Production of (S)-CHBE by Asymmetry Reduction with Whole Cells of Yeasts and E.coli as Catalyst
|Keywords||Asymmetric reduction 4 - chloroacetoacetate ethyl acetate ( S ) -4 - chloro-3 - hydroxybutyrate Mulan Candida Biocatalysis E coli M15 ( pQE30 - car )|
(S) -4 - chloro-3 - hydroxybutyrate ((S)-CHBE) is an important chiral pharmaceutical intermediates can be used for the synthesis of HGM-CoA reductase inhibitors and beta-1, 4 - dihydropyridine calcium channel blocking agent drugs has important application value. From laboratory preserved seven yeast strains screened the strain - Mulan Candida (the Candida magnoliae) has a higher ability to restore and chiral selectivity. Optimize the reduction reaction conditions, the results show that 25 g / L Magnolia Candida yeast at 30 ° C, 10g / L of glucose as a the auxiliary substrate buffer solution (pH6.0) can 12.1g / L 4 - chloroacetyl ethyl acetate (COBE) reduction of (S)-CHBE The product yield was 87.5%, enantiomeric excess value (ee) is 76.4%. When the cells increased to 45 g / L product yield and ee values ??were increased to 99.2% and 85.2%, respectively. After the substrate concentration is increased, the single-phase reaction substrate and product inhibition. Substrate was added portionwise can be partially lifted substrate inhibition, but product inhibition persists. Study the Candida magnoliae two phase system to mitigate the effects of product inhibition, the asymmetric reduction reaction, and optimize the reduction reaction conditions. The results showed that, in the two-phase system of water / Titanium Dioxide, 30 ° C when adding a buffer solution of 50g / L glucose, 100 g / L of magnolia Candida in the dropwise addition, the substrate under the conditions of 60.5g / L COBE reduced to (S)-CHBE, the product of a rate of 93.8%, 85.7% ee value. In addition, heat-treated Mulan Candida catalytic COBE stereoselective reduction reaction. The fresh magnolia Candida strains by heat treatment (55 ° C, 30 min) after having a high degree of stereoselectivity, and not subject to reduction conditions, the product (S) type, the ee value is greater than 99%. This process without adding any commercial coenzyme NADP - / NADPH and glucose dehydrogenase (GDH). In the two-phase system of water / Titanium Dioxide, heated by 55 ° C for 30min after treatment of Candida magnoliae (100g / L), 60.5g / L COBE restore for (S)-CHBE product yield was 58.5% its value is greater than 99% ee. Finally, the constructed a the higher carbonyl reductase enzyme activity of the E. coli strain E. coli M15 (pQE30-car) and higher glucose dehydrogenase activity in E. coli strains of E. coli M15 (pQE30-gdh0310). ' Optimized culture conditions and asymmetric reduction reaction conditions, 30 ° C when 50 g / L of E. coli M15 (pQE30-car) and 50 g / L of E. coli M15 (pQE30-gdh0310) in 100 mM, pH 6.0 phosphate buffer able to 12.1g / L COBE reduction of (S)-CHBE The product yield was 78.4%, and its value is greater than 99% ee. In the two-phase system of water / Titanium Dioxide 100 g / L of E. coli M15 (pQE30-car) and 100 g / L of E. coli M15 (pQE30-gdh0310,) in pH6.0 phosphate buffer (50g / L glucose) (100 r / min) Oscillation Reaction of 60.5g / L COBE converted to (S)-CHBE product yield was 42.0%, and its value is greater than 99% ee.