Study On The Immobilization Of Penicillin G Acylase For Cephradine Preparation

Cephradine,a member of the first generation cephalosporins,is widely used clinically due to its anti-inflammatory action.The traditional method for the preparation of cephradine is“acyl chloride”or“acid anhydride”method,which contains many disadvantages such as tedious steps,long operation period,strict operation conditions and serious pollution.So it is important to develop a green and efficient route to synthesize cephradine.This paper mainly focused on the preparation and immobilization of the penicillin G acylase?PGA?from the recombinant strain B.subtilis WB800/pPZW103-PGA,as well as its catalytic properties and applications for the production of cephradine in the packed bed reactor.Firstly the immobilization of PGA was studied using different epoxy resins and the resin of ES-103B was chosen as the best matrix.The optimal conditions were as follows:the ratio between the resins and enzyme solution was 1:14 and the initial enzyme activity was 2.5 U/ml,Co²⁺was added to a final solution of 0.5 mM and equal volume ammonium sulfate?pH 7.0?3M?was also added.Then the system was stirred at 100 rpm for 12 h.Finally,the PGA was immobilized with a specific activity of 9.3 U/g.The catalytic conditions for the synthesis of cephradine were investigated.It showed that the optimal temperature and pH was 15?and 7.5,respectively.The appropriate ratio between 7-ADCA and DHPGM was 1:2.The maximum productivity of 55%was reached with20 g/l of immobilized PGA and 30 mM of substrate concentration.The stability experiments showed that the immobilized PGA had a good operational stability and 85%of the initial activity was remained after 50batches.The maximum reaction rate of 11.5?mol/min·g was obtained when the substrate concentration was 75 mM,the reaction would be inhibited when the substrate concentration was higher than 75 mM.Finally,the synthesis of cephradine in a packed bed reactor?PBR?was investigated.The optimized operation parameters were as follows:50ml substrate solution with 30 mM 7-ADCA and 60 mM DHPGM was recycled through the PBR with a feeding rate of 50 ml/min.The H/D of the PBR was 6 and the additional amount of immobilized PGA was 6 g.It took an hour to get the maximum productivity of 55%in the optimum conditions.Only 7%of the activity of the IPGA was decreased after reused 50 batches with reaction 1h at each batch.In addition,the optimal substrate concentration was increased from 75 mM to 100 mM at PBR.