Novel Epoxide Hydrolase: Microorganism Screening And Applications In Chiral Synthesis

1. Optically pure epoxides are versatile chiral compounds in organic synthesis. Various chemical and biological methods for synthesis of chiral epoxides have been developed. Resolution of racemic epoxides by epoxide hydrolases is a promising way of achieving this. Epoxide hydrolases require no co-factor and, depending on the substrate, may show high enantioselectivity as well as high activity. As a consequence, they can be used to prepare enantiopure epoxides and/or vicinal diols from cheap racemic epoxides.2. Using double-wavelength spectrophotometry in microtiter plate, the correlation between the absorption and epoxide concentration was studied in phosphate buffer and bacterial cell suspension. It was found, by comparison, that the two regressive curves in the two systems parallel with each other. And it was inferred and proved that epoxide concentration in cell suspensions could be calculated using the regressive curve in the buffer system as standard curve. Based on the double-wavelength spectrophotometric method to determinate epoxide, epoxide hydrolase activity could be assayed quickly and conveniently.High performance capillary electrophoresis (HPCE) is used widely in enantiomer analysis. Using 5 % sulfated -β- cyclodextrin as chiral selector, and 25 mmol/L phosphate solution (pH2.5) as running buffer, the baseline separation of phenylethanodiol enantiomers was achieved by capillary zone electrophoresis under separation voltage of-25 KV at 25℃.With the combination of epoxide assay by double-wavelength spectrophotometry in microtiter plate and chiral resolution of phenylethanodiol by HPCE, a fast and effective system was established for screening of microbial strains with high enantioselective epoxide hydrolase activity.3. It has been very important to discover novel epoxide hydrolases. Enantiopure glycidates are extremely important compounds in chiral drugs synthesis. In particular, some glycidates are key intermediates for Taxol and Taxotere side chains, Diltiazem and Bestatin, et al. Therefore, microbial strains were screened capable of producing epoxide hydrolase with high enantioselectivity towards glycidates.Considering that glycidates have both oxirane ring and ester bond, styrene or styrene oxide was used as model substrate for epoxide hydrolase-producing strains screening. For the first time, a bacterial strain BZS21 was screened with high enantoselectivity to trans ethyl 3-phenyl glycidate (EPG). The strain could hydrolyze racemic EPG, with 100% ee of (2R,3S)-EPG remained. And compared with most known epoxide hydrolases, the strain showed complementary enantioselectivity for styrene oxide hydrolysis. In addition, a strain Aspergilus niger HNG5 was also screened, which could hydrolyzed racemic benzyl glycidyl ether (BGE) with (R)-BGE remained.The strain BZS21 was identified as Pseudomonas pseudoalcaligenes according to its morphological and physiological characteristics, and also 16S rRNA analysis.4. The enantioselectivity of P. pseudoalcaligenes BZS21 was strongly influenced by the media composition. With 1 % sucrose as carbon source, 0.2 % beef extract, 0.2 % peptone and 1.5 % urea as mixed nitrogen source, the strain produced epoxide hydrolase with the highest enantioselectivity. However, the growth and enantioselcetivity of the strain did not correlate with each other.The cells suspended in 100 mmol/L phosphate buffer (pH 8.0) containing 10% DMF (v/v) as cosolvent produced (2R,3S) -EPG with 95% ee and 26% yield in 12h from 0.2% (w/v) of the racemic mixture.Though P. pseudoalcaligenes BZS21 showed esterase activity to P-naphthyl acetate, the esterase was proved not to be responsible for enantioselective hydrolysis of EPG5. The applications in various reaction systems were studied using an epoxide hydrolase preparation from P. pseudoalcaligenes BZS21.(1) EPG was resolved in some biphasic systems. When 20-25 % of isooctane was contained in the system, the enzyme showed high enantioselectivity.(2) The enantioselectivity of the enzyme could be altered by various surfactants. Ion surfactants were not good to enantioselectivity or activity of the enzyme. On the other hand, non-ion surfactants, including Tween 60 and Tween 80, could improve the enantioselectivity. In particular, when 2 % of Tween 60 was used in the emulsion system, up to 78 mmol/L of EPG was hydrolyzed with the remaining substrate of 98% ee and 33% yield, which was of significant potentials for chiral epoxide preparation.(3) For cyclodextrin-induced emulsion systems, 30 mmol/L of hydroxypropyl-P-cyclodextrin could improve the enantoselectivity significantly.6. Using an epoxide hydrolase preparation from Aspergillus niger HNG5, 200 mmol/L of racemic BGE was resolved in 3 h with remaining (-K)-BGE of 100% ee and 22% yield o That suggested that epoxide hydrolase of Aspergillus niger HNG5 could be used in the resolution of high concentration of BGE.