Immobilization Of Penicillin G Acylase And Its Applicatin Of Production Of(S)-2-chloroohenyl Glycine

(S)-2-chlorophenyl glycine[?S?-2-amino-?2-chlorophenyl?ethanoic acid]is a very important pharmaceutical intermediate,one of its maintain use is to synthesis the Clopidogrel.Traditionally,the approach for synthesis of?S?-2-chlorophenyl glycine is using tartaric or camphor sulfonic to formate diastereomeric salts then followed by fractional crystallization.However,these methods have many shortages including long time cost,low productivity,low purity of chirality and so on,so it may be very meaningful to create a routing to avoid these inadequates.In this work,we develop a strategy for immobilization of Penicillin G acylase?PGA?form B.subtilis,which can produce?S?-2-chlorophenyl glycine.This paper is focused on the process of immobilization,the characteristics of immobilization PGA and the possibility of using immobilized PGA to produced?S?-2-chlorophenyl glycine in a packed bed reactor.Firstly,a domestic epoxy resin LX-1000EP?C?is chiosed as carrier to immobilize the PGA by multipoint covalent binding.The optimal conditions for immobilization are determined as follows:25?,pH 7.0,the proportion of resin and enzyme solution is 1:10?V/m?,0.5 mM of Co²⁺ and 1.5 M of ammonium sulphate are added to the enzyme solution?31 U/mL?before immobilization.The immobilization system is stired by electric mixer for 6-12 h and finally gets the immobilized PGA with 75 U/g wet support.The PGA is proved to multipoint covalent with the support by the SDS-PAGE analyses of the supernatants.The characterize of the immobilized PGA is investigated.The optimal reaction temperature is 65?,the half-time of the immobilized PGA at 30?,40? and 50? are 61.59 day,49.94 day and 9.6 day,respectively.The immobilized PGA can maintain its activity almost unchanged when the pH increases from 7.0 to 10.6 and the activity is two times than its original activity when the sodium phosphate buffer is replaced by glycine-NaOH buffer at the same pH value.Substrate specificity of the immobilized PGA is also studied.Each kind of substrate is resoluted at different levels and all the products are S enantioselectivity and the e.e.value are above 99.9%.The immobilized PGA is also employed to produce P-lactam antibiotics and 6-aminopenicillanic acid?6-APA?,an important ?-lactam intermediate.The react condition is 25? and keeps pH at 6.0,7.0 and 8.0,the proportion of the ?-lactam nuclei and side-chain is 1:2.The best conversion of caphalexin,cefadroxil,cefaclor and cephradine is 73.3%,40%,90%and 90%,respectively.The 6-APA has a 16%conversion rate in pH 8.0,2%MIBK aqueous systems.The react condition of using immobilized PGA to produce?S?-2-chlorophenyl glycine is also investigated.The max initial rate is 159 ?M/?min.g?at 100 mM substrate and when the concentration of substrate more than 100 mM will display inhibitory effect on the activity of immobilized PGA.?S?-2-chlorophenyl glycine showes competitive inhibition while the vice product phenylacetic acid has very little inhibition effect on the immobilized PGA.The Km and Vm of immobilized PGA at 30? are 20.64 mM and 3.852 mM/min,respectively.The inhibition constant of substrate and product?S?-2-chlorophenyl glycine are 282.34 mM and 14.97 mM,respectively.Finally,a packed bed reactor?PBR?is used to produce?S?-2-chlorophenyl glycine.The optimized operation parameters are examined as follows:50 mL,100 mM of substrate is recycled through the PBR with a feeding rate of 18 mL/min,the H/D of the PBR is 8 and the immobilized PGA content is 8.0 g.It only needs 18 min to get 98%conversion of substrate in the optimized condition.The immobilized PGA resolution of substrate in PBR is semi-continuously operated for 50 batches and it is worth noting that the activity of immobilized PGA is almost unchanged.