The Effect Of PtsG Gene Deficient In Escherichia Coli On The Expression Of CPC Acylase Gene

Cephalosporin C acylase is an enzyme that can hydrolyze the side chain of the Cephalosporin C to form 7-aminocephalosporanic acid.In this study,the enzyme gene was transformed into Escherichia coli BL21(DE3).After optimizing the fermentation medium,the enzyme activity was improved.In the study process,we tried to improve the enzyme activity of cephalosporin C acylase through the following two ways.In the first way,we utilized the Vitreoscilla hemoglobin(VHb)to enhance the uptake of oxygen and biomass utilization ability of the cell,for promoting cell growth and synthesis of metabolites under microaerobic conditions,than improved the cephalosporin C acylase activity.In this paper,the combination of Vitreoscilla Hemoglobin gene(vgb)and Cephalosporin C acylase gene(CPCacy)was used to investigate the effection of VHb on the activity of CPCacy in Escherichia coli.In the process of optimizing the medium,it was found that the glycerol of carbon source had a significant effect on the enzyme activity.With the increase of glycerol concentration,the OD600 value of the fermentation liquid was similar to the diauxic growth curve.Glucose is the first utilization carbon sources when the medium containing glucose,then the utilization of other carbon sources could be limited.If we can find a way to limit the bacterial to absorb the glucose,that will promote the utilization of other carbon composition effectively.In the secend way,we through the method of phage P1 transduction in this study,we had replaced the pts G gene in Escherichia coli BL21(DE3).The pts G gene encodes the specific glucose transporter II CBGlc in phosphotransferase system,when it had been replaced that would reduce bacteria translocation of glucose utilization,and mobilize other related enzymes to synthesize bacterial metabolic pathways such as protein,improve the utilization of biomass on other kinds of carbon the source composition of the culture,increase the density of bacteria fermentation liquid,enhance the synthesis of exo genous protein.Finally,more CPC acylase had been obtained and hihger enzyme activity had been increased after the fermentation.The four expression vectors p ET-28a-CPCacy,p ACYCDuet-1-CPCacy,p ETDuet-1-CPCacy and p RSFDuet-1-CPCacy were transformed into the BL21(DE3)which was a strain with no resistance and no pts G gene,then obtained four recombinant bacteria.Comparative study on the cephalosporin C acylase activity,we found that the pts G-type strains were higher than that of the pts G+ type strain,the most obvious improvement of four strains is the p RSF-1-CPCacy(pts G-)which was 2.86 times of its pts G+ type.Further studies on the optimization of carbon sources showed that the addition of 0.3% glycerol or glucose in the LB medium could significantly improve the activity of the enzyme in all kinds of Engineering bacteria.In the pts G+ type,the effect of adding glucose on the activity of the enzyme was better than that the effect of adding glycerol,while in the pts G-type,the effect of adding glycerol on the enzyme activity was more obvious.After we added the glycerin and teminated the fermentation,about the expression vector for pts G deficient p ACYCDuet-1-CPCacy engineering bacteria,found that its enzyme activity was 4.197U/m L,and which was 6.59 times aa much as the activity of its pts G+ type strain without additional carbon sources added to LB.We have succeed that the pts G-deficient BL21(DE3)was applied to the expression of cephalosporin C acylase,and we obtained the obvious effection by optimizing the composition of carbon source.It affords us a new way to promot the CPCacy activity in Escherichia coli.at the same time,further optimization of culture medium composition and other nutrient conditions and other factors,will be expected to be further enhance the CPCacy activity and improve the feasibility of the production in industrial fermentation of cephalosporin C acylase.