FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
COMPLETE SPECIFICATION (See section 10; rule 13)
"A PROCESS FOR PRODUCTION OF WATER SOLUBLE
MELANIN USING A STRAIN OF THE FUNGUS
GLIOCEPHALOTRICHUM"
MYKO TECH PRIVATE LIMITED
313, Vainguinnim Valley,
Dona Paula
Goa-403 004, India
A Company incorporated under the Companies Act 1956
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED:

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
A PROCESS FOR PRODUCTION OF WATER SOLUBLE MELANIN USING A STRAIN OF THE FUNGUS GLIOCEPHALOTRICHUM
FIELD OF THE INVENTION
The present invention relates to the production of melanin for various pharmaceutical,
cosmaceutical and industrial applications. It particularly relates to a process to produce water soluble melanin for the purpose. This invention further relates to the production of water soluble melanin from a strain of the fungus Gliocephalotrichum simplex MTCC No: 5489,. by growing the fungus in a culture medium, harvesting the cell-free culture medium, extracting and purifying the melanin from the culture medium and the fungal biomass and using the melanin.
BACKGROUND OF THE INVENTION AND PRIOR ART
Melanins are dark brown heteropolymeric, high molecular pigments and are found in animals,
plants, bacteria, fungi and protists (Hill, Z.H. 1992. The function of melanin or six blind people examine an elephant. BioEssays 14: 49-56; http://en.wikipedia.org/wiki/Melanin). In humans, melanins are found in the skin, hair, certain parts of the brain and the retina. Plant melanins are found in the black pigment in the seed coat of water melon seeds, ripening bananas and date fruit. Fungal melanin is present in their cell walls.
Melanins are generally black or brown pigments, although other colours may occur. Melanins are formed by the oxidative polymerization of phenolic and /or indolic compounds and are generally produced by the oxidation of L-tyrosine by tyrosinase or laccase to L-dihydroxyphenylalanine (L-DOPA) and dopaquinone, which further form indoles, benzthiazoles and amino acids (Aghajanyan, A.E., A.A. Hambardzumyan, A.S. Hovsepyan, R.A. Asaturian, A.A. Vardanyan and A.A. Saghiyan, 2005. Isolation, purification and physicochemical

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
characterization of water soluble Bacillus thuringensis melanin. Pigment Cell researchA8; 130-135; Plonka, P.M. and M. Grabacka. 2006. Melanin synthesis in microorganisms -biotechnological and medical aspects. Acta Biochimica Polonica. 53: 429-443).
Melanins derived from L-DOPA are referred to as EumelaninS and are characteristically black or brown. Yellow or reddish melanins are called Pheomelanins and incorporate cysteine with L-DOPA. Brownish melanins formed from homogentisic acid by tyrosinases are called Pyomeianins. Pigments derived from acetate via the polyketide synthase pathway are generally black or brown and referred to as Dihydroxynaphthalene (DHH) melanins.
irradiation, lytic enzyme attack and a number of other environmental conditions. Melanins also have radio protective and antioxidant pioperties, are adsorbents of radio nuclides and heavy metals and can effectively protect living organisms from ultraviolet radiation. They are used in medicine, pharmacology and in cosmetic preparations. (Butler, M., R. Gardiner and A. Day,
2005. Fungal melanin detection by use of copper sulphide-silver. Mycologia 97(2), 312-319;
Plonka, P.M. and M. Grabacka. 2006. Melanin synthesis in microorganisms - biotechnological
and medical aspects. Acta Biochimica Polonica. 53: 429-4^3; Dastager, S.G., Wen-Jun Li,
Dayanad A., Shu-Kun Tang, Xin-Peng Tian, Xiao-Yang Zhi, Li-Hua Xu and Cheng-Lin Jiang.,
2006, Separation, identification and analysis of pigment (melanin) production in Streptomyces.
African Journal of Biotechnology. 5(8), 1131-1134). Water soluble melanins inhibit the
replication and pathogenesis of the viruses which cause AIDS (e.g., AIDS-viruses) (Montefiori,
D.C., and Zhou, J.Y. 1991. Selective antiviral activity of synthetic soluble L-tyrosine and L-
dopa melanins against human immunodeficiency virus in vitro- Antiviral Research 15:11-25).

