In this article we will discuss about the production of various vitamins.
1. Vitamin B12 (Cyanocobalamine):
G.R. Minot and W.B. Murphy (1926) studied that liver extracts cure pernicious anaemia in human beings. In 1936, both of them shared a Nobel Prize in medicine for this discovery.
After this, E.L. Ricke and L. Smith (1948) isolated and crystallized vitamin B12 from liver extracts. This vitamin is present in a very small amount in every animal tissue including human blood 2 × 1014 µg/ ml) but it is synthesized exclusively by microorganisms such as Butyribacterium rettgeri.
Bacillus megaterium, Streptomyces olivaceus, Micromonospora sp., Klebsiella pneumoniae etc. High yields have been obtained from Propionibacterium freudenreichii, P. shennanii and Pseudomonas denitrificans.
The most important industries manufacturing this vitamin are: Farmitalia S.P.A. (Italy), Glaxo Lab., Ltd. (England); Merck & Co., Inc. (USA). Rhome Poulenc S.A. (France); Roussel UCLAF (France); G. Richter Pharmaceutical Co. and Chinoin in Hungary.
Vitamin B12 consists of a base structure corrin and a tetrapyrrole ring which differs from the porphyrin ring system in that the methane bridge between rings A and D is missing. The molecular biology and genetic engineering techniques have improved strains of Rhodopseudomonas protamicus (hybrid between Propionibacter rubber and Rhodopseudomonas spheroides) produces 135 mg/litre vitamin B12.
Thus, vitamin B12 is not a single compound, but a group of closely chemically related cobamides. These cobamides are also called pseudo B12 group. They consist of cobalt porphyrin nucleus to which is attached ribose and phosphate.
Various cobamide differ in the purine, Benz imidazole, or other base, found in the nucleotide like portion of the molecule. Vitamin B12 analogue having other heterocyclic bases (purines or substituted benzimidazoles) are either spontaneously produced by microorganisms or are produced after the addition of these substances in the culture medium. The chemical process requires about 70 reactions, hence not viable process (Table 20.1).
(i) Fermentation:
The nutritionally rich crude medium with glucose as a major carbon source is used in a two-stage process with added cobalt chloride (10-100 mg lit-1).
In a preliminary anaerobic phase (2-4 days), 5-deoxyadenosylcobinamide is mainly produced; in a second phase, which is aerobic (3-4 days), the biosynthesis of 5, 6-dimethyl Benz imidazole takes place, so that 5-de-oxy-adenosyl cobalamine (B12) can be produced.
This compound is completely intracellular and bound to the cell which after heat treatment released in solution form after 10 min at 80-120°C at pH 6.5-8.5. This process is applicable in case of Propionibacterium freudenreichii ATCC 6207 and P. shermanii, ATCC3673.
While using Pseudomonas denitrificans, there is one-stage process that occurs during the entire fermentation. Similar to the previous process, cobalt is added but here we also add 5, 6-dimethylbenzimidazole as supplement.
It has also been observed that addition of betaine induce the yield. In place of glucose, other cheap C sources such as hydrocarbon and higher alcohol are also found promising. Methanol proved better carbon source (Fig. 20.26).
(ii) Uses:
Vitamin B12 is produced by intestinal microorganisms. However, humans obtain vitamin B12 from food, since the B12 synthesized by microorganisms in the larger intestinal tract cannot be assimilated.
For scoine and poultry feeds, 10-15 mg vitamin B12is added per ton of feed, since animal protein can be replaced with less expensive vegetable protein if the vegetable protein is fortified with viz. B12. It has also a role to play in biological nitrogen fixation.
2. Riboflavin (Vitamin B2):
Kuhn, Gyorgy and Wagner Jauregg in 1933 isolated riboflavin (also called lactoflavin) from whey of milk where it is present in free riboflavin form. It is also present in other foods (liver, heart, kidney, or eggs) as flavoproteins which contain the prosthetic group FMN (flavin mononucleotide) or FAD (flavin adenine dinucleotide).
Several microorganisms namely, Clostridium acetobutylicum, Mycobacterium smegmatis, Mycocandida riboflavina, Candida flareri, Eremothecium ashbyii and Ashbya gossypii are used in commercial production and has got an ability to resist against riboflavin accumulation.
Riboflavin is produced also by chemical synthesis but biotransformation of glucose to D- ribose by mutants of Bacillus pumilus and Subsequent chemical conversion to riboflavin produced 50% of world wide production. It is an alloxazine derivative which consists of a pteridine ring condensed to a benzene ring. The side chain consists of a C5-polyhydroxy group, a derivative of ribitol (Fig. 20.27).
Riboflavin is produced in the following steps:
(a) Media preparation and biosynthesis of riboflavin:
For Riboflavin production, basic medium consists of corn steep liquor 2.25%, commercial peptone 3.5%, soybean oil 4.5% but it can be supplemented further by addition of different peptones, glycine, distiller’s soluble, or yeast extract.
The glucose and inositol increase the production of riboflavin. The medium should be kept at 26-28°C at pH 6.8 for 4-5 days incubation. After inoculation the submerged growth of Ashbya gossypi is supported by insufficient air supply. The excess air inhibits mycelial production and reduces the riboflavin yield. The fermentation progresses through three phases.
