Production Of Ethyl (S)-4-Chloro-3-Hydroxybutyrate By Biotransformation

Oxidoreductases are the most popular biocatalyst used in biocatalytic processother than hydrolases. Chiral hydroxyl chemicals are produced by oxidoreductase orrelated microorganisms by reducing the potential chiral carbonyl chemicals. In thisthesis, the asymmetric reduction of ethyl 4-chloroacetoacetate (COBE) to opticallyactive ethyl (S)-(-)-4-chloro-3-hydroxybutyrate (S-CHBE) catalyzed bymicroorganisms in aqueous phase was reported. The main contents includemicroorganisms screening, fermentation, biocatalysis, and product purifying. Theresults are shown as follows.Gas chromatography(GC) was established as an analytical method for detectingCOBE and CHBE in bioconversion. At the same time, chiral capillary GC combiningderivative method was used to determine e.e. value of CHBE. Aureobasidiumpullulans SW0202 producing COBE reductase with high enantioselectivity wasisolated from the microorganisms preserved in our laboratory. The conditions forenzyme production of the strain were studied, and the results showed that the optimalmedium were as follows (g/L): maltose 30.0, yeast extract 20.0, peptone 3.0,(NH4)2SO4 5.0, KH2PO4 2.0, and MgSO4·7H2O 0.7;the optimum fermentationtemperature and pH were 28℃ and 6.0, respectively;the optimum medium volumewas 70mL/500mL The organism was cultured under the optimum conditions for 24h,about 1,007 U/L and 16.78g dry cells/L were obtained.The asymmetric reduction of COBE to optically active CHBE catalyzed by A.pullulans SW0202 in aqueous phase was investigated. The reduction was conductedin K2HPO4-KH2PO4 buffer. The conversion conditions were optimized, showing thatthe optimal conditions were as follows: the initial cell concentration was 8g(wetweight)/25mL, the initial COBE concentration was 20g/L, pH7.0, the temperature 30℃, and the molar conversion rate reached up to 79.6%. In addition, the investigationof immobilized cells was operated. The cells of A. pullulans SW0202 entrapped inκ–carrageenan showed higher molar yield and more suitable to the K2HPO4-KH2PO4buffer system. The optimum immobilized cells reaction conditions are as follows:immobilized cells concentration 20g/25mL, pH7.0, and the temperature 30℃.Besides, the methods for CHBE separation and purification were also described.Solvent extraction, vacuum distillation, silica gel column chromatography(if need)acted as the major separation and purification method.At last, the product structure was characterized by GC-MASS,NMR andpolarimeter. The results showed that: the product purity is 98.23%,[ α ]D18(product)=-20.5(c=0.9028g/100mL, CHCl3){Aldrich standard sample[ α ]D18=-20.8(c=0.9132g/100mL,CHCl3)}, 1H-NMR(D2O,400MHz) δ (ppm),1.31(3H, m, CH3), 2.66(2H, m, CH2COO), 3.63(2H, m, ClCH2), 4.20(2H, m,COOCH2), 4.29(1H, m, CH).On the basis of above work, the author thinks that these problems should bestudied furtherly listed below: 1) to improve enzyme activity through mutagenesis,and genetic reconstruction;2) to discuss substrate adding process;3) biocatalystimmobilization;and 4) to investigate water-organic medium used in these process.