Vitamin B12 by Streptomyces spp

Vitamin B12, also called Cobalamin, is a water- soluble vitamin, an essential micronutrient that has a key role in the normal functioning of the brain and nervous system, and the formation of red blood cells. It is involved in the metabolism of every cell of the human body, especially affecting DNA synthesis, fatty acid and amino acid metabolism.

For humans, there is a daily requirement of 2-3 micrograms. 

 It is synthesized only by microorganisms. Humans should obtain it from food, since it is produced by microorganisms in large intestine, from where it cannot be assimilated.

Vitamin B12 is naturally present in foods of animal origin, including fish, meat, poultry, eggs, and dairy products. Those who consume only small amounts of animal products or are vegetarians must compensate for this.

Deficiency diseases

People who lack intrinsic factor produced by stomach parietal cells cannot absorb Vitamin B12 from food in the intestine, so develop pernicious anemia. It is an autoimmune disease, cause permanent damage to nerves and other organs, and stomach cancer in the long run.

Vitamin B12 helps in the production of healthy red blood cells that carry oxygen around the body. Not having enough vitamin B12 makes the body produce larger than normal red blood cells (not quite effective as normal RBS’s), described as megaloblastic or macrocytic, resulting in megaloblastic or macrocytic anemia.

Once diagnosed, vitamin B12 deficiency can usually be treated successfully with B12 injections and sometimes with B12 tablets.

History

First reported by George Whipple, George Minot and William Murphy -from liver juice, in 1926. Found that liver extracts cure pernicious anemia in humans.- Nobel Prize in Physiology or Medicine in 1934  - “for their discoveries concerning liver therapy in cases of anaemia,”

Alexander R. Todd, at the University of Cambridge,  extracted the anti-pernicious anemia factor from liver extracts, purified it, and named it vitamin B₁₂. In 1955, Todd helped elucidate the structure of the vitamin, for which he was awarded the Nobel Prize in Chemistry in 1957. The complete chemical structure of the molecule was determined by Dorothy Hodgkin, based on crystallographic data in 1956, and she was awarded the Nobel Prize in Chemistry in 1964. 

Five people have been awarded Nobel Prizes for direct and indirect studies of vitamin B₁₂: George Whipple, George Minot and William Murphy (1934), Alexander R. Todd (1957), and Dorothy Hodgkin (1964).

Structure of Vitamin B12

It is the largest and most structurally complicated vitamin and can be produced industrially only through bacterial fermentation

A molecule of cobinamide linked to a nucleotide-

Nucleotide- Atypical nucleotide where, in place of purine/pyramidine, there is 5,6-dimethylbenzimidazole, connected to sugar and phosphate group

Cobinamide- A corrin ring with a central cobalt atom and various attached side groups -the ring consists of 4 pyrrole subunits (like a porphyrin ring in chlorophyll)

Vitamin B12 is the only known essential biomolecule with a stable metal-carbon bond, that is, it is an organometallic compound. The cobalt can link to:

 

• a methyl group - as in methylcobalamin
• a 5'-deoxyadenosine at the the 5' positon - as in adenosylcobalamin - coenzyme B12
• a cyanide group - as in cyanocobalamin/ commercial Vitamin B12 - as supplied from drug companies

Cyanocobalamin, is the industrially produced stable Cobalamin form which is not found in nature.

Vitamin B 12 analogues

• Pseudo Vitamin B 12- similar to cobinamides -produced by different algae, promote their growth.
• Found along with Vitamin B 12in food sources, supplements and preparations from algae and cyanobacteria
• Less biologically active/inactive in humans
• Have heterocyclic bases–purines/substituted benzimidazoles in place of 5,6-dimethylbenzimidazole

Industrial production of B12

Vitamin B12 is entirely produced on a commercial basis by the fermentation, using selected microorganisms.

Microorganisms

Streptomyces griseus, was the commercial source of vitamin B12 for many years. The species Pseudomonas denitrificans and Propionibacterium shermanii are more commonly used today

The microorganisms that may be employed in the industrial production process are :

i.                    Streptomyces griseus

ii.                  Streptomyces olivaceus 3.3mg / L

iii.                Bacillus megaterium 0.45 mg/L

iv.                Bacillus coagulans 

v.                   Pseudomonas denitrificans 60 mg/L

vi.                Propionibacterium freudenreichii 20 mg/L

vii.              Propinibacteriun shermanii 30-40 mg/L          

Hybrid strain made by protoplast fusion technique between Protaminobacter ruber & Rhodopseudomonas spheroides, Rhodopseudomonas protamicus -135 mg/L.

Fermentation

Submerged culture process, completed in 3-5 days.

The process includes

Ø  Inoculum preparation

Ø  Formulation & Sterilization of the medium

Ø  Fermentation

Ø  Recovery

A.    Preparation of Inoculum:

Pure slant culture of S. olivaceus is inoculated in 100-250ml of inoculum medium and the flask is incubated with mechanical agitation. This flask culture is then subsequently used to inoculate larger inoculum tanks. (2 or 3 successive transfers are made to obtain required amount of inoculum cultures.)

Media used in preparation of inoculum is Bennett’s agar-pH 7.3

Component	Amount (g/L)
Yeast extract	1.0
Beef extract	1.0
N-Z-Amine A(Enzymatic hydrolysate of casein)	2.0
Glucose	10.0
Agar	15.0
D/W	1000 L

 B.     Production Medium:

• Most of the B12 fermentation processes use glucose as a carbon source. Medium contains carbohydrate (glucose), proteinaceous material, and source of cobalt and other salts like calcium carbonate.
• Carbon source can be glucose, corn steep liquor, beet molasses, soya bean meal, distiller’s solubles (dissolved and fine particles left after solid grains are strained off from the residues after alcoholic fermentation)
• Nitrogen source may be ammonium phosphate, ammonium hydroxide,
• It is necessary to add cobalt as precursor (2-10 ppm) to the medium for maximum yield of cobalamin.
• Temperature of 27°C and starting pH 7.0. pH falls due to rapid consumption of sugar, then rises after 2-4 days due to lysis of mycelium. pH 5 is maintained with H₂SO₄ and sodium sulphite.
• Aeration and agitation provided- aeration at optimum rate of 0.5 volume air/volume medium/min.
• Excessive aeration can cause foaming. Antifoam agents like soya bean oil, corn oil, lard oil and silicones can be used.
• Prevention of contamination- contamination results in reduced yields, so it is essential to maintain sterility. Equipments must be sterile and all transfers are carried out under aseptic conditions.

Typical yield with Streptomyces olivaceus 1-3 mg / L

 C.    Recovery:

During fermentation, most of cobalamin is associated with the mycelium; boiling mixture at pH 5 liberates the cobalamin quantitatively from mycelium.

Broth containing cobalamin is subjected to further process to obtain crystalline B12.

1. Filtration of broth to remove mycelium.
2. Filtered broth is treated with cyanide to bring conversion of cobalamin to cyanocobalamin.
3. Adsorption of cyanocobalamin from the solution is done by passing it through adsorbing agents (activated charcoal, Fuller's Earth) packed in a column.
4. Cyanocobalamin is then eluted from the adsorbent by the use of an aqueous solution of organic bases or solutions of Na-Cyanide and Na- thiocyanate.
5. Extraction is carried out by countercurrent distribution between cresol, amylphenol, or benzyl alcohol and water or a single extraction into an organic solvent (e.g. Phenol) is carried out.
6. Chromatography on alumina and final crystallization completes the process.

Recovery process designed depending on the type of product required

Uses

(1)   Food preservative

(2)   Feed supplement

(3)    Medicinal/pharmaceutic applications