Stability Of Epoxide Hydrolase And Its Application In The Preparation Of Chiral Epichlorohydrin In Biphasic System

Chiral epichlorohydrin is an important C3 chiral synthon for applications in medical,chemical,material and many other industries.It had been one of the most effective ways for synthesis of chiral epichlorohydin that a stereoselective hydrolysis of the racemic epichlorohydrin(rac-ECH)by epoxide hydrolase.Based upon the work of the epoxide hydrolase screening,the construction of genetic engineering bacteria and the molecular modification,the mutant enzyme was used as the research object in this paper.Bio-resolution of epichlorohydrin by epoxide hydrolase in ionic liquid system was mainly studied,the chemical glycosylation modification for improving the stability of epoxide hydrolase was researched,and the cross-linking immobilization of the whole cell epoxide hydrolase was investigated.The specific results are as follow:First,the resolution of epichlorohydrin by whole cell epoxidehydrolase in the ionic liquid/buffer biphasic system was studied.[BMIM][PF6] was selected to construct a biphasic system for bio-resolution of epichlorohydrin.Buffer/[BMIM][PF6] volume ratio 4 : 1,buffer pH 8.0,and reaction temperature 35 °C were preferred in this work.When the cell concentration was 0.02 g/ml,the optimal substrate concentration was 1 mol/L,the reaction time was about 45 min,the e.e.value and yield were 98.4% and 38.2% for(R)-ECH,respectively.Besides,the time-space yield and catalyst efficiency was 0.51 mol/L/h and 23.9 mmol/g.When the cell concentration was 0.04 g/mL,the optimal substrate concentration was 1.2 mol/L.Second,in order to improve its stability,the epoxide hydrolase was chemically modified by polysaccharide.The results showed that the best glycosylation method was through coupling the amylated dextran with the enzyme by EDAC.The thermostability,p H stability and denaturant tolerance of the epoxide hydrolase modified with dextran 70 k Da were investigated.This modified enzyme showed improved the above properties compared to its native free enzyme,among which the half-life of the modified enzyme at 50 °C increased by 28%.Further,The catalytic reaction of the modified enzyme in the [BMIM][PF6]/buffer biphasic system was studied.At last,The immobilization of whole cell epoxide hydrolase was studied and PEI-GA crosslinking was selected as a better method.Theoptimum immobilization conditions were obtained as follow: PEI concentration 3%(v/v),GA concentration 1%(v/v),diatomite concentration 0.6%(w/v).The optimum reaction temperature and pH of immobilized cell were 40 °C and 8.0.Besides,the thermal stability,pH stability and operational stability of immobilized cell were significantly improved compared with free cell,which still retained 93% of the initial activity after 8 cycles reaction.Further,synthesis of(R)-ECH using immobilized cell was studied in aqueous phase and [BMIM][PF6]/buffer biphasic system.