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
It is important for melanins to be water soluble to enable their biotechnological applications. Sunscreens containing water soluble melanin protect against harmful UV radiation, which is a major causative factor of melanoma and other cancers of the skin (Pawelek, J., A. Chakraborty, M. Osber, J. Bologna (1992) DHICA Conversion: A possible control point in melanin biosynthesis. Pigment Cell Res. 5:180). Melanin is also used as a substitute for para-aminobenzoic acid (PABA). another compound that absorbs UV light. PABA can become toxic under certain conditions, which may lead to adverse reactions in some individuals. Melanin has also been used in paints, varnishes, and other surface protection formulations to provide greater UV protection to these surfaces. Water soluble melanin may be used in solid plastic films of polyvinyl alcohol to be used in conjunction with other plastics to make laminated sheets or lenses. Such laminates can be used as filters to protect against photochemical damage from electromagnetic radiation (Gallas, .1. and Eisner, M. 2006. Melanin polyvinyl alcohol plastic laminates for optical applications US Patent 7029758). Melanin binds a number of chemicals and drugs, thereby making it useful in the detection of low levels of compounds and metabolites, or in the elimination of toxic target.
Mammalian melanins are highly insoluble and require severe treatments such as boiling in strong alkali, or the use of strong oxidants such as hydrogen peroxide, which often damage the melanins. Therefore, they do not have a commercial potential. Melanin is commercially extracted from cuttle fish (http://www.tightrope.it/nicolaus/12.htm). However, this technology depends on irregular supply of natural material and also is expensive (http://www.encyclopedia.com/doc/lG2-2896600098.htrnl).
Water soluble melanin may be produced synthetically or isolated from natural sources. Various

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
technologies described in the prior art to produce water soluble melanins are described below:
The following patents provide processes to produce soluble, synthetic melanins.
United States Patents 5,216,136, 5,218,079 of 1993, 5,225,435 of 1993, 5,227,459, 5,384,116 of 1995, 5,744,125 of 1998 and 6315988 of 2001 provide various processes to produce synthetic melanin, such as by combining dopachrome and 5,6-dihydroxyindole (or allowing dopachrome to spontaneously form 5,6-dihydioxyindole) and an appropriate enzyme or by combining 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid or by incubating 5,6-dihydroxyindole-2-carboxylic acid alone, or with 3-amino-tyrosine, by procedures involving oxidative polymerization of monomeric precursors of melanin and/or co-monomers, and by polymerizing a dihydroxyindole-carboxylic acid in an aqueous reaction medium, the improvement which comprises effecting the polymerizatiori in the presence of a sulfhydryl compound, or for preparing protein- and/or peptide-bound melanin, which is soluble in an aqueous solution at pH 2 to 11 and temperature 0 DEG C. to 50 DEG C, by the steps of reacting dihydroxyphenylalanine or tyrosine with an oxidant enzyme in the presence of an acidic protein and/or peptide having a pi of 3-6.
However, in vitro synthesis using various precursors, as is given in the above patents is generally more expensive than natural melanins that can be produced in high quantities by suitable organisms. Besides, synthetic melanins involve the use of chemicals that are often environmentally unfriendly.
Natural, water soluble melanins have been described as below,

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
A water soluble melanin has been reported in a mutant strain of Bacillus thuringensis (Aghajanyan, A., A. Hambardzumyan, A. Hovsepyan, R. Asaturian, A. Vardanyan and A. Saghiyan, 2005. Isolation, purification and physicochemical characterization of water soluble Bacillus thuringenesis melanin. Pigment Cell research.18; 130-135). However, the actual yield of melanin is not known from this publication.
Bacteria, belonging to the group Actinomycetes synthesize and excrete melanin, or melanoid pigments into the culture medium (Dastager .S.G.,Wen-Jun Li, Dayanad A., Shu-Kun Tang, Xin-Peng Tian, Xiao-Yang Zhi, Li-Hua Xu and Cheng-Lin Jiang., 2006. Separation, identification and analysis of pigment (melanin) production in Streptomyces. African Journal of Biotechnology. 5(8), 1131-1134). However, the yields of melanin in such cases is very low.
United States Patent 5,814,495 of 1998 provides a process for producing melanins by Streptomyces, a bacterium belonging to the Actinomycetes by manipulating the constituents of the growth medium, and/or fermentation conditions, and/or genetically engineering microorganisms. However, their Claims 1 and 2 on production of melanin from naturally occurring Streptomyces do not specify the amounts of melanin produced by the organism.
The above patent also discloses a process for overproducing melanin in Streptomyces by genetically engineering the said organism. Examples 2 to 15 relating to these claims on genetically engineered microorganisms shows that a maximum of 3.0 g dry weight melanin / L culture medium was produced. These Claims, therefore, pertain to a genetically modified organism with a yield of only 3.0 g / L melanin. Besides, production of compounds from genetically engineered organisms requires a number of regulatory clearances.

