Ethnobotanical Leaflets 12: 827-31. 2008.
Isolation, Identification and Characterization of
Melanin Pigment Production in Streptomyces from
S.Venkatesan1, R. Ganesan2 and K. Muthuchelian2
1Dept of Microbiology,
2Centre for Biodiversity
and Forest Studies,
Corresponding Author: S.venkatesan,
Dept of Microbiology,
����������� Six strains among 25 streptomyces isolates produce a diffusible dark brown pigment on both peptone-yeast extract agar and streptomyces isolation agar. They also show the +ve reaction to L-tyrosine substrates. The pigment has been referred to be dark brown water soluble pigment, as melanin. The different carbon and N2 sources, which influence the pigment production in the streptomyces isolates, were also investigated. The melanin formation in the streptomyces sp is the key feature for the classification of the streptomyces group.
Key words: Streptomyces, melanin, pigment. L-asparagine, L-arginine.
����������� Actinomycete also synthesizes and excretes dark pigments, melanin (or) melanoid, which are considered a useful criterion for taxonomical studies. Melanin compounds are irregular, dark brown polymers that are product by various microorganisms by the fermentative oxidation, and have the radioprotective and antioxidant properties that can effectively protect the living organisms from UV radation (vinarov et al, 2002). Melanins are frequently used in medicine, pharmacology and cosmetic preparations.
����������� Biosynthesis of melanin with tyrosinase transform the tyrosine into L-Dopa which in further converted into dopachrome and autooxidized to indole �5,6-Qu inone. The later it is polymerized spontaneously into DOPA � Melanin that gives dark brown pigment.
����������� Soil samples from different
locations of the megamalai forest,
����������� The general criteria used for streptomyces identifications (Table 1) are morphology, aerial mycelium, substrate mycelium, spore morphology, production of diffusible pigments, production of melanin pigment, and utilization of various carbon and nitrogen sources (Simon et al., 1999).
����������� Melanin formation was tested on Peptone � yeast extract agar and streptomyces isolation agar. 10ml of suitable liquid media were dispensed in test tubes and inoculated with one loop full of the spores of the streptomyces are subjected to stationary stage at 27oC for seven days. Melanin pigment was estimated by taking 2 ml of the culture and 1ml of 0.4% substrated solution (L-Tyrosine (or) L-dopa). The reaction mixture was incubated at 37oC for 30 min for L-tyrosine and 5 min for L-Dopa and red coloration resulting from dopachrome formation was observed and read spectrophotometer at 480nm. When there was no coloration within these periods, the reaction mixture was further incubated for as Long as 2 hrs. After incubation melanin was then formed within 30 min (Scribners et al., 1973).
Effect of carbon sources on melanin formation
����������� The basal medium of the following composition was used with 1% glycerol, starch, dextrin, lactose, sucrose, fructose (or) glucose as the sole carbon source. 2.0 g of NaNO3, 1.0 g of K2HPO4, 0.5 g of mgso4.7H2O, 0.5 g of Kcl, 0.01 g of Feso4.7H2O in 1000 ml of distilled water (pH 7.2).
Effect of Nitrogen sources on melanin formation
����������� The effect of nitrogen sources (L-asparagine, L-arginine, L-citrulline, L-histidine, glycine, L-Lysine, L-proline (or) L-tyrosine) on the melanin pigment production in streptomyces was studied with the same basal medium using 1% of glycerol as the carbon source. All carbon and nitrogen sources were prepared in 10% solution, sterilized with bacteriological filter, and added to the basal medium to give the final concentration of 1%
����������� The effect of carbon sources on the melanin pigment production by the streptomyces spp. is recorded in Table 2. Starch is the most effective carbon sources for the production of melanin, followed by glycerol and fructose. The comparative efficiency of various nitrogen sources for the production of melanin by the streptomyces is reported in Table 3. The amino acids like citrullin, argine, Lysine and proline were found to be the most effective nitrogen sources. Strain SV 6 is most effective in producing the melanin pigment.
����������� Pigmentation of streptomyces is distinct enough to allow ready delineation in most streptomyces cultures when combined with other fundamental features, such as colour of the surface aerial mycelium after sporulation, sporophore morphology and spore surface. Sometimes it is quite difficult to determine, whether the diffusible pigments produced are melanoid (Dark brown) (or) merely a brown substance, especially when complex organic media is employed. The present study reveals that the method of testing melanin production by L-tyrosine (or) L-dopa as a substrate may be the good criterion for the identification and classification of streptomyces.
Table 1. The Physiology and Biochemical characterization of Streptomyes isolates.
Table 2. Effect of Carbon Source on Melanin formation by Streptomyces.
Table 3. Effect of Nitrogen Source on Melanin formation of Streptomyces.
Armen, E.A. Artur,A.H. Anichka, S.H. Rafael, A.A. Andranik,A.V.and Ashot, A.S. (2004). Isolation, purification and physiological characterization of water �soluble Bacillus thurengiensis melanin Pigment. Cell Res 18: 130-135.
Cochrane, V.W. (1961). Physiology of Actinomycetes. Annu Rev. Microbiol 15:1-26.
Grant, W.D. Mwatha, W.E. Jones, B.E. (1990).Alkaliphiles ecology, diversity and application .FEMS Microbial. Rev. 75:255-270.
Gottlieb, D. (1961). An evaluation of criteria and procedures used in the description and characterization of Streptomyces: A cooperative study .Appl. Microbiol 9: 55-65.
Heim, A.H. Lechavalier, H. (1956). Effect of Iron, Zinc, Manganese and Calcium on the growth of various strains of Streptomyces. Mycologia 48:628-636.
Mencher, J.R. Heim, A.H. (1962). Melanin biosynthesis by Streptomyces lavendulae. J Gen Microbial 28: 665-670.
Shirling, E.B. Gottlibe, D. (1966). Methods for characterization of Streptomyces species. Int. JI Syst. Bacterial. 16: 313-340.
Shirling, E.B., Gottlieb, D. (1970). Report
of the intemational Streptomyces
Project �five Years of collaborative research. The Actinomycetales.
Gustav, Fischer. Verlag.
Scribner�s, E. lll. Terry Tang, Bradley, S. G. (1973). Production of a sporulation pigment by Streptomyces venezuelae. Appl. Microbial 25(6): 873-879.
Meirelles, L. Marta, H. Branquniha, A. Beateiz, V. (1999). Influence of growth medium in protease and pigment producation in Streptomyces cyaneus. Mem. Inst.Oswaldo. Cruz. 94 (2): 173-177.
Arai, T. Mikami, Y. (1972).Choromogenecity of Streptomyces.Appl. Microbiol. 23:402-406.
Zonova, G.M. (1965).Melanoid pigments of Actinomycetes. Microbiologiya 34: 278-283.