重组青霉素G酰化酶拆分制备（S）-邻氯苯甘氨酸

对产青霉素G酰化酶的重组枯草芽胞杆菌发酵产酶条件进行优化，确定优化后的发酵条件：可溶性淀粉10g/L、蛋白胨12g/L、酵母粉3g/L、NaCl10g,／L；pH7．5、培养温度37℃、装液量80mL（500mL三角瓶）、培养28h，青霉素G酰化酶的表达水平由最初的7．34U／mL提高至18．23U／mL。以表达青霉素G酰化酶的枯草芽胞杆菌发酵液为酶源，在水相中对映选择性催化N-苯乙酰-（R，S）-邻氯苯甘氨酸制备（S）-邻氯苯甘氨酸，当底物浓度为100mol／L时转化4h，转化率达44．2％。对底物浓度为80mmoL／L反应液中的（S）-邻氯苯甘氨酸进行分离，达到理论收率的94．29％（以N-苯乙酰-（R，S）-邻氯苯甘氨酸的0．5倍摩尔量为理论产率），e．e．值大于99．9％。170℃条件下，N-苯乙酰-（R）-邻氯苯甘氨酸与苯乙酸共熔消旋为N-苯乙酰-（R,S）-邻氯苯甘氨酸可用于循环拆分。

Preparation of (S)-2-chlorophenyl glycine using penicillin G acylase

Cultivation conditions of recombinant Bacillus subtilis expressing penicillin G acylase were investigated. The optimized fermentation conditions were as follows： soluble starch 10. 0 g/L, peptone 12 g/L,yeast extract 3 g/L,and NaCl 10 g/L;fermentation temperature 37 ℃ ,flask column 80 mL in 500 mL flask,and inoculation time 28 h. Under the optimal conditions, the penicillin G acylase activity increased from 7. 34 U/mL to 18. 23 U/mL. （S）-2-chlorophenylglycine was resolved by enantioselective catalytic of N-phenylacetic-（R,S）-2-chlorophenylglycine in aqueous phase for the presence of penicillin G acylase from recombinant Bacillus subtilis. When the substrate concentration was 100 mmol/L and the reaction time was 4 h, the conversion reached 44.2%. （S）-2-chlorophenylglycine was isolated from the reaction mixture of 80 mmol/L substrate concentration in enantiomerically pure form （ 〉 99. 9% e. e. ） and 94. 29% yield. N-phenylacetic-（R）-2-chlorophenylglycine was racemized at 170 ℃ for recycling.