Application Of Immobilized Penicillin G Acylase In The Enzymatic Synthesis Of Cefaclor

Cefaclor which belongs to the the second generation of oral cephalosporins is widely applied.It can kill many kinds of gram-positive and gram-negative bacterium strongly.In this paper,enzymatic synthesis of cefaclor from 7-aminodesacetoxymethyl-3-chlorocephalosporanic acid(7-ACCA)and phenylglycine methyl ester hydrochloride(PGME·HCL)with penicillin G acylase(PGA)in phosphate buffer was studied.Recombinant Escherichia coli JM109/pET24 a was cultivated in M9 medium,IPTG as inducer.The free penicillin G enzyme can be obtained by ultrasonication.Then we used this enzyme solution as catalyst to synthesize cefaclor.The effect of pH,temperature,substrates concentrations,enzyme to 7-ACCA and acyl donor to nuclephile ratios on the conversation of 7-ACCA to cefaclor was investigated.The optimized catalytic conditions were as follows: PGME·HCL:7-ACCA=2:1,7-ACCA 210 mM,33.3U/mmol 7-ACCA,pH 7.0,15?C.Under the optimal conditions,the conversation of 7-ACCA to cefaclor can reach96.73% and the volumetric productivity(P)was 243.77mM/h.Kinetic study of this PGA synthesizing cefaclor,cefalexin and cefadroxil was also investigated.We chose a domestic epoxy resin LX-1000EP(C)as carrier to immobilize the PGA from recombinant E.coli JM109/pET24 a by multipoint covalent binding.Then we used this immobilized enzyme as catalyst to synthesize cefaclor.The conversion of 7-ACCA to cefaclor attained 99% via optimising reaction conditions such as p H,temperature,substrates concentrations,enzyme to 7-ACCA and acyl donor to nuclephile ratios.It is the highest conversation compare to those already reported.Substrate specificity of this immobilized PGA synthesizing cephalosporin antibiotics(cefaclor,cefalexin,cefadroxil,cefradine)was also investigated.The stability of this immobilized PGA was studied lastly in this paper.This immobilized PGA showed no activity loss in 4?C after 30 days.It also had good pH stability between pH 5.5 to 8.5 and good temperature stability in 20?C and 30?C.After 50 sequential batch reactions to synthesize cefaclor,the conversation can still attain more than 94%.It means that stability of the immobilized PGA was very high and this biocatalyst is potential for large scale production of cefaclor.