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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, Tamil Corresponding Author: S.venkatesan,
Dept of Microbiology, Issued 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 Isolation ����������� Soil samples from different
locations of the megamalai forest, 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 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% RESULTS ����������� 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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