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.
出处
《生物加工过程》
CAS
CSCD
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)
