【作者】 王雷；

【导师】 王云山；

【作者基本信息】 河北大学 ， 微生物学， 2006， 硕士

【摘要】 钴胺素广泛应用于医药和食品行业，目前主要由微生物发酵生产。本论文从应用角度出发，初步研究了氮源、DMB等因素对脱氧腺苷钴胺素发酵的影响以及羟基钴胺素的制备，期望为钴胺素的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几个方面研究： 1．氮源对腺苷钴胺素发酵的影响 考察了多种有机氮源对费氏丙酸杆菌发酵生产脱氧腺苷钴胺素的影响，实验结果表明以玉米浆作为氮源时发酵单位最高，而胰蛋白胨和酸水解酪素发酵单位最低。以酵母提取物、胰蛋白胨、酸水解酪素做为复合氮源，发酵单位较高。 2．5，6-二甲基苯并咪唑(DMB)对腺苷钴胺素发酵的影响 费氏丙酸杆菌进行厌氧合成脱氧腺苷钴胺素时，需添加前体DMB。采用高效液相色谱法检测发酵液中的钴啉醇及脱氧腺苷钴胺素的合成情况，在此基础上考察了DMB添加时间以及浓度对发酵的影响，确定了DMB的最佳添加时间和添加量。 3．钴胺素的光解研究及快速检测方法的建立 脱氧腺苷钴胺素和甲基钴胺素对光线十分敏感，光照处理后会完全转化为羟基钴胺素，羟基钴胺素对光线相对稳定。采用高效液相色谱法研究了脱氧腺苷钴胺素和甲基钴胺素的水溶液在不同光源及不同照度下的光解情况，结果表明随着光能量的增加光解速度加快。利用脱氧腺苷钴胺素的光解性质，开发了一种检测发酵液中钴胺素含量的新方法。将含有钴胺素的细胞破碎液完全光解后，测定光解后的产物羟基钴胺素，以此来确定钴胺素的产量。此方法简便快速、重复性好，可应用于发酵生产钴胺素的各个环节。 4．大孔树脂吸附法提取纯化羟基钴胺素 比较了4种大孔吸附树脂对羟基钴胺素的静态吸附、解吸性能，考察了温度和pH对树脂的吸附和解吸性能影响。结果表明CAD45树脂对于羟基钴胺素具有良好的吸附能力，并且使用乙醇溶液可达到良好的解吸效果。CAD45树脂使用湿法装柱，研究树脂对羟基钴胺素的动态吸附和洗脱过程。更多还原

【Abstract】 Cobalamin is widely used in medicine and food industry, which is produced mainly by fermentation. In this paper, the influcens of several organic nitrogen sours and 5,6-dimethylbenzimidazole on deoxyadenosylcobalamin fermentation was studied. The production of hydroxycobalamin was also researched. The study in this paper was based on industry applications, which have special valuable reference to improvement of cobalamin fermentation.1. Influence of nitrogen sources on deoxyadenosylcobalamin fermentationEffect of nitrogen sources on the deoxyadenosylcobalamin fermentation of Propioniba -cterium Jreudenreichii was investigated. Among nitrogen sources investigated, corn steep liquor is the best organic nitrogen source and the deoxyadenosylcobalamin production was high when corn steep liquor was used.2. Influence of 5,6-dimethylbenzimidazole(DMB) on deoxyadenosylcobalamin fermentationThe biosynthesis of deoxyadenosylcobalamin in Propionibacterium jreudenreichii anaerobic cultures requires addition of 5,6-dimethylbenzimidazole.Studies on deoxyadenosylcobalamin fermentation using DMB as the precursor was carried out. Reference the phase of fermention at which DMB is to be added to the fermentation medium to obatain its maxium ultilization, resulting in maxium cobalamin productivity. And the optium dosage of DMB addition was studied.3. Research on cobalamins light stability and a rapid method to determine cobalamin The light stability of cobalamins was studied. As a result, Hydroxycobalamin is stable inexperimental time. The photolytic behavior of deoxyadenosylcobalamin and methylcobalamin in water solution was investigated with high performance liquid chromatography.The results indicated that the photolytic cleavage rate increased with the light intensity, according to which a new method was developed to determine the concentration of cobalamin fermentation broth. The samples were completely photolyzed after cell disruption. The content of cobalamin was obtained by determining the content of the hydroxycobalamin. The method shows many advantages, such as rapidness, high accuracy and low sample quantity needed, over traditions methods. The developed method may be used in the field of cobalamin fermentation.更多还原