Studies On The Catalytic Properties Of Epoxide Hydrolase 2 From Vigna Radiata (VrEH2) And Its Preliminary Molecular Modification

Epoxide hydrolases (EHs) can catalyze the hydrolysis of epoxides into its corresponding diols through adding a water molecule to epoxide rings without any metal ion or cofactor. Due to the high enantioselectivity and stereoselectivity, epoxide hydrolases have drawn great attention on catalyzing bioresolution of racemic epoxides in decade years. Epoxide hydrolases from Vigna radiata (VrEH1 and VrEH2), which can enantioconvergently hydrolyze rac-pNSO into (R)-pNPG, had been cloned, expressed and characterized. In this thesis, large amount of recombinant VrEH2 was obtained by fermentation, the catalytic properties of recombiant VrEH2 was further studies after purification, a novel chemoenzymatic method to obtain optical pure (R)-pNPG was constructed, the conditions for the kinetic resolution of (R)-2-((2,3-dimethylphenoxy)methyl)oxirane using recombinant VrrEH2 were optimized. Finally, the solubility of recombinant VrEH2 was improved and the key amino acids influencing the activity of VrEH2 were identified by homology modeling and molecular modification. The details of the thesis were as follows:1. Crude enzyme powder of recombinant VrEH2 (14 g) with a specific activity of 44 U/g was prepared by fermentation of E. coli BL21. After one-step Ni affinity chromatography, the specific activity was 1.95 U/mg (44 times higher than crude enzyme) and the activity recovery yield was 31.9%. The optimum temperature and pH of purified VrEH2 were characterized as 30 ℃ and pH 6.5, respectively. VrEH2 had the opposite regioselectivity toward (S)-pNSO (87.2% to Ca) and (R)-pNSO (98.4% to Cp), which made it able to catalyze the enantioconvergent hydrolysis of pNSO at 100% conversion affording (R)-p-nitrophenyl glycol (89.4%ee).2. Considering the acid catalyzed hydrolysis of (S)-pNSO had better regioselectivity (91.5% to Cα) and much higher rate than VrEH2 (87.2% to Cα), the chemoenzymatic synthesis route of (R)-pNPG from racemic pNSO by enantioconvergent hydrolysis of pNSO with VrEH2 catalyzing the first stage hydrolysis of (R)-pNSO and H2SO4 catalyzing the second stage hydrolysis of (S)-pNSO was constructed. The enantiopurity of the resultant (R)-pNPG (89.4% ee) was higher than that (84.8%ee) obtained by using VrEH2 alone. The reaction time of synthesis (R)-pNPG (89.9%ee) was shorten to merely 4 h,1/4 that using VrrEH2 alone. After chloroform recrystallization, ee of (R)-pNPG was up to 99.0% with 71.5% overall yield.3. In order to expand the substrate spectrum of VrEH2, derivatives of phenyl glycidyl ether was selected as substrates for the determination of its activity and enantioselectivity. The activities of VrEH2 toward phenyl glycidyl ethers were higher than those toward styrene oxides but with poor stereoselectivity. Hence, surfactant and organic solvent was applied to the system to improve the stereoselectivity. Tween-80 can improve the enantioselectivity. Kinetic resolution 2-((2,3-dimethylphenoxy)methyl)oxirane was conducted in such reaction system, the resulting yield of product was 27.5% and the ee value could reach to 98.0%.4. Combining with homology model and site-directed mutation, the solubility of mutant VrEH2 (G3E/V4I, M), which had similar catalytic properties of VrEH2, was improved. By alanine scanning of residues around the active site, three amino acids (P185, L261 and F298) were determined to influence the activity of mutant VrEH2, which can lay the foundation for subsequent protein molecular modification.