Studies On The Enhanced Production Of Polysialic Acid And Preparation Of Sialic Acid

Sialic acid is a family of of neuraminic acid, which exist mostly the cell membrane surface of mammals and a few bacteria widely found in animal tissues and microorganisms. As an important component of cell membrane glycoconjugates, sialic acid plays a very important role in the regulation of a variety of vital biological processes. Polysialic acid is a polymer of sialic acid linked byα-2,8 orα-2,9- glycosidic (ketosidic) bonds which is the capsular polysaccharide of a few of bacterias. It is supposed to be one of the main preparation methods for sialic acid by hydrolysis of the polysialic acid.Pyruvate is the main prerequisite for the synthesis of polysialic acid. Effect of sodium pyruvate addition on the production of polysialic acid by E. coli CCTCC M208088 was investigated. The results showed that the quantities and addition-time of sodium pyruvate had significant impact on the synthesis of polysialic acid. In order to maximize the synthesis efficiency of polysialic acid, the best strategy for sodium pyruvate addtion was obtained in the shake flask fermentation. The optimal time and concentration for sodium pyruvate addition were 0 h and 6 g/L. At the end of the fermentation, polysialic acid production reached 3470 mg/L, and showed 73% increase as compared with the control. The final polysialic acid production reached 6630 mg/L when the strategy was confirmed in a 30 L fermentor, increasing by 53% as compared with the control.Effect of oxidative stress on the biosynthesis of polysialic acid by E. coli CCTCC M208088 in the shake flask fermentation was investigated. The results showed that H₂O₂ stress could effectively induce the accumulation of polysialic acid. The addition time, the quantity and addition methods of H₂O₂ had significant impact on the production of polysialic acid. By adoping three-points H2O2 stress (4.0 mmol/L at 4 h, 8.0 mmol/L at 8 h, 16.0 mmol/L at 12 h), the highest polysialic acid yield of 2705 mg/L was obtained, and increased 35% as compared to the control. The final polysialic acid production reached 6630 mg/L when the strategy was confirmed in a 30 L fermentor, increased by 53% as compared with the control.In addition, the technology of sialic acid preparation by hydrolysis of polysialic acid was improved in the study. Hydrolysis method of polysialic acid was optimized, and various methods for sialic acid purification were compared. Finally, the preparation of sialic acid monomer by polysialic acid hydrolysis was determined: polysialic acid was dissolved in 0.05 mol/L H₂SO₄ and incubated at 80℃for 4 h. The hydrolysate was directly mixed with five times volume of glacial acetic acid, and then let standing at 4℃for 3 days. The sialic acid crystal was obtained by filtering and washing with acetic acid. The final product of sialic acid monomer with 95% purity was obtained by vaccum drying.