Mechanism On Influence Of Acetate And K₂HPO₄ On Biosynthesis Of Molecular Weight Stability Of Polysialic Acid

Polysialic acid(PSA)is a linear polyanionic polysaccharide composed of N-acetylneuraminic acid linked by ?-2,8 glycosidic bonds,usually with a degree of polymerization ranging from 8~400 dp.PSA has potential usage in cosmetics,biomedicines,tissue engineering,etc.Escherichia coli K1 and K235 are commonly used for the PSA fermentative production,and the process of shake flask fermentation required a higher titer of K2HPO4.In reactor,cultivations required control of pH at neutral condition.Especially in the later stage,the production of polysialic acid and molecular weight will decline rapidly due to the accumulation of organic acids,resulting in the instability of molecular weight of polysialic acid.In this thesis,the influence of the acetic acid and potassium on the PSA biosynthesis process and molecular weight stability of PSA were investigated.Firstly,the influence of acetic acid accumulation on the biosynthesis of PSA was studied.Different carbon sources were used for PSA fermentation.The yield and molecular weight of PSA were the highest when sorbitol was used as carbon source.When xylose was used,a lot of pyruvic acid occurred.As a result,the concentration of acetic acid in the later period was higher with glucose or sorbitol as carbon sources.Furhter,exogenous acetic acid stress was applied.When the concentration of acetic acid was below at 30.0 mmol·L-1,the cell can grow well,with remarkable reduction in PSA molecular weight.The yield and molecular weight of PSA declined a lot when the addition of acetic acid was 30.0~60.0 mmol·L-1.The growth of cell was totally limitted when the addition of the acetic acid exceeded 90.0 mmol·L-1.Secondly,the influence of phosphates on the biosynthesis and molecular weight stability of PSA were studied.Different phosphate in the initial medium was used and Na2HPO4 was found to inhibit cell growth due to high osmotic pressure from Na+,leading to low PSA yield and small molecular weight of PSA.In addition,different initial concentrations of K2HPO4 were tested in a 7 L reactor.Results showed that the yield and molecular weight of PSA were higher when initial K2HPO4 was 5.0 g·L-1,but the molecular weight of the polysialic acid was decreased steadily after 40 h of cultivation.Further,the pH was controlled by KOH solution,and the molecular weight of PSA remained stable at the later stage of fermentation,but the yield of PSA was not improved.The pH control strategy was further changed.The broth pH was controlled by ammonia water before 16 h of cultivation in the 7 L reactor.After 16 h,KOH solution was switched for pH control and a certain amount tryptone was supplemented in the feed sorbitol.As a result,the PSA yield and molecular weight increased and remained stable at the later phase for a comparable long time.This indicates that the presence of a certain amount of phosphate and potassium ions contribute to the PSA biosynthesis and molecular weight stability.Finally,the stability of PSA molecular weight in different metal salt solution was studied.The PSA was dissolved in the different salt solution and the monovalent metal ion could increase the estimated value of PSA molecular weight and the estimated values were proportional to the concentration of salt.Among all these salts and concentrations used,10.0~25.0 mmol·L-1 K2HPO4 increased the PSA molecular weight significantly.However,the influence disappeared when K2HPO4 was higher than 50 mmol·L-1.Then,the Zeta-potential of the PSA in different salt solution was measured.Interestingly,the Zeta-potential of the PSA solution increased with the concentration of metal ions.Again,K2HPO4 posed the significant influence on the change of the Zeta-potential.When the concentration of K2HPO4 was higher than 50.0 mmol·L-1,the Zeta-potential of PSA solution was positive.With the observation of UV-visible(UV-vis),circular dichroism spectroscopy(CD)and transmission electron microscopy(TEM),it was found that potassium ion could affect the spatial structure of polysialic acid in aqueous solution.Accordingly,we speculate that the monovalent metal ion such as K + and the like can interact with the sialic acid residue C1 in the PSA.The ionic bond and the electrical neutralization reduce the mutual repulsion between the PSA macromolecules;the aggregation of the two PSA molecules by K2HPO4 is favorable for the stability of PSA marcromolecule.