摘要
钴胺素是人体组织代谢过程中必需的维生素,具有广泛的生理作用。广泛应用于医药和食品行业。目前钴胺素主要由微生物发酵生产。本论文从应用角度出发,初步研究了DMB、丙酸等因素对腺苷咕啉醇酰胺(关键中间产物)、菌体生长及脱氧腺苷钴胺素(adenosylcobalamin,简称ADO)生物合成的影响,探索了解除有机酸(丙酸)抑制的发酵工艺过程,期望为钴胺素发酵生产工艺的改进提供参考借鉴。本论文主要进行了以下两方面的研究。
1.丙酸杆菌厌氧合成维生素B12研究
在费氏丙酸杆菌(propionibacterium freudenreichii)发酵生产维生素B12的研究中,分离到脱氧腺苷钴胺素生物合成途径中一种关键而稳定的中间产物。该物质在维生素B12发酵过程中大量积累,在研究中发现该中间产物腺苷钴啉醇酰胺(CBI)与5,6-二甲基苯并咪唑(DMB)来源的前体α-吡咯核糖(α-ribozale)结合后可转化为脱氧腺苷钴胺素(ADO)。光谱和质谱分析该物质应是脱氧腺苷钻胺素生物合成途径中的一种重要中间产物—腺苷钴啉醇酰胺(adenosylcobinamide,简称CBI)。对此中间产物的分析可深入了解厌氧合成脱氧腺苷钴胺素的代谢途径。进一步以CBI为核心,考察不同时间添加DMB对菌体生长及ADO合成的影响,从而运用代谢调控手段优化DMB的添加时间。从而优化发酵工艺,指导发酵生产维生素B12的生产实践。批次发酵条件下,考察了菌体的CBI合成量,ADO产量和菌体的生长曲线,可确定DMB对菌体生长的抑制作用并不明显,但可抑制CBI在细胞内的生物合成,因此可影响单位菌体产量。
2.反应与分离耦合的发酵工艺研究
初步探索了用离子交换树脂吸附和更换新鲜培养基两种方法解除有机酸抑制对费氏丙酸杆菌发酵生产VB12的影响。初步探索了发酵过程中定时对丙酸进行分离的发酵工艺,可有效解除有机酸对细胞生长的抑制,实现了细胞的连续生长,为连续发酵的生产工艺提供了借鉴。
进一步考查了弱碱性阴离子交换树脂ZGA330对费氏丙酸杆菌发酵液中丙酸的静态和动态吸附性能。树脂ZGA330对含有12.6g/L丙酸的发酵液中丙酸的静态吸附量最大,吸附量为44.9g/L；动态吸附过程中对发酵液中主要营养成分糖、氨基酸吸附很少,而对丙酸吸附量高达205.5g/L。成功地通过树脂吸附将发酵系统中的丙酸分离出去,实现了维生素B12的高密度发酵。研究结果为丙酸的发酵与吸附分离耦合过程研究提供了基础,丙酸吸附分离后的发酵液继续进行维生素B12发酵生产可以解除丙酸的抑制作用,提高了维生素B12的产量。
National forms of cobalamin compounds are the necessary vitamins in the process of organization metabolize in human body and they have a broad of physiological functions. Cobalamin is widely used in medicine and food industry and it has been produced in the process of microbial fermentation. In this paper, the effects of DMB、propionic acid on intermediate biosynthesis、biomass and adenosylcobalamin biosynthesis were studied. The fermentation process of removal propionic acid was also explored. The study in this paper was based on industry applications, which have special valuable reference to improvement of cobalamin fermentation.
1.Research on Deoxyadenosylcobalamin Biosynthesis of Propionibacterium freudenreichii
An important intermediate of deoxyadenosylcobalamin biosynthesis by anaerobic pathway of Propionibacterium freudenreichii was separated from the broth. It has been qualitatively analyzed by spectrum and ESI MS-GC analysis and further determined as a key intermediate of deoxyadenosylcobalamin:adenosylcobinamide which can be transformed to deoxyadenosylcobalamin when infused 5,6-dimethylbenzimidazole (DMB) to the fermentation medium. It is hope that the intermediate could be a key factor which guides the infusion of DMB and further optimize the fermentation process.
The effects of 5,6-dimethylbenzimidazole(DMB) on the growth and deoxyadenosylcobalamin biosynthesis of propionibacterium freudenreichii are explored in this part. In the anaerobic pathway,An important intermediate adenosylcobinamide(CBI), which can be transformed to deoxyadenosylcobalamin when infused DMB to the fermentation medium. When the CBI as a key factor, the effection of DMB addition at different phases of fermentation, the CBI biosynthesis curve, the deoxyadenosylcobalamin yield and cell growth curve were measured in the batch fermentation process. The infusion of DMB at the phase of 90-100h of incubation has been found optimum for maximizing metabolic efficiency of the culture, and there was no obvious restrain effection on cell growth.
2.The research on coupling fermentation technics
The two methods that changing for fresh broth and adsorbing by resin ZGA330 to take propionic acid off in vitamin B12 (VB12) fermentation broth were evaluated. The fermentation process of removal of propionic acid was also explored. It can efficiently relieve the inhibition of cell growth and realize the continuous grow of biomass. And the research results can be the foundation for the technology of continuous fermentation.
The adsorption effects of ZGA330 of weakly basic anion exchange resins for propionic acid (PA) in vitamin B12 (VB12) fermentation broth were evaluated. The result was that the static adsorption capacity on resin ZGA330 was 44.9 g/L dry beads at 12.6 g/L of PA. Dynamic state exchange trials, including the adsorption for glucose, amino acids and PA in the fermentation broth, were also determined which indicated that the adsorption was minimal for glucose and amino acids that were main nutrient compositions of the fermentation broth. The dynamic adsorption capacity was 205.5 g/L dry beads. By the removal of propionate acid with ZGA330, high concentration of VB12 was obtained comparing to the batch fermentation. The VB12 concentration increased from 9.1 mg/L to 13.1 mg/L. VB12 concentration was 0.44 fold of those in the batch fermentation respectively. To be concluded, it can provide theory of fermentation process coupling with separation and the cell concentration and productivity of VB12 will be greatly improved by the removal of propionate.
引文
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