Study On Fermentation And Purification Of Polysialic Acid

Polysialic acid is a homopolymer of sialic acid withα-2,8 orα-2,9 ketosidic linkages or a mixture ofα-2,8 andα-2,9 which are found in bacterial capsular homopolysaccharide. Polysialic acid is not only a promising scaffold material for the application in tissue engineering or other biomedical applications, but also polysialic acid has potential application in controlled-drug release as polysialic acid can prolong the active life and improve the pharmacokinetics of peptides and proteins. Polysialic acid can be hydrolyzed into sialic acid. Sialic acids and its derivatives were obtained after purification and conversion by chemical and enzymatic methods.Production of polysialic acid by batch fermentations with Escherichia coli K235 at different agitation rates was investigated. Based on that maximal values of specific growth rate and specific production rate of polysialic acid obtained at different agitation rates in different culture stages, a two-stage agitation rate controlling strategy was proposed, in which agitation rate was controlled at 500 r/min in the first 15 h and then switched to 700 r/min afterwards. As a result, the polysialic acid reached 3966 mg/L, as compared to that with constant agitation rate of 500 r/min or 700 r/min, increased by 31.8 % and 49.3 %, repectively. Combined the two-stage agitation rate controlling strategy with fed-batch fermentation technology, Polysialic acid was improved to 5108 mg/L with Polysialic acid yield on sorbitol of 0.12 g/g.Different initial sorbitol concentrations were studied to determine their effects on microbial growth and substrate consumption. It was concluded that high concentration, high yield and high productivity of both cell and polysialic acid could not be consolidated in a batch process.The effects of different cultivation modes such as single short feeding, batch feeding, constant-rate feeding on polysialic acid production were also inverstigated. High production of biomass. and polysialic acid were all realized in these cultivation modes of sorbitol feeding. As a whole, single short feeding culture was the most suitable approach to attain not only high concentrations but also high yields and high productivities of Polysialic acid under which the maximum Polysialic acid reached 6638 mg/L.Based on the previous work, polysialic acid was extracted from fermentation broth. Refining effect was studied by columns chromatography and ultrafiltration technique. The optimal refining processes were as follows: the crude polysialic acid passed ultrafiltration membrane (100 KDa), then were purified by DEAE Fast Flow column chromatography and Sephacry S-200 HR column chromatography. The purity of polysialic acid product was 99.71 %, with the protein no detected and the bacterial endotoxins at 100-500 EU/mg PSA.