Study On Enzyme Activity Stability Of Penicillin Gacylase During The Process Of Enzymatic Synthesis Of Cephalexin And Its Regeneration

Cephalexin is the first generation of cephalosporin antibiotics for oral use.It has the advantages of broad antimicrobial spectrum,strong bactericidal power,good acid resistance and good gastrointestinal absorption.This antibiotic is listed in the catalog of essential medicines in China and other countries.Compared with the traditional chemical method,the enzymatic synthesis of cephalexin has the advantages of mild reaction condition,simple process,no group protection,clean and safety.At present immobilized penicillin G acylase(PGA)is used to synthesize cephalexin in China and other countries.Immobilized PGA is expensive.How to keep enzyme activity of PGA to be stable for a long time so as to achieve maximum reuse number of immobilized PGA,is one of the most concerned problems in the current enzymatic synthesis of cephalexin.In this study,the factors which cause the decrease of PGA enzymatic activity,improvement of the enzymatic activity stability of immobilized PGA and the regeneration of inactivated immobilized PGA were investigated.Study on the factors causing the decrease of enzymatic activity stability of immobilized PGA.7-ADCA and PGM had a strong inhibitory effect on immobilized PGA.The inhibitory effect of PGM was almost independent its concentration.Cephalexin showed a product inhibition,and its inhibition was caused mainly by its hydrolysates.When reaction temperature was ? 30°C and pH of reaction solution was 6.5,immobilized PGA presented perfect enzyme activity stability.Added disodium hydrogen phosphate-citric acid buffer solution could improve the stability of immobilized PGA.Study on improvement of the stability of immobilized PGA penicillin.The effects of metal ions,surfactants,sugars,amino acids and alcohols on immobilized PGA were investigated.Ca2+ had activation on immobilized PGA,and its enzyme activity was increased by 10.65%.The addition of fructose,polyvinyl alcohol and isopropanol could significantly increase the activity of immobilized PGA.Lysine and glycine had a great activation on the immobilized PGA,which increased the activity of PGA more than 34%,while the tryptophan and folic acid had obvious inhibitory effect.Isopropanol,L-lysine,Ca2+,polyvinyl alcohol were added to the reaction solution,no synergistic effect between them was observed.When the enzymatic synthesis of cephalexin was carried out,0.5%isopropanol was added into the reaction solution,and 16 reuse number of immobilizedPGA was achieved,which was 45.45% longer than the control.The enzymatic stability of immobilized PGA was remarkably improved.Reactivation of immobilized PGA was carried out.Deionized water washing and acid water(pH 6)washing had no reactivation on immobilized PGA,while alkaline(pH 8)washing had a reactivation effect.The enzyme activity increased by 5.7% by washing with alkaline water at 30°C for 60 min.Ultrasound treatment(frequency of 28 KHz,15 min)increased the eznyme activity of inactivated PGA by 5.67%.The unfolding-refolding method was ineffective for regeneration of inactivated immobilized PGA.Direct reactivation(30% ethylene glycol+100 mM7-ADCA)could reactivate immobilized PGA,and made its activity increase by 13.81%.The optimal reactivation method of immobilized PGA was as follows: immersing with 30% ethylene glycol-100 mM7-ADCA solution for60 min,then washing with alkaline water for 60 min,finally ultrasonic treatment at28 KHz for 15 min.The recovery rate of immobilized PGA was upto 77.75%,and the reactivation result was perfect.