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
United States Patent 5,837,505 of 1998 provides a process of producing melanin by Escherichia coli which was transformed with a vector containing DNA sequences from Streptomyces spp. This patent discusses the production of melanin from a genetically modified organism. Besides, the yield of melanin thus produced does not exceed 4 g liter medium. Besides, production of compounds from genetically engineered organisms requires a number of regulatory clearances.
European patent EP 1052290 provides a process for producing melanin by culturing the fungus Podospora anserina in a medium containing carbon, phosphate and protein sources pH 5-7 and extracting melanin from the biomass. The process does not deal with melanin freely secreted into the culture medium.
In US Patent 6,090,588 of 2000, the production of a melanin-like compound by introducing an inactivated virus into a culture of bacteria is disclosed. After introduction, the inactivated virus causes a phenotypic change in the bacteria. When introduced into a media containing tyrosine, these modified bacteria produce a melanin-like compound, pyomelanin. The bacteria will continue to produce pyomelanin until they are removed from the tyrosine source. However, this patent does not concern the production of high yields of water soluble melanin, but only a process to produce them.
United States Patent 6,576,268 of 2003 discloses a process of production of vegetable melanin and its use in industry and pharmacology. This patent discloses a method to extract melanin from raw materials of vegetable origin. However, production of vegetable melanins is subject to natural variabilities in the plant and seasonal availability of plants. The above patent also does not indicate absorbance of the said melanin in the UV range of 200 — 350 nm.

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
Cabrera-Valladares, N. et al. (2006. Expression of the melA gene from Rhizobium etli CVN42 in Escherichia coli and characterization of the encoded tyrosinase. Enzyme Microbial Technology 38: 772-779) have cloned the tyrosinase coding gene, mel A from the nitrogen-fixing bacterium Rhizobium etli and transformed the bacterium Escherichia coli W3110 to produce eumelanin. Production of water soluble eumelanin from this recombinant strain was optimized to produce 6 g of melanin per litre of culture filtrate (Lagunas-Munoz, VH et al. 2006. Optimum melanin production using recombinant Escherichia coli. Journal of Applied Microbiology 101: 1002-1008). This process uses recombinant strains. Production of melanin from such a strain requires stringent regulatory clearances. Besides, the maximum yield was only 6.0 g per litre culture filtrate.
Whereby it is desirable to provide for water soluble melanin from naturally occurring microbial sources in amounts in excess of 6 g per litre of culture medium and which would avoid the problems/disadvantages noted above and overcome other problems encountered in conventional methods.
SUMMARY OF INVENTION
The above problems are overcome in the present invention which relates to the production of melanin which is water soluble and is obtained by growing a strain of the fungus Gliocephalotrichum simplex MTCC 5489, in a culture medium, harvesting the cell-free culture medium, extracting and purifying the melanin from the culture medium and the fungal biomass, the yield of the said melanin being at least 6.6 grams per litre, and using the melanin,
The objects, advantages and novel features of the invention may be realized and obtained by

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
means of the instrumentalities and combinations particularly pointed out in the detailed description.
OBJECT OF INVENTION
It is therefore an object of present invention to disclose a process for production of water soluble melanin.
Yet another object of the patent is to disclose a process to produce water soluble melanin from a naturally occurring microbial source.
A further object of this invention is to disclose a process to produce water soluble melanin from a strain of the fungus Gliocephalotrichu simplex bearing the Accession No. 5489. It is another object of the present invention wherein the fungus Gliocephalotrichum simplex MTCC 5489 is isolated from natural sources.
Yet another object of the present invention is to disclose a process to produce water soluble melanin from a strain of the fungus Gliocepluilotrichum simplex MTCC 5489 which is freely secreted into the culture medium and also adhering to the biomass in amounts of up to 7 g per liter of culture medium, by growing the fungus in a suitable nutrient medium.
Yet another object of the present invention is to extract the water soluble melanin from the culture filtrate and the biomass of Gliocephalotrichum simplex MTCC 5489 by first separating the two by standard procedures such as centrifugation and filtration, repeatedly washing the biomass with water to extract water soluble melanin adhering to the biomass, adding the fraction thus obtained to the culture filtrate containing water soluble melanin, precipitating the dissolved

