Screening Of Limonene Epoxide Hydrolase Mutants And Analysis Of Their Enzymatic Properties

Epoxide hydrolases were a group of function related enzymes that catalyze the selective ring opening of racemic and racemic epoxides to produce enantiomeric diols and residual epoxides.Limonene epoxide hydrolase(LEH)was a new kind of epoxide hydrolase subfamily separated from Rhodococcus erythropolis DCL14.It could specifically catalyze the hydrolysis of limonene epoxide,indene oxide and1-methylcyclohexene oxide and other substrates.It had the potential to replace Jacobsen catalyst due to its mild catalytic conditions,independent of cofactors,easy cloning and exogenous expression.In this study,FRESCO strategy was used to construct a mutation library of limonene epoxide enzyme,and a high-throughput and rapid screening method was established with the help of gas chromatography-mass spectrometry.Molecular docking technology and bioinformatics software chimera predict the protein structure and analyze the enantioselectivity of different mutant residues to the substrate epoxicosahexane,and explore the enzymatic properties of ideal mutants in organic mediaWe screened out(S,S)-selective mutants BE3(ee> 86%),(R,R)-selective mutants BG5(ee> 70%)and high conversion rate mutants BF6 and BG9.The optimum temperature of the mutants BE3 and BG5 with the highest stereoselectivity were 50 ℃ and 40 ℃,respectively.After 90 minutes of heat treatment,the temperature stability of the natural enzyme was improved by 5℃,and the optimal p H range of BG5 was expanded from 5-9 of the natural enzyme to 4.2-10.0.Ile80 and l103 near the catalytic center of limonene epoxide hydrolase were important sites closely related to the stereoselectivity of the enzyme,while high conversion mutants affected the catalytic efficiency by changing the distance between N-loop and C-loop.Limonene epoxide hydrolase could exert catalytic activity in a variety of organic media,and the most suitable medium was acetonitrile,the most suitable concentration was 4.48%～10%.The screened mutants with high stereoselectivity and high conversion rate had good catalytic properties and had the potential to replace chemical catalysts for industrial synthetic pharmaceutical intermediates.