Vitamin B-12: Cyanocobalamin

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Mayur D. Chauhan

1. Vitamin B 12 M.Sc. Biotechnology Part II ( Sem III) Paper III - Unit III Mumbai University By: Mayur D. Chauhan 1

2. Vitamins Vitamins are organic compounds which are required in smaller amounts. Vitamins are not synthesized in sufficient quantities inside the body. Thus they must be obtained through diet. Hence the name Vitamin. 2

3. Types of Vitamins 3

4. Types of Water soluble Vitamins Vitamin B: B 1 - Thiamine B 2 - Riboflavin B 3 - Niacin B 5 - Pantothenic Acid B 6 - Pyridoxine B 7 - Biotin B 9 - Folic Acid B 12 - Cyanocobalamin Vitamin C: Ascorbic acid 4

5. Types of Fat soluble Vitamins Vitamin A – Retinol Vitamin D – Cholecalciferol Vitamin E – Tocopherol Vitamin K - Phylloquinone 5

6. History of Vitamin B 12 Cyanocobalamins are important biological compounds which are active as hematopoietic factor in mammals. These factors are basically a group of glycoproteins which help the blood cells to grow and mature. Cyanocobalamins act as growth factors for many microbial and animal species. 6

7. It was first isolated by Rickes et al. and Smith as pure crystals in 1948. Hodgkins et al. revealed it’s structure in 1955 by using X-ray crystallography. Synthesis of this vitamin is very difficult as it required 70 steps. Thus fermentation processes are used to decrease the number of steps and improve the production. 7

8. Structure of Vitamin B 12 It belongs to the family of Cobaltcorrinoids Cobalamins have X and a Y radial. X radical: 5,6 – dimethylbenzimidazole (DBI) Y radical: CH 3 – Methylcobalamin / Mecocobalamin CN – Cyanocobalamin OH – Hydroxocobalamin / Vitamin B 12a 5’ deoxyadenosyl – Cobamamide or Coenzyme B 12 8

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11. Other Examples Pseudovitamin B 12 – X radical is Adenine Factor III – X radical is 5-hydroxybenzimidazole Etiocobalamin – When nucleotide moiety is removed from cobalamins , we obtain etiocobalamin . It is designated as incomplete. 11

12. Sources of Vitamin B 12 It is originated from microorganisms Vitamin B 12 is present in every animal tissue. Example: 1mg/kg in beef liver Materials rich in Biomass like activated sludges and broths of antibiotic-producing Streptomyces, were used for isolating Vitamin B 12 either in crude form for animal feed or in a pure state for medicinal use. 12

13. General Production of Vitamin B 12 Microorganisms were tested regarding their abilities to produce Vitamin B 12 Based upon the yield of the vitamin, 2 P ropioni bacteria and 1 Pseudomonas bacterium were selected. 13

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16. Best strains are spontaneous or mutants screened for their resistance to different agents, cobalt or manganese ions, antibiotics etc. Mutants are induced in the organisms for producing cobalamins . Induction is carried out by UV rays or X-rays. Chemical agents like N-methyl-N’- nitrosoguanidine , nitrosoethylurea , ethyleneimine , dimethylsuulfate and mustard gas. 16

17. Certain essential elements are added for the vitamin synthesis. These include cobalt ions, DBI, and other precursor compounds like Glycine, amino- levulinic acid, aminopropanol . 17

18. Isolation of Vitamin B 12 18

19. Isolation and Conversion To isolate Vitamin B 12 , whole broth or the harvest is heated at 80-120 o C for 10-30 minutes at pH 6.5-8.5. Conversion to cyanocobalamin is obtained by treating the heated broth with cyanide or thiocyanate solution often in presence of Sodium nitrite or Chloramin B. If the cyanide treatment is postponed to a later stage in order to reduce tedious handling, conversion to a more stable sulfito-cobalamin is advisable. 19

20. Extraction Procedures Adsorption on such different supports as Amberlite IRC50, Alumina, Carbon and elution by Hydroalcoholic or H ydrophenolic mixtures. Extraction from aqueous solutions by phenol or cresol alone or in mixture with benzene, butanol , carbon tetrachloride or chloroform. Precipitation or Crystallization from various solutions by evaporation, dilution with appropriate solvents. Impurities are removed by common precipitating agents such as calcium, or zinc hydroxide. 20

