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Vitamin B12

M.Sc. Biotechnology Part II (Sem III)Paper III - Unit III

Mumbai UniversityBy: Mayur D. Chauhan

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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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Types of Vitamins

2 types

Fat SolubleWater Soluble

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Types of Water soluble Vitamins• Vitamin B:

B1 - ThiamineB2 - RiboflavinB3 - NiacinB5 - Pantothenic AcidB6 - PyridoxineB7 - BiotinB9 - Folic AcidB12 - Cyanocobalamin

• Vitamin C: Ascorbic acid

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Types of Fat soluble Vitamins

• Vitamin A – Retinol• Vitamin D – Cholecalciferol• Vitamin E – Tocopherol• Vitamin K - Phylloquinone

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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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• 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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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/

MecocobalaminCN – CyanocobalaminOH – Hydroxocobalamin/ Vitamin B12a

5’ deoxyadenosyl – Cobamamide or Coenzyme B12

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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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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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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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• 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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• 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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Isolation of Vitamin B122. Extraction of a crude product (80%) for animal feeding

3. Purification to obtain purity of 95-98%

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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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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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Fermentation Processes from Carbohydrates

Production by Propionibacteria

Production by Pseudomonas

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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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• 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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• 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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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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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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• 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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Fermentation using other Substrates

• Alcohols – More attention was paid on methanogenic bacteriaThey produce 2 mg/L per 8 gm of methanol per litre.

• Hydrocarbons

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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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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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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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Methylcobalamin

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

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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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