解酶AuEH2催化对甲基苯基缩水甘油醚的对映选择性  

Improving the Enantioselectivity of an Epoxide Hydrolase towards p-Methylphenyl Glycidyl Ether by Site-directed Mutagenesis

作　　者：苏永君 胡蝶 胡博淳 李闯[3] 文正 章晨 邬敏辰 SU Yong-jun;HU Die;HU Bo-chun;LI Chuang;WEN Zheng;ZHANG Chen;WU Min-chen(School of Pharmaceutical Science,Jiangnan University,Wuxi 214122,China;Wuxi School of Medicine,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学药学院,无锡214122 [2]江南大学无锡医学院,无锡214122 [3]江南大学生物工程学院,无锡214122

出　　处：《中国生物工程杂志》2020年第3期88-95,共8页China Biotechnology

基　　金：江苏省自然科学青年基金(BK20180622);中国博士后面上基金(2018M630522);江苏省普通高校研究生科研创新计划(SJCX19_0790)资助项目。

摘　　要：环氧化物水解酶可催化外消旋环氧化物的动力学拆分或对映归一性水解制备手性环氧化物或邻二醇,具有广阔的应用前景。为提高宇佐美曲霉环氧化物水解酶(Au EH2)催化外消旋对甲基苯基缩水甘油醚(rac-pMPGE)的对映体选择率(E)。通过分子动力学模拟(MD)选取相互作用频率最高的位点A250替换为其他19种氨基酸;选取对映选择性显著提高的突变体测定其动力学参数(Km和kcat)及区域选择性系数(βS和βR),并利用重组大肠杆菌全细胞拆分racp MPGE。突变体AuEH2A250H的E值从12.7提高至38.4,重组菌比活力为51.9U/g湿细胞;其水解(S)-pMPGE的kcat/Km从10.0mmol/(L·s)提高至12.8 mmol/(L·s),而水解(R)-pMPGE的kcat/Km从1.13mmol/(L·s)降低至0.35mmol/(L·s);全细胞拆分20mmol/L rac-p MPGE获得(R)-pMPGE的ees为>99%,产率从33.0%提高至40.7%。A250位点的突变对Au EH2的对映选择性和酶活力具有显著影响;高对映选择性的Au EH2突变体在制备高光学纯的(R)-pMPGE中具有应用潜力。Epoxide hydrolase can be used in the kinetic resolution or enantioconvergent hydrolysis of racemic epoxides for prepare optically pure epoxides or vicinal diols,which has broad application prospects.To improve the enantioselectivity of Aspergillus usamii epoxide hydrolase(Au EH2)towards racemic p-methylphenyl glycidyl ether(rac-p MPGE).According to the protein-ligand finger-print(IFP)in the molecular dynamics simulation,the key residue site A250 with the highest interaction frequency towards(R)-p MPGE was selected,and then replaced by other 19 residues by site-directed mutagenesis.A mutant with the improved enantioselectivity was obtained and purified by affinity chromatography.Furthermore,the kinetic parameters and regioselectivity coefficients towards(R)-and(S)-p MPGE of the purified mutant were measured,respectively,and the recombinant E.coli whole cells was applied to the kinetic resolution of rac-p MPGE.The mutant Au EH2 A250 Hpossessed the highest E value of 38.4,which was remarkably higher than that of Au EH2(12.7).The specific activity of E.coli/aueh2 A250 Hwas determined to be 51.9 U/g wet cells.The kcat/Kmfor(S)-p MPGE of the purified Au EH2 A250 Hwas increased from 10.0 mmol/(L·s)to 12.8 mmol/(L·s),while decreased from 1.13 mmol/(L·s)to 0.35 mmol/(L·s)for(R)-p MPGE.Furthermore,using the whole cells of E.coli/aueh2 A250 Has biocatalyst,the kinetic resolution of 20 mmol/L rac-p MPGE was performed at 25℃for 1 h,obtaining(R)-p MPGE with>99%eesand 40.7%yield.The results indicated that the mutations at A250 played an essential role in regulating the activity and enantioselectivity of Au EH2.The mutant with the improvement of enantioselectivity of Au EH2,which has potential for industrial application on the preparation of(R)-p MPGE.

关 键 词：环氧化物水解酶 理性设计 定点突变 对映选择性 对甲基苯基缩水甘油醚 

分 类 号：Q814.9[生物学—生物工程]