Optimization Of PNPase Fermentation In Recombinant E. Coli BL21

In this paper, we researched the effects of medium composition on the fermentation of E. coli BL21 in expressing purine nucleoside phosphorylase (PNPase) by complex medium and synthetic medium. In complex medium,we found that the optimal C/N ratio for cell growth and protein express is 3:1;the mineral element K has an important role in promoting cell growth、percentage of target protein and total enzyme activity; the variation of organic and inorganic nitrogen ratio make no different on cell growth, but have a significant role in protein expression which persents a trend that the less organic nitogen the less protein exression. In synthetic medium,microelemet is absolutely necessary for high cell density culture; the optimal C/N ratio is 10:1. Compare complex medium with synthetic medium, we got the same final biomass at optimal C/N ratio, but got big differ in protein expression. This might be caused by the difference of organic and inorganic nitrogen, so we make a comparion. It was found that inorganic nitrogen is essential for high cell density culture. when inorganic nitrogen is absent,the cell can’t grow to high density, also the protein production are very low. Add inorganic nitrogen to medium,the cell will grow nomally to high density, but it must be get available organic nitrogen (suitable organic and inorganic nitrogen ratio). In order to intensive study the relationship between nutrition supply (especially nitrogen resource) and cell growth、protein production, an IP-HPLC method was established for the determination ofα-ketoglutarate in E.coli. Based on this method, we monitored the variation ofα-ketoglutarate on different medium. The result show that the variation ofα-ketoglutarate can reflect the nutrition conditions(rich or pool) by alteration its concertration(maintein a constant level or accumulation). This result is also verified by John A and Jeremy A.