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
melanin from the pooled fractions by acidifying the pH of the culture filtrate to 1.5 to 2.0, removing the precipitated melanin by standard procedures such as centrifiigation or filtration and drying the precipitated melanin by standard procedures such as heat drying, spray drying and freeze-drying.
Yet another object of the present invention is to dry the precipitated melanin Gliocephalotrichum simplex MTCC 5489 by standard procedures such as heat drying, spray drying and freeze-drying.
In yet another object of the present invention, the dried melanin obtained from Gliocephalotrichum simplex MTCC 5489 is resolubilized by dissolving it in water at a pH of 11 to 12.0, after which the solution containing melanin may be adjusted to pH between 2.0 and 12.0 as required by addition of acid, dialysis, etc.
Yet another object of the present invention is to use the melanin obtained as above as medical, cosmaceutical and industrial ingredient.
The foregoing object of the present invention is achieved by the present invention.
DETAILED DESCRIPTION OF THE INVENTION
In order to produce melanin, the fungus is typically grown in a suitable liquid culture medium that will promote the production of water soluble melanin. Such a melanin production culture medium contains an organic carbon source, a nitrogen source, tyrosine and copper sulphate. Additional minerals may be added. Examples of organic carbon sources are glucose, starch, glycerol, lactose, peptone, yeast extract and corn steep liquor. Nitrogen sources may be organic, such as, but not exclusively peptone. Examples of inorganic nitrogen sources are salts of

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
ammonium, nitrate and urea. Minerals may be added in the form of magnesium, phosphate, sulphur etc. Further, the culture is provided with tyrosine, which is required for melanin formation, as well as copper sulphate, which is required for enzymatic conversion of tyrosine to melanin by the organism. Any suitable pH of the culture medium for optimal production, ranging from 4.0 to 8.0 may be chosen.
A typical procedure is to first grow the fungus in Petri plates containing culture medium with agar, such as Malt Extract Agar for about 4 days and inoculating the actively growing edge of the fungal colony in a liquid medium such as but not restricted to malt extract broth for a further period of about 4 days. This is the inoculum medium. The biomass of the fungus produced in this inoculum medium is used to inoculate the melanin production culture medium. Typically, a volume from the inoculum medium containing fungal biomass corresponding to 5 % of the production culture medium is used as inoculum. The culture is grown at any suitable temperature ranging from 20 to 35 deg. C. The cultures are grown under shaken conditions ranging from 60 to 200 rpm. The cultures are grown for the required number of days, typically from 3 to 6 days tilt the culture filtrate becomes black in colour owing to dissolved melanin.
In order to separate the melanin from the culture filtrate, the culture filtrate may be separated from the biomass by any one of the conventional methods, such as centrifugation and filtration. Melanin in the culture filtrate may be precipitated by lowering the pH of the liquid using hydrochloric acid, or acetic acid. In a preferred method, aluminium hydroxide at a final concentration of 1 % may be added to precipitate the melanin. The precipitated melanin may be separated from the supernatant liquid by using methods well known in prior art, such as centrifugation and filtration. The melanin may then be dried by air-drying, freeze-drying or any

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
of the other well known methods. In addition to the culture filtrate, water soluble melanin may be further extracted from the fungal biomass that was separated as given above. The biomass is suspended in water for 3 to 24 hours, whereby melanin still adsorbed to the biomass is released into the water in a dissolved form. This melanin may again be precipitated as above from the liquid and pooled to the melanin fraction collected from the culture filtrate. In a preferred method, melanin present in the culture filtrate, as that extracted in a dissolved form from the fungal biomass may be concentrated using any of the conventional methods, such as ultrafiltration or rota-evaporation. The concentrated solution of melanin may then be used for precipitating the melanin by acidifying the liquid or by adding aluminium hydroxide as above.
Powdered melanin obtained in the above manner may be redissolved by suspending the powder in water and adjusting the pH to 11 or above. Melanin is then immediately solubilized. This solution may be dialyzed in order to remove salts and the solution may be adjusted to pH from 3 to 10 as required.
The above description is illustrated below by way of examples.
Example 1
The fungus Gliocephalotrichum simplex MTCC 5489 was cultured from soil under thick vegetation along the roadside to Tambdi Surla, Goa. About 1 g of the soil sample was suspended in 10 ml of sterile water in culture tubes and a series of one to ten dilutions of the soil were prepared further in sterile distilled water in tubes. An aliquot of 0.1 ml of the soil dilution was poured in Petri plates and overlaid with Potato Dextrose Agar (PDA) medium, corresponding to standard pour plate methodology for isolating soil microorganisms. Fungal colonies appearing on the Petri plates were examined under a stereo zoom microscope and transferred to fresh

