Production Of (S)-CHBE By Asymmetry Reduction With Whole Cells Of Yeasts And E.coli As Catalyst

Optically active D-(5)-4-chloro-3-hydroxybutanoate ethyl ester ((S)-CHBE) is a key chiral intermediate in the enantioselective synthesis of slagenins B and C as well as in the total synthesis of HMG-CoA reductase inhibitors, and can be converted into 1,4-dihydropyridine-type blocker. The aim of this work was to produce (5)-CHBE from ethyl 4-chloro-3-oxobutanoate (COBE) by whole cell catalyzed reduction.Among the seven yeast strains investigated, Candida magnoliae exhibited the highest activity and stereo-selctivity, and was selected as the catalyst for the reduction of COBE to (5)-CHBE in the subsequent study.Optically active (S)-CHBE was prepared firstly by stereoselective bioreduction of COBE in aqueous phase with Candida magnoliae. The results showed that 99.2% of 12.1 g/L COBE was reduced to (5)-CHBE with 85.2% e.e. value by 45 g/L cells of Candida magnoline at optimal conditions. Both of substrate inhibition and product inhibition were found in aqueous phase reaction. Dividing the addition of substrate to several fraction decreased substrate inhibition in certain extent.To reduce product inhibition, the asymmetric reduction of COBE was carried out in a water/n-butyl acetate two-phase system by using whole-cells of Candida magnoliae. At optimal conditions, 60.5g/L COBE was reduced to (S)-CHBE with an e.e. value of 85.7% and a yield of 93.8% by 100 g/L cells of Candida magnoline. In comparison with mono-phase system, the yield of (S)-CHBE was increased by 10 times at the same substrate concentration.Heat treatment of the whole cells Candida magnoliae was able to enhance the enantiomeric excess of product further. The acetone-dried cells, air-dried cells and fresh cells were treated with heat, receptively. Then they were used for the reduction of COBE to (S)-CHBE. The results showed that heat treated fresh cells lead to the highest yield and the enantiomeric excess. When these cells were heated at 55℃ for 30 min, the enantiomeric excess of the reduction product increased to 99% upwards, while the yield remained at 99% upwards. The biotransformation reaction was completed effectively without any addition of glucose dehydrogenase or NADPH/NADP+.At optimal conditions in the two-phase system, 60.5 g/L COBE was reduced to (S)-CHBE with a yield of 58.5% and an e.e. value of 99% by 100 g/L cells of Candida magnoline.E. coli M15 (pQE30-car) and E. coli M15(pQE30-gdh0310) were recombinant strains harboring the car gene from Candida magnoliae and the gdh gene from Bacillus megaterium, respectively. By using E. coli M15 (pQE30-car) and E. coli M15 (pQE30-gdh0310), with respect to the specific activities of CAR and GDH, 8.2 fold and 12.3 fold enhancement over the corresponding original strain were achieved, respectively. The cells of these two strains were used together to construct a reaction system with cofactorregeneration. Under the optimized conditions, the reaction was completed effectively without any addition of glucose dehydrogenase or NADPH/NADP+. An optical purity of 99% (e.e) was obtained and the yield of (5)-CHBE reached 96.6% when initial concentration of COBE was 6.05g/L.When the total addition amount of COBE was enhanced to 60.5 g/L, an e.e. value of 99% and a yield of 42.0% was achieved by using 100 g/L cells of E. coli M15 (pQE30-car) and 100 g/L cells of E. coli M15 (pQE30-gdh0310).