Study On The Preparation Of Polysialic Acid By Microbial Fermentation

Polysialic acid (PSA) is a polymer of sialic acid linked with?-2, 8-and/or?-2, 9-glycosidic (ketosidic) bonds. PSA has many advantages, such as poor immunogenicity, biodegradable and so on, which is considered as the most ideal material used in the control-release drugs and scaffolds in biomedical applications. In addition, various sialo-products have been derivated by means of treating PSA with degradation methods or enzymatic catalysis, and these derivatives can subsequently be used in pharmaceutical, food and health-care industries.Bacterial fermentation is the only way to produce PSA up to now, and Escherichia coli CCTCC M208088 was adopted in this study. The aim of this thesis is to efficiently prepare PSA by optimizing processes including fermentation and purification. At first, a series of strategies aiming to strengthen PSA synthesis by Escherichia coli CCTCC M208088 were employed to improve the fermentation profiles by taking several aspects into concern which included cultivation conditions, microbial metabolic characteristics, biological stresses and PSA releasing efficiency. Then, a comprehensive and integrated purification process was established based on properties of the PSA fermentation broth. Outlines of this thesis are shown as follows:(1) The cultivation conditions (pH and dissolved oxygen control strategy) for PSA synthesis by E. coli CCTCC M208088 were optimized. Ammonia water was adopted for pH control through optimization, and PSA production were significantly increased from 1.92 g/L to 3.03 g/L. Meanwhile, the residual phosphate in the broth was decreased from 19.31 g/L to 1.72 g/L, which significantly reduced the difficulty of the subsequent PSA purification and wastewater treatment. Besides, the effects of agitation speed and dissolved oxygen level on PSA biosynthesis were investigated in shake flasks and fermentors, which proved that PSA biosynthesis favored high-level dissolved oxygen (DO) environment. Based on the effects of various agitation rates on PSA production, a two-stage strategy for PSA production was carried out: low stirring rate of 500 r/min was conducted during pre-fermentation (before 12 h) and then switching to high stirring speed (700 r/min) from 12 h forwards. With this strategy, PSA production reached 3.92 g/L.(2) The fermentation modes for PSA synthesis by E. coli CCTCC M208088 were optimized. The effects of batch fermentation and fed-batch fermentation modes (pulse fed-batch, constant feeding rate fed-batch, variable feeding rate fed-batch and exponential feeding rate fed-batch) on the PSA fermentation were determined. The results showed that batch fermentation could improve PSA synthesis, while fed-batch culture was more preferable to bacterial growth. Therefore, a combination of fed-batch and batch culture was proposed: fed-batch fermentation (exponential feeding rate fed-batch) was carried out to increase cell density rapidly, as the cell density increased nearly its maximum level (16 h), high concentration of sorbitol was added into the fermentor at once, switching to batch fermentation. As a result, PSA production was increased to 5.70 g/L.(3) The metabolic characteristics of PSA synthesis by E. coli CCTCC M208088 were investigated, and the effect of pyruvate addition on the metabolic flux distribution in E. coli CCTCC M208088 was studied. The results suggested that the addition of pyruvate can apparently strengthen the tricarboxylic acid cycle, and can improve the productivity of ATP. Meanwhile, the carbon flux of pentose phosphate pathway was reduced to a certain degree, and the competition between cell growth and product synthesis was weakened, thereby, enhanced the efficiency of PSA biosynthesis. Based on these facts, a new strategy combining addition of pyruvate with the optimized combination of fed-batch culture and batch culture fermentation strategy was developed, resulting in PSA production of 6.92 g/L.(4) Biological stresses and facilitating releasing strategy were adopted to strengthen PSA synthesis by E. coli CCTCC M208088. The effects of various surfactants on release and biosynthesis of PSA by E. coli CCTCC M208088 were investigated. The results showed that Tween-60 can promote the release of PSA, accordingly, promoting the biosynthesis of PSA. Tween-60 addition strategy was optimized and confirmed in PSA batch fermentation in 7 L fermentor. As a result, PSA yield was increased by 10.9% by adding 0.4 g/L Tween-60 at 12 h. Moreover, the effect of H2O2 stress on PSA biosynthesis was investigated. The results revealed that addition of H2O2 to 7.5 mmol/L at 4 h, 15 mmol/L at 8 h, 15 mmol/L at 12 h and 25 mmol/L at 16 h can maximally increase PSA production. This strategy was confirmed in a 7 L fermentor with batch fermentation, as a result, PSA production increased by 19.8% as compared to the control.(5) In order to establish PSA purification process, the stability and separation characteristics of PSA as well as the methods for removing the main impurities from the fermentation broth were studied. The condition for PSA maintaining stability was determined: the heat treatment tempreature <100?, pH 5-10. Furthermore, the optimal cetyl pyridinium chloride (CPC) precipitation procedure was determined: addition of 3 g CPC/g PSA, pH of 6-7, NaCl of 0.1 mol/L or less and temperature lower than 40?. The suitable condition for ethanol precipitating PSA was determined: the volume of ethanol added was three times that of treatment solution and 0.68 moL/L NaCl was added. The suitable operating condition for the perlite filtrating and eliminating impurities was determined: the pH of treatment fluid modified to pH 10, simultaneously; 80 g/L perlite should be adopted.(6) Separation and refining process for PSA from the fermentation broth was proposed. To separate PSA from the broth, Fermentation broth was pretreated at 80?for 30 minutes, followed by centrifugation to remove biomass. The supernatant was precipitated with ethanol, and then the precipitate was dissolved in water. The mixture was filtered with perlite filter and the filtrate was then desalted by ultrafiltration. The resultant PSA solution was precipitated by CPC, and the PSA-CPC precipitate was dissolved in 0.8 mol/L NaCl solution. Then PSA was precipitated with ethanol again. Finally, PSA was obtained after vacuum-drying. The purity of PSA product obtained was higher than 95% at 50-60% recovery rate. To obtain pharmaceutical grade PSA, the refining process for the PSA sample was tested: PSA sample was sequently treated by ultrafiltration, ion exchange chromatography and gel filtration, and then through subsequent desalination, condensation, and freeze-drying. In the refined PSA, no protein was detected and endotoxin content was lower than 100 EU/mg. The characteristic IR and NMR spectra of refined PSA were similar to the spectra of?-2, 8 glycosidic linked polysialic acid reported.(7) Pilot production of PSA was verified on 500 L scale. PSA production reached 5.50 g/L, and purity of the obtained PSA reached 95% almost. PSA production, productivity and production scale were the top level reported.