Production Of Vitamin B₁₂ By Propionibacterium Freudenreichii Ex-situ Cell Transformation Process

Propionibacterium freudenreichii yields by-product propionic acid when synthesis vitamin B₁₂（Ado-cbl）, and propionic acid can certain feedback inhibition for the growth of bacteria. It is inportant to reduce the propionic acid feedback inhibition as same as increasing the economical efficiency of fermentation process. In order to realize the Propionibacterium freudenreichii semi-continuous fermentation, the feasibility of synthesis of vitamin B₁₂ by ex-situ cell transformation was investigated by this research. When fermentation by a certain time, the bacteria cells was isolated from the fermented liquid to another reaction system.Then the 5,6-Dimethylbenzimida-zole（DMB） was added for the synthesis of vitamin B₁₂,which could realize the semi-continuous fermentation on the basis of fed-batch fermentation.So, the the feasibility of synthesis of vitamin B₁₂ by ex-situ cell transformation was investigated in this article by researching the relationship between intermediate Ado-cbi and product vitamin B₁₂. The cell separation time, the separation method, ex-situ cell transformation system had been optimized, for the Propionibacterium freudenreichii semi-continuous coupling fermentation process.1 The detection of metabolic intermediates Ado-cbi.The HPLC detection method was optimized, the elution conditions were as follows: 0 to30 min, 5-24 % acetonitrile gradient elution. The intermediates Ado-cbi detection peak was obtained by researching the relationship between intermediate ado-cbi and product vitamin B₁₂. Then the HPLC-MS was used to detect the molecular mass of detection peak which as same as Ado-cbi 1239.3. The real-time detection of metabolic intermediate Ado-cbi and vitamin B₁₂ can be realized at the same time.2 The DMB control strategy based on the metabolic intermediates Ado-cbi.Through the DMB adding time and adding amount research, DMB optimum adding time was confirmed for fermentation 84 h and the optimum adding amount was 0.9 mg/L. Three DMB adding strategies were explored as follows: DMB was added at the beginning offermentation, DMB adding tactics and DMB continuous adding strategy. In the early transformation, DMB in certain concentration range influenced the vitamin B₁₂ transformation rate. DMB was added at the beginning of fermentation would greatly reduce the production of vitamin B₁₂. The optimal DMB adding strategiy was adding enough DMB（0.9 mg/L） directly at the time of transformation.3 The establishment and the optimization of Propionibacterium freudenreichii ex-situ cell transformation.The semi-continuous coupling fermentation process can significantly improve the economical efficiency of Propionibacterium freudenreichii. In order to avoid the negative effect of DMB addition on Propionibacterium freudenreichii semi-continuous fermentation,the feasibility of synthesis of vitamin B₁₂ by ex-situ cell transformation was investigated.Also, the ex-situ cell transformation process was optimized from the aspects of cell seperation time, transformation system and DMB addition mode. The details were as follows:when the fermentation process was carried out for 84 h, cells were removed from the fermentation broth by centrifugation. The supernatant was used to resuspended the cells and a5 times concentration suspension was obtained. Then a final concentration of 4.5 mg/L DMB was added to synthesis vitamin B₁₂ for 48 h at the optimal temperature 30 ℃ and optimal pH7.0. The Vitamin B₁₂ yield can reached 108.06 mg/L, with a conversion efficiency of 2.26mg/（Lh）.