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
culture tubes containing slants of PDA medium. One such fungus producing colonies was Gliocephalotrichitm simplex. The colony was maintained on PDA tubes. The culture has been deposited at the Microbial Type Culture Collection of the Institute of Microbial Technology, Chandigarh under the Budapest Treaty and has been given an accession number MTCC 5489.
Example 2.
When grown in liquid medium made of Malt Exract Broth on a shaker at 120 rpm, the culture filtrate of the fungus turned dark brown after 6 days. In order to check if the dark brown colour of the culture filtrate was due to water soluble melanin, the fungus was grown in a culture medium containing tyrosine. The following experimental protocol was followed. The fungus was first grown in Petri dishes containing Malt Extract Agar medium. After about 5 days, a small portion of the growing edge of the fungal colony was inoculated into a 250 ml Erlenmeyer flask containing 100 ml of Malt Extract Broth medium. This was the medium used as inoculum for the experiments. Experimental media in flasks were inoculated with 5 % by volume of the inoculum. Cultures were grown at a room temperature of approximately 25 C on an orbital shaker at an rpm of 120 for the experiments. For this experiment using tyrosine, the composition of the experimental medium was: 2 % glucose, I % peptone, 0.5 % yeast extract and 100 ml of distilled water. Tyrosine was added to one set of flasks at a concentration of 0.5 %. Cultures were grown for 5 days on a shaker at 120 rpm at a room temperature of 25 - 28 C. Culture filtrates of the fungus grown in the presence of tyrosine turned black in colour after 5 days growth, while no black colour was noticed in flasks containing no tyrosine. The culture filtrate was separated from the fungal biomass by centrifugation and the pH was adjusted to pH 1.5 using I N Hydrochloric Acid. The precipitated melanin was centrifuged and the clear supernatant was discarded. The precipitate was repeatedly washed with distilled water and was

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
then freeze-dried to yield a black powder. The above experiment indicated that the black powder was melanin. A known weight of melanin was added to a tube containing a known volume of distilled water and the pH was adjusted to 12.0 using 1 N sodium hydroxide. Different dilutions of this melanin solution were then prepared and a standard graph was prepared using a UV/Visible Spectrophotometer at a wavelength of 270 nm,
Example 3.
In order to further ensure that the melanin was induced in the presence of tyrosine, the amount of melanin produced was estimated in Malt Extract Broth with 2.5 % tyrosine and without tyrosine. The experimental protocol provided in Example 2 was used to grow the fungus and extract the melanin. At the end of the growth period, the amount of melanin present in culture filtrates of both the treatments was estimated spectrophotometrically, using the standard graph, as given in Example 2. Malt Extract Broth without tyrosine yielded 0.2 g per Liter culture of tyrosine, while Malt Extract Broth containing 2.5 % tyrosine yielded 1.26 g per Liter of culture medium. This experiment showed that the pigment was induced in the presence of tyrosine and that it was melanin.
Example 4.
The melanin was resolubilized as given in Example 2. A suitable dilution of this melanin solution was prepared and an absorbance spectrum of the solution was analysed between 200 and 700 nm. The solution showed the typical UV absorbance of melanin and an absence of absorbance in the visible range of 400 to 700 nm (Fig. 1). The spectrum further indicated that the black powder was melanin.

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
Example 5.
In order to confirm that the pigment was melanin, two qualitative tests were performed (Babitskaya VG, VV Shcherba and NV Ikonnikova. 2000. Melanin complex of the fungus Inonotus obliqutts. Applied Biochemistry and Mic robiology 36: 439-44.), Dilutions of the melanin solution obtained as in Example 2 were used. Test 1: To a 0.1 % solution of 1 % melanin, 1 ml of 1 % potassium permanganate was added and kept for 15 minutes. The melanin solution turned green from brown and subsequently precipitated. Test 2: An amount of 0.01 g of melanin as dissolved in 10 ml of 2 % sodium hydroxide solution. An amount of 1 ml of hydrogen peroxide was added to this. Another solution was kept as control to which no hydrogen peroxide was added. The absorbance of these was estimated at 2 and 24 hours. The results are given in Fig. 1.


NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
The bleaching of the pigment with hydrogen peroxice and the reaction with potassium permanganage indicate that the pigment was melanin.
Example 6.
In order to examine whether copper sulphate, which is known to be essential for production of tyrosinase involved in melanin synthesis was required or not, Gliocephalotrichum simplex MTCC 5489 was cultured in the experimental culture medium containing 1 % glucose, 1 % peptone, 0.1 % yeast extract, 0.5 % tyrosine. Culture protocols were as given in Example 2. One set of flasks contained in addition to the above ingredients of the culture medium, also 0.0005 % copper sulphate. Melanin was extracted and weighed as given in Example 2, The culture containing copper sulphate produced 0.18 g per Litre melanin, while the medium without copper sulphate produced only 0.1 g per Litre melanin, thus suggesting that tyrosinase was involved in the production of the black pigment, indicating that the pigment was melanin.
Example 7.
In order lo optimize levels of glucose in the medium and the amount of fungal inoculum that needed to be inoculated in the experimental flasks, the following experiment was earned out. Experimental protocols were basically the same as in Example 2. The basal experimental medium contained 1 % peptone, 0.1 % yeast extract and 0.5 % tyrosine. Glucose was added at concentrations of 1 and 2 % to two different sets of experimental flasks. The two sets were subdivided into two further sets, one set receiving 5 % and the other set receiving 10 % inoculum. The following results were obtained.


NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
The results showed that glucose above 1 % was inhibitory to melanin production and that a 5 % inoculum would suffice.
Example 8.
Media optimization for optimal production of melanin by Gliocephalotrichum simplex MTCC 5489 was studied using 9 different media, as follows.

The results indicated that a culture medium number 2, containing 1 % Peptone, 2.5 % tyrosine, 1 % glucose, 0.1 % yeast extract, 0.05 % KH2P04, 0.02 % MgS04, 0.0005 % CuS04 and 0.01 % FeS04 was the best, providing 4.6 g melanin per litre culture medium.

NAME OF THE APPLICANT: MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
Example 9.
It was earlier observed that the centrifliged and pelleted biomass continued to ooze out water
soluble melanin. In order to further increase the yield of the compound, melanin was extracted
from both the culture filtrate and the mycelial biomass. Basic experimental protocols provided in
Example 2 were followed. The organism was grown in a culture medium provided in Example 7,
comprising 1 % peptone, 2.5 % tyrosine, 1 % glucose, CUSO4, KH2PO4, MgS04 and FeS04.
Melanin was extracted after 6 days from the culture filtrate using the same protocols as given in
Example 2. The pelleted biomass was suspended in distilled water overnight to recover water
soluble melanin oozed out from the biomass. This solution was pooled with the culture filtrate.
Melanin from this pooled extract was precipitated as given in Example 2. The total yield of
melanin obtained this way amounted to 6.6 g per Liter melanin.

NAME OF THE APPLICANT; MYKO TECH PRIVATE LIMITED PROVISIONAL APPLICATION NO: 2545/MUM/2008
We claim:
1. A process for producing water soluble melanin.
2. A process as in Claim 1, wherein the melanin is obtained from the fungus Gliocephalotrichum simplex , with the accession No. MTCC 5489
3. A process as in Claims 1 and 2, wherein the yield of the water soluble melanin produced from the fungus is at least 6.6 g per litre culture medium.
4. A process as in Claims 1 to 3, wherein the fungus Gliocephalotrichum simplex MTCC 5489 is cultivated in any suitable liquid culture medium containing an organic carbon source, an organic or inorganic nitrogen source, tyrosine and copper sulphate for 3 to 7 days.
5. A process as in Claims ) to 4, wherein the water soluble melanin secreted into the culture medium is precipitated and separated by acidifying the culture medium to a pH of 2.0, preferably a pH of 1.5 using hydrochloric acid.
6. A process by which the water soluble melanin secreted into the culture medium is precipitated and separated by acidifying the culture medium to a pH of 2.0, or preferably a pH of 1.5 using acetic acid.
7. A process by which the water soluble melanin secreted into the culture medium is precipitated and separated by the addition of 1% alum to the culture medium.
8. A process by which the water soluble melanin obtained from Gliocephalotrichum simplex MTCC 5489 is used in various applications such as cosmetics, optics, pharmacology and industrial applications.