Molecular Modification To Epoxide Hydrolase And Its Application In The Resolution Of Epichlorohydrin

Enantiopure epichlorohydrin is an important C3 chiral building blocks for applications in medical,pesticide,chemical,material and many other industries.With the market price of epichlorohydrin falling,the kinetic resolution of racemic epichlorohydrin(rac-ECH)by epoxide hydrolase(EH,EC.3.3.2.3)had been one of the most effective ways for synthesis of chiral epichlorohydin.In this paper,molecular modification to epoxide hydrolase gene from Agrobacterium radiobacter was designed.Firstly,the molecular docking experiment of(S)-ECH into the EH active site to identify active site residues that are close to the substrate.Eight amino acid residues(I108,A110,D131,I133,Q134,H215,Gly245 and T247),which might be suitable targets for engineering the activity and enantioselectivity of EH towards rac-ECH,were selected for site-saturation mutagenesis and eight single-site libraries were created.Six single mutants:I108L,D131 S,Q134T,H215 Y,G245A and T247 K affected EH activity and enantioselectivity towards rac-ECH.Then,combinatory mutagenesis libraries were constructed.The best mutant T247K/I108L/D131 S showed significantly higher activity towards substrate rac-ECH with a 5.4-fold higher specific activity(315.2 U/mg)and increased the yield of(R)-ECH from 31.8% to 39.2%.Enzymatic properties of wide-type and the mutant T247 K,I108L,D131 S,T247K/I108 L and T247K/I108L/D131 S indicated that: All mutants had an optimum temperature of 40�C and a p H optimum of 8.5-9.0,similar to that of the wild-type enzyme.However,these mutants displayed higher thermostability,with half-lifes at 50�C of more than 2-13-fold as long as that of the wild-type enzyme.Kinetics analysis confirmed the enhanced catalytic efficiency and enantioselectivity(E value)of muatants toward racemic epichlorohydrin(rac-ECH)compared to the wild-type enzyme.The best mutant T247K/I108L/D131 S exhibited a 4.4-fold higher Kcat/Km value(2.05 s-1m M-1)and 2.1-fold higher E value towards rac-ECH than the wild-type EH.In order to illustrate the differents in activity and enantioselectivity between the wide-type EH and the best mutant T247K/I108L/D131 S,we modeled the S-and R-ECH into the active site of wide-type and mutant EH.In terms of wild-type Ar EH,the distances between O?1 on the catalytic Asp107 with the primary C in the substrate S-and R-ECH were 4.08 ? and 4.73 ?,respectively.For mutant T247K/I108L/D131 S,the distances were 2.94 ? and 5.12 ?,respectively.The nucleophilic attack on this primary carbon becomes easier in virtue of the proper orientation and distance for(S)-ECH,oppositely for(R)-ECH.So mutant T247K/I108L/D131 S showed significantly higher activity and enantioselectivity towards substrate rac-ECH.The biotransformation of rac-ECH using whole cells of mutant T247K/I108L/D131 S as biocatalyst was investigated.The optimal reaction conditions are as follows(100m L): 0.2 M carbonate buffer(p H 9.0),35?C and Tween 40(5% v/v).20g/L Whole cells of mutant T247K/I108L/D131 S was used to catalyze 660 m M rac-ECH under the optimal reaction condition.The e.e.value and yiled of(R)-ECH reached 100% and 40.2%,respectively,at 25 min.