(b) First phase:
In this phase, rapid growth occurs with small quantity of riboflavin production. The utilization of glucose occurs resulting into decrease in pH due to accumulation of pyruvate. By the end of this phase, the glucose is exhausted and growth ceases.
(c) Second phase:
Sporulation occurs in this phase. The pyruvate decreases in concentration. Ammonia accumulates because of an increase in deaminase activity. The pH reaches towards alkalinity.
(d) Third phase:
There is a rapid synthesis of cell-bound riboflavin (FMN and FAD). This phase is accompanied by rapid increase in catalase activity subsequently cytochromes disappear. As the fermentation completes, the autolysis takes place which releases free riboflavin into the medium as well as retained in the nucleotide form. It is also observed that certain purines also stimulate riboflavin production without simultaneous growth stimulation.
The riboflavin is present both in solution and bound to the mycelium in the fermentation broth. The bound vitamin is released from the cells by heat treatment (1h, 120°C) and the mycelium is separated and discarded. The riboflavin is then further purified. The crystalline riboflavin preparation of high purity have been produced using Saccharomyces fermentation with acetate as sole C source.
Uses:
It is essential for the growth and reproduction of both humans and animals and, thus, it often is recommended as a feed additives for the animal nutrition. The riboflavin deficiency in rats causes stunted growth, dermatitis, and eye damage. Ariboflavinosis is a disease in humans caused by riboflavin deficiency.
Steroids Biotransformation:
These are complex organic compounds, as shown in Fig. 20.28. Since the several chemical reactions involved in transformation of basic 4-membered ring into various other products, hence biological transformation is important due to the reason that only few steps are required in getting products.
For example 32 reactions or steps were required to get cortisone from deoxycholic acid, by Sasset (1946). Hence, it proved to be uneconomical. Now-a-days, soybean for stigma sterol, diosgenin from the Dioscorea plant were found to be easy and economical sources.
Microbial preparations of many steroids by their enzyme action at the specific site led to the synthesis of novel varieties of steroids. Such processes are more viable and specific.
Steroid transformation is different with that of microbiological process due to the fact that in later that organic acids, solvents, antibiotics are synthesized from the Ingredients in the medium which also serve as substrate for growth and reproduction of microorganisms.
(i) Microorganisms:
Several fungi, such as Rhizopus Curvularia lunata Aspergillus sp, Penicillium sp., Gliocladium sp., Fusarium solani and yeasts are some of the important organisms of steroid biotransformation. Besides, bacteria namely Corynebacterium simplex and actinomycete Nocardia restrictus are known for biotransformation.
(ii) Production:
The production of steroids can be earned out by the following steps:
(a) Cultivation of microbes:
The appropriate microbe is grown in a way to get maximum growth in a short period of time. Generally, glucose or sucrose are recommended as carbon source and com steep liquor as nitrogen source.
(b) Incorporation of steroids and inhibitors into the medium:
A suitable amount of steroid (0.25 to 1.0 g lit-1) to be transformed is first dissolved with the desired solvent (solvent should not inhibit with the microbe) and then is to add in the medium after the growth of the organism. The desired inhibitors are added to inhibit the undesired enzyme activities.
(iii) Transformation of Steroids:
Microbes transform the steroid in a few hours to several days.
Separation and purification:
For this, regular sample is to be analysed chromatographically using TLC. The spots are eluted and their quantity can be measured spectrophotometacally. For this, the transformation product is extracted with a suitable solvent and then purified by using column chromatography or crystallization.
The structure is determined by using classical organic chemistry methods.
Some important transformations are shown as below (Table 20.2):
Some Important Bio-Transformations:
Steroids are organic molecules which have in common a per hydro-cyclopentaphenanthrene nucleus. Steroids are named because they are related to sterols which are abundant in nature e g cholesterol (found in brain and nerve tissue), stigma sterol (in vegetable oils), ergosterols (in yeast) sapogenms (diosgenin and hecogenin in plants) etc.
The sapogenins are extremely useful starting matenals for sex hormone synthesis and later on for corticoides and contraceptive drugs. It is observed that an agar plate exposed on the window yielded a culture of the genus Rhizopus. When this culture was grown using progesterone as a substrate, they found that it unexpectedly converts progesterone to 11 α-hydroxy progesterone in higher yield (Fig. 20. 29).
The chemical synthesis of steroid hormones is quite difficult due to the reason that it requires several steps. The process is quite costly and may provide low yields because of certain rather difficult chemical steps in the process. For example the total chemical transformation of the cattle bile steroid deoxycholic acid to cortisone requires 37 chemical steps (Fig. 20.30).
These reactions often proceed with apparent base when mediated by microorganisms. Certain microorganisms can introduce hydroxyl groups at any of several of the carbon atoms of the steroid molecule.
Actually, in this process the hydroxylation occurs at 11P position. The reaction is given in Fig. 20.31.
In this case, carbon 11 of the steroid nucleus is of particular interest, because an oxygen atom at this point required for biological activity such as that required for the treatment of rheumatoid arthritis and introduction of oxygen atom at this point is difficult by chemical means.
These days steroid biotransformation is carried on in submerged cultures in large fermenters. These transformations differ from the conventional process in that the products are not synthesized from the medium ingredients. Steroid precursors are added into the culture towards the end of growth phase. In recent years, purified enzymes have also been tried in place of organisms in steroid transformation.