Halohydrin dehalogenases (HHDHs) have attracted much attention due to their ability to synthesize enantiomerically enriched epoxides and β-haloalcohols. However, most of the HHDHs exhibit low enantioselectivity. Here, a HHDH from the alphaproteobacteria isolate 46_93_T64 (AbHHDH), which shows only poor enantioselectivity in the catalytic resolution of rac-PGE (E = 9.9), has been subjected to protein engineering to enhance its enantioselectivity. Eight mutants (R89K, R89Y, V137I, P178A, N179Q, N179L, F187L, F187A) showed better enantioselectivity than the wild type. The best single mutant N179L (E = 93.0) showed a remarkable 9.4-fold increase in the enantioselectivity. Then, the single mutations were combined to produce the double, triple, quadruple, and quintuple mutants. Among the combinational mutants, the best variant (R89Y/N179L) showed an increased E value of up to 48. The E values of the variants N179L and R89Y/N179L for other epoxides 2-7 were 12.2 to > 200, which showed great improvement compared to 1.2 to 10.5 for the wild type. Using the variant N179L, enantiopure (R)-PGE with > 99% ee could be readily prepared, affording a high yield and a high concentration.

中文翻译：

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通过蛋白质工程，卤代醇脱卤酶对不对称环氧化物开环反应的对映选择性显着提高。

卤代醇脱卤酶（HHDHs）由于其能够合成对映异构体富集的环氧化物和β-卤代醇而备受关注。但是，大多数HHDH表现出低对映选择性。在这里，来自α变形细菌分离株46_93_T64的HHDH（AbHHDH）在rac-PGE的催化拆分中仅表现出较弱的对映选择性（E = 9.9），已经进行了蛋白质工程改造以提高其对映选择性。八个突变体（R89K，R89Y，V137I，P178A，N179Q，N179L，F187L，F187A）显示出比野生型更好的对映选择性。最佳的单个突变体N179L（E = 93.0）显示出对映选择性显着提高9.4倍。然后，将单个突变合并以产生双，三，四和五元突变体。在组合突变体中，最好的变体（R89Y / N179L）的E值增加了多达48。对于其他环氧化物2-7，变体N179L和R89Y / N179L的E值是12.2至> 200，与1.2至10.5相比，显示了很大的改进。对于野生型。使用变体N179L，可以容易地制备具有> 99％ee的对映体（R）-PGE，具有高收率和高浓度。