Biocatalytic Production Of Chiral Aryl Alcohol And Hydroxyl Acid By Asymmetric Reduction

Chiral aromatic alcohol and hydroxy acid are important intermediates for the synthesis of optically active molecules and are widely applied in food, pharmaceutical, agricultural, fine chemicals areas. In this thesis, the synthesis of (R)-2-chloro-1-(3-chlorophenyl)ethanol and 2-hydroxy-3-phenylpropionic acid by asymmetric reduction was studied, and could potentially be applied in the industry production.Using 2-chloro-(3-chlorophenyl)ethanone as a model substrate, the fermentation and bioreduction conditions for the asymmetric reduction by Candida ontarioensis were optimized to be: 30 oC, initial pH of 6.5, rotation speed of 180 r/min, and cell concentration of 20. After 72 h of reaction, the e.e. and yield of product were 99.5% and 99% respectively at substrate concentration of 10 g/L. C. ontarioensis whole-cell showed high reaction activity and enantioselectivity towards various halogeno-, amino-, and nitro-substituted acetophenone, but not for keto ester substrates.Using C. ontarioensis whole cells pretreated with 4 g/L CTAB at 4 oC for 20 min, the product e.e. and yield reached 99.9% and 97.5% at substrate concentration of 30 g/L within a significantly shortened reaction time of 24 h. The reaction system was scale up to 120-mL and the product (R)-2-chloro-1-(3-chlorophenyl)ethanol was purified by silica gel column chromatography. The product (R)-2-chloro-1-(3-chlorophenyl)ethanol was obtained in 99.0% purity, 99.9% e.e., and a yield of 66.7%. Then (R)-2-chloro-1-(3-chlorophenyl)ethanol was characterized by NMR spectra.A yeast strain A.6.3 was selected for the synthesis of 2-hydroxy-3-phenylpropionic acid among 28 microorganisms tested in aqueous reaction system. The fermentation and bioreduction conditions of A.6.3 were also optimized. The optimum fermentation medium is: 20 g/L glucose, 20 g/L corn powder, 2.5 g/L KH2PO4, 1.5 g/L MgSO4?7H2O; and the optimal reaction conditions are: initial pH of 7.0, temperature of 30 oC, cell concentration 100 g/L. The inhibitory effect of substrate was investigated and substrate concentration 20 g/L was selected for the further study. Using the A.6.3 whole-cell pretreated with 10%(v/v) ether for 5 min at 20 oC, a product yield of 86.9% was reached .In order to obtain the biocatalysts with higher enzyme activity and stability for the preparation of 2-hydroxy-3-phenylpropionic acid, the recombinant E. coli BL21(DE3)/pET-20b-ScCR, BL21(DE3)/pET-20b-KlCR, harboring carbonyl reductase from Saccharomyces cerevisiae and Kluyveromyces sp., were constructed by genome mining. The specific activity of BL21(DE3)/pET-20b-ScCR, BL21(DE3)/pET-20b-KlCR were 0.24 U/mg and 0.31 U/mg, respectively.