Cloning And Expression Of Penicillin G Acylase And Application Of Resolution Of Racemic 2-chlorophenyl Glycine

Optically active 2-chlorophenyl glycine is one of the most important pharmaceutical intermediates with broad use.It can be used for synthesis of antiplatelet medication clopidogrel.Penicillin G acylase has highly stereoselective towards acyl chains containing benzene ring.We obtain enantiomerically pure(S)-2-chlorophenyl glycine,which is based on the principle of enantioselective hydrolysis of N-phenylacetyl derivatives of 2-chlorophenyl glycine with Penicillin G acylase,R-configuration remains untreated and can be racemized for recycling and create a routing of stereoselective catalyzed production of(S)-2-chlorophenyl glycine by penicillin G acylase.In this paper,the penicillin G acylase gene was cloned in Escherichia coli and Bacillus subtilis.Firstly,penicillin G acylase gene was cloned into pCDFDute-1 and the recombinant plasmid was introduced into E.coli BL21(DE3).Through the reaction of colony PCR and analysed by SDS-PAGE,the recombinant E.coli BL21(DE3)/pCDFDute-PGA was successfully constructed but has lower PGA activity.Then,the penicillin G acylase gene was cloned into pPZW103 to construct recombinant plasmid pPZW103-PGA and the recombinant plasmid was introduced into B.subtilis WB800.SDS-PAGE indicated that the recombinant B.subtilis WB800/pPZW103-PGA was successfully constructed and can express of penicillin G acylas in extracellular.Cultivation conditionss of recombinant B.subtilis expressing penicillin G acylase were investigated.The optimized fermentation conditions were as follows:soluble starch 10.0 g/L,peptone 12.0 g/L,yeast extract 3.0 g/L,NaCl 10.0 g/L;pH 7.5,fermentation temperature 37?,initial pH 7.5,flask column 80 mL in 500 mL flask,inoculation time 28 h.Under the optimal conditions,the penicillin G acylase activity increased from 7.34 U/mL to 18.23 U/mL,which was 2.48 times higher than the initial numerus.The penicillin G acylase was purified and characterized in this paper.The PGA was purified to 4.6 folds with a yield of 55.5%through ammonium sulfate precipitation,DEAE IEX,t-Butyl HIC.The purified enzyme consisted of two dissimilar a and P subunits with molecular masses of approximately 24.2 and 61.4 kDa.The enzyme was higher activity at a pH range of 9.0-11.0.The optimal reaction temperature was 40 ?.The half-life of PGA at 40 ? and 50 ? were 75.4 h and 24.8 h respectively.Fe2+?Ag+?Al3+ and SDS inhibited the activity of PGA and Co2+ stimulated the activity of PGA.Tween-80 and sorbitol had a little influence on enzyme activity.Substrate specificity of PGA was also studied.The PGA had high activity and good S enantioselectivity,the e.e.of all products were above 99.9%.The kinetics parameters of five substrates were determined.The catalytic mechanism of penicillin G acylase torwards five substrates was explain by molecular docking and also explain the difference of enzymatic activity towards the five substrates.The influence of reaction conditions on bio-resolution of N-phenylacetyl derivatives of 2-chlorophenyl glycine to produce(S)-2-chlorophenyl glycine by PGA were also investigated.When pH was 10.0,40 ? and enzyme concentration 0.126 mg/mL,the substrate concentration was 110 mM,48.11 mM of product was obtained in enantiomerically pure form(>99.9%e.e.).In fed-batch transformation,180 mM of substrate concentration was added and the final product concentration reached 79.53 mM.The(S)-2-chlorophenylglycine was isolated and purified from reaction mixture with total yield of 47.15%.The sample was characterized by polarimeter,'H NMR and HPLC analysis and the results demonstrated that chemical and optical purity of the sample were both above 99%.N-phenylacetic-(R)-2-chlorophenylglycine can be racemized for recycling,100%of racemization rate and 94.7%of racemic yield.Also phenylacetic acid was separated with yield of 69.7%.