In this article we will discuss about:- 1. Introduction to Streptomycin 2. Chemical Structure of Streptomycin 3. Biosynthesis 4. Fermentation Process 5. Uses of Streptomycin.
Introduction to Streptomycin:
Streptomycin, produced by streptomyces griseus is active against Gram (-) ve bacteria and against tuberculosis bacterium, Mycobacterium tuberculosis. However, it proved to be useful in the treatment of infections caused by Gram (+) ve specially resistant to penicillin. It is also useful in the control of plant diseases caused by bacteria as it acts systemically in plants.
One of the disadvantages of streptomycin is its neurotoxicity due to which hearing impairment and balance maintenance is lost in man due to prolonged streptomycin treatment at high dosage. Its reduction to dihydrostreptomycin results in the decreased toxicity.
For this reason in recent times only dihydrostreptomycin is being produced due to ready development of resistance against streptomycin. It is used mostly in conjunction with para aminosalicyclic acid or isoniazid (isonicotinic acid hydrazide) which minimizes resistance build up in sensitive microorganisms.
Chemical Structure of Streptomycin:
Streptomycin and dihydrostreptomycin is an aminoglycoside antibiotic and basic compound which is available as hydrochloride, C21H39N7O12. 3 HCl, as a crystalline hydrochloride double salt with calcium chloride or as phosphate or sulphate and dihydrostreptomycin as the hydrochloride or sulfate. The chemical structure of streptomycin is given in Fig. 6.10.
Unit of streptomycin activity is equal to one microgram of the free base. Use of precursor does not increase yields of streptomycin.
Biosynthesis of Streptomycin:
Streptomycin is directly derived from glucose. Though the enzymes involved in the synthesis of N-methyl glucosamine are not yet known, it is expected that about 28 enzymes take part in the conversion of glucose into streptomycin as precised in Fig. 6.11.
Fermentation Process of Streptomycin:
Industrially streptomycin is produced by submerged culture method, whose flow sheet is given in Fig. 6.12.
When Woodruff and Mc Daniel (1954) suggested medium consisting of soyabean meal (1%), glucose 1% and sodium chloride (0.5%), Hocken hull (1963) recommended the medium consisting of glucose (2.5%), soyabean meal (4.0%), distillers dry soluble (0.5%) and sodium chloride (0.25%) and pH 7.3-7.5 for production of streptomycin by S. griseus.
(i) The Inoculum Production:
Spores of S. griseus maintained as soil stocks or lyophilized in a carrier such as sterile skimmed milk, is employed as stock culture. The spores from these stock cultures are then transferred to a sporulation medium to provide enough sporulated growth to initiate liquid culture build-up of mycelial inoculum in flasks or inoculum tanks. After sufficient mycelial growth, it is fed to production fermenter.
(ii) Preparation of the Medium:
A production medium contains carbon source and nitrogen source. Glucose is one of the best carbon sources which helps in the greater yield of streptomycin, because it provides basic carbon skeleton for the streptomycin production. Apart from glucose, fructose, maltose, lactose, galactose, mannitol, xylose and starch can also be used as carbon source. Polysaccharides and oligosaccharides generally give low yields.
Peptones, soya extracts, meat extract, the residue from alcohol distillation, ammonium salts, nitrates and glycine may be used as nitrogen source. Magnesium, calcium, potassium, boron and molybdenum may be used as mineral source along with sulphates, phosphates and chlorides.
Phenylacetic acid, L-naphthalene acetic acid may be added as growth stimulating compounds. It is better to add proline into the medium which helps in high streptomycin production. Fats, oils and fatty acids may also be used along with glucose. If necessary antioxidants such as sodium sulphate or starch or agar may also be added into the medium. There is no need of precursor in the production of streptomycin.
Sterilized liquid medium with all the above substances is fed to the production fermenter. Appropriate volume of inoculum (4-5%) is introduced into it. The optimum fermentation temperature is in the range of 25 to 30°C and the optimum pH range is between 7.0 and 8.0. High rate of streptomycin production, however, occurs in the pH range of 7.6 to 8.0.
The process of fermentation is highly aerobic and lasts approximately for 5 to 7 days and passes through 3 phases:
(a) The First Phase:
It takes about 24 hours to 48 hours. Rapid growth and formation of abundant mycelium occurs during this phase. The pH rises to 8.0 due to release of ammonia into medium, due to proteolytic activity of S. griseus. Glucose is utilized slowly and little production of streptomycin is witnessed.
(b) The Second Phase:
It lasts for 2 days. Streptomycin production takes place at a rapid rate without increase in the mycelial growth. The ammonia released in the first phase is utilized, which results in the decrease of pH to 7.6-8.0. Glucose and oxygen are required in large quantity during this phase.
(c) Third Phase:
Cells undergo lysis, releasing ammonia and increase in the pH, which falls again after a period of continuous streptomycin production. Requirement of oxygen decreases and the contents of the medium including sugar get exhausted. Finally streptomycin production ceases. A yield of 1200 micrograms per milliliter of streptomycin is obtained.
(iv) Harvest and Recovery:
After completion of fermentation the mycelium is separated from the broth by filtration. Streptomycin is recovered by several methods.
But the one which is generally employed is described below:
The fermentation broth is acidified, filtered and neutralized. It is then passed through a column containing a cation exchange resin to adsorb the streptomycin from the broth. The column is then washed with water and the antibiotic is eluted with hydrochloric acid or cyclohexanol or phosphoric acid. It is then concentrated at about 60°C under vacuum.
The streptomycin is then dissolved in methanol and filtered and acetone is added to the filtrate to precipitate the antibiotic. The precipitate is again washed with acetone and vacuum dried. It is purified further by dissolving in methanol. The streptomycin in pure form is extracted as calcium chloride complex.
(v) Byproduct Vitamin B12:
Vitamin B12 is produced as a byproduct which will not affect adversely the yield of streptomycin.
Uses of Streptomycin:
1. Streptomycin is active against Gram (-) negative bacteria. It is used in the treatment of tuberculosis caused by Mycobacterium tuberculi.
2. It is also used therapeutically in the treatment of infectious diseases caused by Gram (-) negative bacteria, specially those organisms which are resistant to penicillin.
3. Prolonged treatment by streptomycin at high dosage can produce neurotoxic reactions such as hearing impairment, loss of balance maintenance in man.