Studies On Fermentation Process Optimization Of K5Polysacchairde And Its K5Lyase From Escherichia Coll

Heparan sulfate (HS) is a drug that has the pharmacodynamics of anticoagulant,antithrombotic, anti-HIV infection and slowing down the cells in vitro apoptosis. The capsularpolysaccharide of E. coli K5can be used as the substrate for the enzymatic synthesis of HS.To improve the production of K5polysaccharide, the fermentation conditions wasoptimizated by orthogonal test, and the optimum medium composition and the optimal cultureconditions was obtained. During the fermentation process of K5polysaccharide, K5lyasedegradation was found in the fermentation broth. Its degradation products were low molecularweight of K5polysaccharide whose average molecular weight was3852Da. According to thisfeature of the K5lyase, the enzyme was used as the tools of producting low molecular weightK5polysaccharide. The K5lyase gene-elma was amplified by PCR, and the expression vectorpET-28a-Elma was constructed. Elma was induced expression in E. coli BL21, the enzymewas purified by Ni2+-NTA affinity chromatography and G-75molecular sieve chromatography,then the enzymatic properties of purified enzyme was studied. At the same time K5polysaccharide lyase gene was knockout in E. Coli K5，and we studied the impact of theabsence of K5lyase in K5polysaccharide production. The results were as following.(1) We got optimization of fermentation conditions by orthogonal test, the best mediumcomposition: ρ （MgSO47H20）2.0g/L, ρ(maltose)20g/L, ρ (peptone)15g/L,MnCl4H2O0.1g/L, KH2PO42g/L, K2HPO49.7g/L, sodium citrate0.5g/L. The best liquidvolume is15mL. And in both flask and5L fermenter, the fermentation process wereresearched and the curve of the fermentation process was drawn. K5polysaccharideproduction is up to1.8g/L.The molecular weight analysis of K5polysaccharide in5Lfermentation show that K5polysaccharide of molecular weight turn low as the fermentationtime extension.(2) The gene of Elma was obtained by PCR amplification and inserted into pET-28a,then the recombinant expression vectors pET-28a-Elma was transformed into E. coli BL21(DE3). After induction with0.2mmol/L IPTG at16℃for5h, Elma were successfullyexpressed, SDS-PAGE analysis determined that the enzyme constituted above30%of thetotal cell proteins. After Ni2+-NTA affinity and G-75chromatography, the purity was morethan95%. Enzymatic activity was expressed as the change of absorbance at232nm per ml ofsample. The change of the polysaccharide molecular can be detected by PAGE electrophoresis.Elma can partially split HA and HS. Its optimal temperature is37℃and optimal pH is7.0.(3) We knockout K5-lys through the Red recombination system. The DNA fragmentwhich has homologous arm and the chloramphenicol resistance was successfully cloned byPCR, then we induced homologous recombination, the E. coli K5△K5-lys was constructed.The E. coli K5△K5-lys K5-lys activity decreased50%than original strain, but it can notgrow in the fermentation medium of high concentration carbon source.