Cyanocobalamin production

CYANOCOBALAMINE IS VITAMIN B12 PRODUCED BY DIFFERENT M.OR.

1. Cyanocobalamin Production Mrs. Praveen Garg VITS College, Satna

2. Introduction Cyanocobalamin  is a manufactured form with a cyano (cyanide) group bound to cobalt. Bacterial fermentation creates derivatives of B 12   which are converted to  cyanocobalamin  by addition of potassium cyanide in the presence of sodium nitrite and heat . It is also known as Vitamin B 12. Vitamin B 12 , also known as cobalamin , is a water-soluble vitamin   involved in the metabolism of every cell of the human body. " Cobalamin " actually refers to a group of compounds ( corrinoids ) with near-identical structure. Cobalamins are characterized by a  porphyrin -like  corrin  nucleus that contains a single cobalt atom bound to a  benzimidazolyl   nucleotide and a variable residue (R) group. 

3. Vitamin B 12

4. It is a cofactor in DNA synthesis, and in both fatty acid and amino acid metabolism. It is important in the normal functioning of the nervous system via its role in the synthesis of myelin, and in the maturation of developing red blood cells in the bone marrow. Vitamin B 12  is the largest and most structurally complex vitamin. The vitamin exists in four near-identical chemical forms ( vitamers ):  cyanocobalamin ,  hydroxocobalamin ,  adenosylcobalamin  and  methylcobalamin .  Function of Vitamin B 12  

5. M.Or . Used for Production of Vitamin B 12 Large scale industrial production of vitamin B 12  occurs via fermentation by M .Or . Pseudomonas denitrificans ,  Propionibacterium shermanii or   Sinorhizobium meliloti   Cyanocobalamin is commercially prepared by bacterial Submerged fermentation process. Fermentation by a variety of  microorganisms yields a mixture of  methylcobalamin ,  hydroxocobalamin  and  adenosylcobalamin . These compounds are converted to cyanocobalamin by addition of potassium cyanide in the presence of sodium nitrite and heat.

6. Cobalamin biosynthetic pathway De novo pathway: Cobalamin can be synthesized de novo in prokaryotes through two alternative routes according to the timing of cobalt insertion and the molecular oxygen requirement. These pathways are the aerobic pathway, which has been studied in  P . denitrificans , and the anaerobic pathway, which has been studied in  S. typhimurium ,  Bacillus megaterium , and  Propionibacterium shermanii . Both routes differ in terms of cobalt chelation and oxygen requirements (the aerobic pathway requires oxygen to promote ring-contraction, while the anaerobic pathway does not require oxygen in this step)

7. Biosynthetic pathway

8. Steps involved in Cyanocobalamine production Formation of medium Sterilization of medium Preparation of Starter culture Anaerobic fermentation Aerobic fermentation Recovery of cyanocobalamine

9. 1. Formation of Medium Carbon Source: Soyabean meal/glucose Beet molasses Corn steep glucose Nitrogen Source : Ammonium phosphate Ammonium Hydroxide Perpared medium is sterilized by autoclaving. The sterilized medium is then used for fermentation. 2. Sterilization

10. Preparation of Starter culture Different m.or . u sed for fermentation of cyanocobalamine . Pseudomonas denitrificans ,  Propionibacterium shermanii S. typhimurium Bacillus megaterium For preparation of starter culture, nutrients and the inoculum are added to sterile fermenter . Anti-foaming agent may be added. Once the desired product is present in the fermenter the contents are drained off and the product is extracted.

11. Anaerobic fermentation Batch and Continuous both fermentation process effective in anaerobic condition. Sterilized medium is filled in stirred tank fermenter . 1% of inoculum (starter culture) is added in to the fermenter . Anaerobic condition is maintained to enhance the production of dimethyl benzimidazole cobalamine (DBC) by P. shermanii .

12. In this process, sterile air is pumped in to the fermenter . The culture is stirred well for proper aeration. Aerobic fermentation is performed for 3-4 days. During this process some amount of DBC and pseudovitamin B12 are produced. DBC and pseudovitamin B12 are precursor of cyanocobalamine . Aerobic fermentation

13. Recovery of C yanocobalamine The cyanocobalamine exist in the form of natural substances such as DBC and pseudovitamin B12 Inside the microbial cells. The culture broth contained 10-23mg vitB12/liter. It is harvested and centrifuged at high speed to get a concentrated mass of cells. The cell mass is treated with a dilute acid and heat stock at 10-30 C. During the treatment precursors of pseudovitamin B12 are released free. Then it is treated with cyanide solution to split the DBC and pseudovitamin b12. As a result cyanocobalamine is released free in the solution.

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