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22. Fermentation Processes from Carbohydrates 22

23. Production by Propionibacteria Several microaerophilic propionibacteria produce cobaltcorrinoids in conventional carbohydrate media correctly supplemented with cobalt without aeration. However production of cobalamins especially adenosylcobalamins requires the supply of DBI (internal of external). Also aeration favours DBI formation. 23

24. Thus Propionibacterium freudenreichii ATCC 6207, Propiobacterium shermanii ATCC 13673 are chosen which can synthesize their own DBI. Fermentation is carried out in 2 seed stage. In the first stage, a practically anaerobic culture is run to almost total depletion of sugar in order to promote the growth of the bacteria and etiocobalamin synthesis. In the second stage, an aeration shift leads to DBI formation and conversion of etiocobalamin to deoxyadenosylcobalamin . 24

25. The stages can be carried batchwise in a same tank or continuously in two connected fermentors . DBI supplementation is only required in the second stage if selected propionibacteria are not able to synthesize their own but due to the inhibitory effect of DBI on the corrinoid biosynthesis, addition of DBI must be done in the first stage. 25

26. Constituents in the Fermenter Glucose or Inverted Molasses with small amount of ferrous, manganous , magnesium salts in addition of cobalt salts. Buffering or neutralizing agents Corn steep liquor (30-70 gm./L) Substituting the carbon and nitrogen sources by skim milk or lactoserum is also possibe . Propionibacteria produce 25-40mg of Vitamin B 12 per litre . 26

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28. Production by Pseudomonas Pseudomonas denitrificans strain is most commonly used. Pseudomonas synthesize cobalamin in aerobic conditions only. This is contrasting to the Propionibacteria. Fermentation is carried out with aeration and agitation in a single vat, batch wise or continuous 28

29. Betaine and choline have favorable effects in activating some biosynthetic stages or altering the membrane permeability. Glutamic acid stimulates the growth of these bacteria. Owing to the cheapness and high betaine and glutamic acid content, beet molasses, a multivalent nutrient of choice is preferred. Optimum temperature is 28 o C and pH is 7.0. Over the years, the vitamin B 12 content has jumped from 0.6 to 60 mg/L with the help of these bacteria. 29

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31. Fermentation using other Substrates Alcohols – More attention was paid on methanogenic bacteria They produce 2 mg/L per 8 gm of methanol per litre . Hydrocarbons 31

32. Production of Vitamin B 12 Derivatives Human serum has; 0-10% - Cyanocobalamin 8-15% - Hydroxocobalamin 22-39% - Deoxyadenosylcobalamin 36-62% - Methylcobalamin Each of these derivatives controls several enzymatic reactions which could play a role in man in the genesis of perniciouus anemia, neuropathy and growth deficiency. 32

33. Hydroxocobalamin Propionibacterium shermanii During the extraction, successive transformations of native cobalamins in their sulfato , nitrato and chloro derivatives are requird before a final hydrolytic treatment by Amberlite IRA 400 (OH - ) to generate hydroxocobalamin . 33

34. 5’- Deoxyadenosylcobalamin Propionibacterium shermanii , Propionibacterium freundenreichii etc Conversion of Cyano or hydroxocobalamins . Coenzyme B 12 is always endocellular . Thus the harvest is subjected to centrifugation, extraction is performed in the cold by an acetone-water mixture or at an 80-100 o C during a short time by a 2% phenol aqueous solution or an ethanol-water mixture. 34

35. Methylcobalamin Detected in whole broths of E.Coli , Streptomyces rimosus , Propionibacteri , Klebsiella . 35

36. Vitamin B 12 Antagonists Substances which inhibit the synthesis of Vitamin B 12 are produced by many organisms. The first is constituted by corrinoids deprived of cobalt and found among photosynthetic bacteria such as Chromatium . Second includes peptides and uncommon amino acids such as N-5-hydroxy-L-arginine, 4-oxo-L-isoleucine. 36

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