The following presentation is only for quick reference. I would advise you to read the theoretical aspects of the respective topic and then use this presentation for your last minute revision. I hope it helps you..!! Mayur D. Chauhan

1. Vitamin B12
M.Sc. Biotechnology Part II (Sem III)
Paper III - Unit III
Mumbai University
By: Mayur D. Chauhan
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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.
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3. Types of Vitamins
2 types
Fat Soluble
Water
Soluble
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4. Types of Water soluble Vitamins
• Vitamin B:
B1 - Thiamine
B2 - Riboflavin
B3 - Niacin
B5 - Pantothenic Acid
B6 - Pyridoxine
B7 - Biotin
B9 - Folic Acid
B12 - Cyanocobalamin
• Vitamin C: Ascorbic acid
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5. Types of Fat soluble Vitamins
• Vitamin A – Retinol
• Vitamin D – Cholecalciferol
• Vitamin E – Tocopherol
• Vitamin K - Phylloquinone
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6. History of Vitamin B12
• 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.
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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.
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8. Structure of Vitamin B12
• It belongs to the family of Cobaltcorrinoids
• Cobalamins have X and a Y radial.
• X radical: 5,6 – dimethylbenzimidazole (DBI)
• Y radical: CH3 – Methylcobalamin/
Mecocobalamin
CN – Cyanocobalamin
OH – Hydroxocobalamin/ Vitamin B12a
5’ deoxyadenosyl – Cobamamide or Coenzyme
B12
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11. Other Examples
• Pseudovitamin B12 – 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.
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12. Sources of Vitamin B12
• It is originated from microorganisms
• Vitamin B12 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
B12 either in crude form for animal feed or in a
pure state for medicinal use.
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13. General Production of Vitamin B12
• Microorganisms were tested regarding their
abilities to produce Vitamin B12
• Based upon the yield of the vitamin, 2
Propioni bacteria and 1 Pseudomonas
bacterium were selected.
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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.
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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.
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18. Isolation of Vitamin B12
1. Solubilization of cobalamin and
conversion to Cyanoobalamin
2. Extraction of a crude product
(80%) for animal feeding
3. Purification to obtain purity of
95-98%
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19. Isolation and Conversion
• To isolate Vitamin B12, whole broth or the harvest
is heated at 80-120o 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.
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20. Extraction Procedures
• Adsorption on such different supports as
Amberlite IRC50, Alumina, Carbon and elution by
Hydroalcoholic or Hydrophenolic 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.
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21. 21

22. Fermentation Processes from
Carbohydrates
Production by Propionibacteria
Production by Pseudomonas
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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.
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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.
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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.
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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
B12 per litre.
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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
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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 28o C and pH is 7.0.
• Over the years, the vitamin B12 content has
jumped from 0.6 to 60 mg/L with the help of
these bacteria.
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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
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32. Production of Vitamin B12 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.
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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.
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34. 5’- Deoxyadenosylcobalamin
• Propionibacterium shermanii,
Propionibacterium freundenreichii etc
• Conversion of Cyano or hydroxocobalamins.
• Coenzyme B12 is always endocellular. Thus the
harvest is subjected to centrifugation,
extraction is performed in the cold by an
acetone-water mixture or at an 80-100o C
during a short time by a 2% phenol aqueous
solution or an ethanol-water mixture.
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35. Methylcobalamin
• Detected in whole broths of E.Coli,
Streptomyces rimosus, Propionibacteri,
Klebsiella.
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36. Vitamin B12 Antagonists
• Substances which inhibit the synthesis of
Vitamin B12 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.
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