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*

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 (AuEH2) 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 (K_m和k_cat) 及区域选择性系数 (β_S和β_R),并利用重组大肠杆菌全细胞拆分rac-pMPGE.突变体AuEH2^A250H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k_cat/K_m从10.0mmol/(L·s)提高至12.8 mmol/(L·s),而水解 (R)-pMPGE的k_cat/K_m从1.13mmol/(L·s)降低至0.35mmol/(L·s);全细胞拆分20mmol/L rac-pMPGE获得 (R)-pMPGE的ee_s为>99%,产率从33.0% 提高至40.7%.A250位点的突变对AuEH2的对映选择性和酶活力具有显著影响;高对映选择性的AuEH2突变体在制备高光学纯的 (R)-pMPGE中具有应用潜力.

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

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 (AuEH2) towards racemic p-methylphenyl glycidyl ether (rac-pMPGE). 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)-pMPGE 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)-pMPGE of the purified mutant were measured, respectively, and the recombinant E. coli whole cells was applied to the kinetic resolution of rac-pMPGE. The mutant AuEH2^A250H possessed the highest E value of 38.4, which was remarkably higher than that of AuEH2 (12.7). The specific activity of E. coli/aueh2^A250H was determined to be 51.9U/g wet cells. The k_cat/K_m for (S)-pMPGE of the purified AuEH2^A250H was increased from 10.0mmol/(L·s) to 12.8mmol/(L·s), while decreased from 1.13mmol/(L·s) to 0.35mmol/(L·s) for (R)-pMPGE. Furthermore, using the whole cells of E. coli/aueh2^A250H as biocatalyst, the kinetic resolution of 20mmol/L rac-pMPGE was performed at 25℃ for 1h, obtaining (R)-pMPGE with > 99% ee_s and 40.7% yield. The results indicated that the mutations at A250 played an essential role in regulating the activity and enantioselectivity of AuEH2. The mutant with the improvement of enantioselectivity of AuEH2, which has potential for industrial application on the preparation of (R)-pMPGE.