Ethnobotanical Leaflets 12: 827-31. 2008.

Isolation, Identification and Characterization of Melanin Pigment Production in Streptomyces from Megamalai Forest Soil

S.Venkatesan¹, R. Ganesan² and K. Muthuchelian²

¹Dept of Microbiology, V.H.N.S.N. College, Virudhunagar, Tamil Nadu, India

²Centre for Biodiversity and Forest Studies, Madurai kamaraj University, Madurai,

Tamil Nadu, India.

Corresponding Author: S.venkatesan, Dept of Microbiology, V.H.N.S.N. College, Virudhunagar, Tamil Nadu, India

Issued 30 October 2008

ABSTRACT

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.

INTRODUCTION

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.

MATERIALS AND METHOD

Isolation

Soil samples from different locations of the megamalai forest, Western Ghats, Tamilnadu, India were collected and screened for pigmented microorganisms especially streptomyces by applying standard serial dilution plate technique, using starch casein Nitrate Agar and streptomyces isolation agar. The plates were incubated at 30^oC for 4 weeks. After incubation typically pigmented, dry, powdery colonies were selected from mixed plate culture and maintained on fresh medium to get pure cultures. The pure cultures were stored at 4^oC until further examination.

Characterization

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

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 27^oC 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 37^oC 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%

RESULTS AND DISCUSSION

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.

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