重组青霉素G酰化酶拆分制备(S)-邻氯苯甘氨酸  

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

作　　者：刘学[1] 薛亚平[1] 柳志强[1] 王亚军[1] 郑裕国[1] 

机构地区：[1]浙江工业大学生物工程研究所教育部生物转化与生物净化工程研究中心,杭州310014

出　　处：《生物加工过程》2013年第1期5-11,共7页Chinese Journal of Bioprocess Engineering

基　　金：国家重点基础研究发展计划(973计划)资助(2011CB710804)

摘　　要：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12 g/L、酵母粉3 g/L、NaCl 10 g/L;pH 7.5、培养温度37℃、装液量80 mL(500 mL三角瓶)、培养28 h,青霉素G酰化酶的表达水平由最初的7.34 U/mL提高至18.23 U/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4 h,转化率达44.2%。对底物浓度为80 mmol/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。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.

关 键 词：青霉素G酰化酶 (S)-邻氯苯甘氨酸 拆分 优化 

分 类 号：O623.736[理学—有机化学]