盤尼西林醯化酵素生產菌在二水相系統中之反應

Abstract

Design composition of the medium and strageies of cultivation have been investiged in this disseration . It is useful not only to reach high densityof biomass but also to increase of penicillin acylase production . Then penicillin acylase inside the cells of E. coli hydrolyze penicillin G inaqeous two-phase system . This system can improve the productivity of 6-APA . The purpose of the dissersion was also on aqeous two-phase systemdesign for biochemical process . Because the recombinent E. coli ATCC 9637 produce penicillin acylaseinhibited by carbon source , we discussed that different carbon source influence penicillin acylase production by E. coli . Simple batchculture is unsuitable because of the necessory to supply high initialconcentrations of nutrients which induce a substrate inhibition . Fed-batchculture appears to be an interesting alternative as it is possible to control the concentrations of substrates and nutrients in suitable ranges during thecultivation . Fed-batch culture is useful not only to reach high densities of biomass within short cultivation time but also to increase of 1.2 fold inpenicillin acylase production . The results exhibited that the production of penicillin acylase is strongly dependent on the growth rate so we think howto reach high cell densities . When we modified the composition of medium and feeding substrate , the cell concentration increased 1.77 fold andpenicillin acylase production by E . coli . increased fold in comparisonwith the initial valve . When the bacteria grow too fast to uptake adequateoxygen , we supply pure oxygen gas to the fermentor in order to obtainhigh density of biomass . under the condition the cell concentrationincreased 2.6 fold and penicillin acylase production by E. coli increased2.5 fold in compare with the initial value . Molecular weight and concentration of polyethylene glycol and concentrationof phosphate buffer influence partition coefficiency of bacteria , 6-APAand PAA in aqeous two-phase system . Higher concentration of polyethylene glycol and phosphate buffer inhibit the activity of penicillin acylaseinside the cells of E. coli . Design and perforance analysis of the best composition of aqeous two-phase system for penicillin acylase inside the cells of E. coli . First , we design the best reaction condition for penicillin acylase inside the cells of E. coli because the activity of penicillin acylase is inhibited by substrate and phosphate buffer . Secondly , we used repeated batch process in aqeous two-phase system for the reaction of penicillin acylase . This system can resolve the problem of product inhibition . By mixing two aqeous solutions of polyethylene glycol and phosphate buffer , an opaque solution is formed which spontaneously seperates into two aqeous phases . When the top phase is removed , the fresh composition of the top phase should be added for remaining the aqeous two-phase system . Using this system extened the half-life of enzyme in order that enzyme was reused more times than before . When different strangies of cultivation were applied and the composition of the composition of the medium was modified , cell concentration increased 2.6 foldand penicillin acylase production by E. coli increased 2.5 fold in comparition with the initial valve . The aqeous two-phase system is superior to immobilized the whole cells because the aqeous two-phase system can overcome severe disffusion restrictions and some difficulties in scale up . Before the conversion rate enzyme began to decrease , enzyme was used ten times which was more than fourfold in comparision with the highest times in the literature .