【作者】 刘金龙；

【导师】 詹晓北；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 博士

【摘要】 聚唾液酸(Polysialic acid)是唾液酸以α-2, 8和(或)α-2, 9糖苷键连接而成的同聚物。聚唾液酸具备良好的生物相容性、非免疫原性和可降解性,是理想的药物缓释和医用支架材料。此外,聚唾液酸经降解或者酶催化可得到一系列的唾液酸类衍生物,这些衍生物多为应用于医药或食品和保健品领域的高附加值产品。微生物发酵法是目前获得聚唾液酸的唯一途径,本研究采用的菌株是大肠杆菌CCTCC M208088 (Escherichia coli CCTCC M208088)。本论文旨在通过对聚唾液酸制备过程中的发酵和提纯工艺进行优化,实现聚唾液酸的高效生产。首先,从E. coli CCTCC M208088合成聚唾液酸的培养条件、培养方式、代谢特性、生理胁迫、释放效率等多个角度出发,实施一系列以强化聚唾液酸合成为目的发酵调控策略;其次,基于聚唾液酸发酵液的特性,构建出一条完整的提取纯化工艺。本论文主要研究内容总结如下:(1)对E. coli CCTCC M208088合成聚唾液酸的培养条件(pH和溶氧调控方式)进行了优化。经过pH调控策略的优化,确定了氨水流加控制pH的策略,结果聚唾液酸产量大幅提高了58%,达到3.03 g/L。与此同时,残留磷酸盐浓度从19.31 g/L左右降到1.72 g/L,大大减轻了后续产物提纯和废水处理的难度。在此基础上,进一步考察了搅拌速率和溶氧浓度对聚唾液酸发酵的影响,发现较高的溶氧条件有利于聚唾液酸的合成。通过对不同搅拌速率发酵结果的比较发现:在500-700 r/min范围内,低搅拌速率利于菌体的生长,而高搅拌速率有利于产物的合成。于是引入了分阶段调控策略,在发酵前期(12 h之前)采用低搅拌速率500 r/min,保证菌体的最适生长状态,而在发酵中期开始(12 h之后)采取高搅拌速率700 r/min,结果聚唾液酸产量进一步提高至3.92 g/L。(2)对E. coli CCTCC M208088合成聚唾液酸的培养方式进行了优化。考察了分批培养和分批补料培养模式(间歇式流加、恒速流加、变速流加、指数速率流加)对聚唾液酸发酵的影响。结果表明,分批培养有利于聚唾液酸的合成,而分批补料培养有利于菌体的生长。为了整合两种发酵方式的优势,提出分批补料-分批培养组合发酵策略:发酵前期采用分批补料的方式(指数速率流加)使菌体浓度迅速增加,当菌体浓度接近最高水平时(16 h),一次性补入高浓度山梨醇转入分批发酵阶段,结果聚唾液酸产量达到了5.70 g/L。(3)研究了添加丙酮酸对E. coli CCTCC M208088代谢网络碳流通量分布的影响。发现添加丙酮酸可以显著加强三羧酸循环,提高菌体细胞的产能效率,同时一定程度上减弱磷酸戊糖途径的碳流通量,弱化菌体生长与产物合成对共用资源的竞争,从而提升了聚唾液酸的合成效率。在此基础上,将丙酮酸添加与组合发酵策略结合起来,聚唾液酸产量提高至6.92 g/L。(4)利用生理胁迫和促释放策略强化E. coli CCTCC M208088合成聚唾液酸的效率。考察了不同表面活性剂处理对聚唾液酸释放和合成的影响,发现吐温-60可以在促进聚唾液酸释放的同时强化聚唾液酸的合成效率。与对照相比,12 h添加0.4 g/L吐温-60可提高聚唾液酸产量10.9%。此外,考察了双氧水胁迫策略对聚唾液酸合成的影响,结果发现在发酵过程中4 h添加7.5 mmol/L,8 h添加15 mmol/L,12 h添加15 mmol/L,16 h添加25 mmol/L H2O2的四点添加策略可使聚唾液酸产量比非胁迫情况下提高19.8%。(5)为了构建聚唾液酸的提取工艺,对聚唾液酸的稳定性、分离特性以及发酵液中主要杂质的去除方法进行了研究。首先,确定了聚唾液酸保持结构稳定的条件:热处理不超过100℃、酸碱处理在pH 5-10范围内。在此基础上,优化了氯代十六烷基吡啶(CPC)络合沉淀聚唾液酸的反应条件:CPC质量浓度为聚唾液酸3倍,NaCl<0.1 mol/L,pH 6.0-7.0,温度<40℃。此外,确定了乙醇沉淀聚唾液酸的适宜条件,即处理液中添加氯化钠至0.68 mol/L,然后加入3倍体积的乙醇。最后,研究了珍珠岩过滤除杂的适宜的操作条件:处理液pH=10,珍珠岩用量为80 g/L。(6)确立了提取和精制聚唾液酸的工艺。为了提取聚唾液酸,发酵液首先经过80℃加热预处理30 min,然后离心去除菌体,得到的上清液用乙醇沉淀,沉淀物用水复溶后,以珍珠岩为助滤剂进行过滤,滤液经过超滤脱盐后再用CPC络合沉淀,形成的CPC-PSA络合沉淀物转移至0.8 mol/L NaCl溶液中复溶解离,然后再次利用乙醇沉淀,沉淀物洗涤后真空干燥即得聚唾液酸。得到的聚唾液酸样品纯度可达95%,总回收率在50-60%之间。为了制备医药级别的产品,聚唾液酸提取样品需要进一步的精制。样品依次经过超滤、离子交换和凝胶过滤层析处理,再经脱盐、浓缩、冷冻干燥,得到最终产品。产品中蛋白未检出,内毒素含量低于100 EU/mg,纯度达到99.9%。精制样品的红外和核磁共振图谱的特征吸收峰与文献报道的α-2, 8糖苷键连接聚唾液酸比对一致。(7)优化的聚唾液酸生产工艺进行了500 L规模的中试放大验证,聚唾液酸产量达到5.50 g/L,分离得到的聚唾液酸纯度达到95%。中试的产量、发酵生产强度以及生产规模均为目前报道的最高水平。更多还原

【Abstract】 Polysialic acid (PSA) is a polymer of sialic acid linked withα-2, 8-and/orα-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 ofα-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.更多还原