Microbial process to produce water soluble melanin, a compound for radiation protection.

A water soluble melanin is produced extracellularly in a most economical manner by a microorganism. The melanin corresponds to eumelanin typical of black, human hair. It has excellent UV light absorbance, protects internal organs against gamma radiation. Studies so far have shown it is safe for use.

Full Description:
Melanins are black to brown pigments present in animals, plants, bacteria and fungi. It is the pigment found in human skin and hair, fungal cell walls and the blackened skins of bananas and date palm.

Melanins are phenolic polymers of which there are three types of melanin (Butler, 2005; Plonka and Grabacka, 2006. Acta Biochimica Polonica 53: 429-443; www.en.wikipedia.org/wiki/Melanin).

(1) Eumelanins (black or brown) are present in human hair and skin and are produced by some bacteria and fungi. These are produced by oxidation of tyrosine to o-dihydroxyphenylalanine (DOPA) and dopaquionone.

(2) Pheomelanins (yellow-red) are present in red human hair. These are initially synthesized just like eumelanins, but DOPA undergoes cysteinylation, directly or by the mediation of glutathione. Phaeomelanins contain sulphur, by virtue of the presence of cysteine.

(3) Allomelanins are the least studied and the most heterogenous group of polymers and are found in many fungi and plants. Fungi generally produce the DHN melanin (Babitskaya et al., 2000), which is produced through oxidation or polymerization of di- (DHN) or tetra-hydroxynaphthalene. Allomelanins may also be produced from homogentisic acid (pyomelanins), γ-glutaminyl-4-hydroxybenzene, catechols (plants), as well as of 4-hydroxyphenylacetic acid.

Chemical characterization: The full chemical structure of melanin is still incompletely understood because these complex polymers are amorphous and not amenable to either solution or crystallographic structural studies (Nosanchuk and Casadevall, 2006). However, there are certain general characteristics. Characterization of melanin is based on qualitative tests, UV-Visible Spectrum, HPLC determination of oxidized melanin,IR spectrum, electron spin resonance signal and NMR.

Uses of melanin:
• Protection against UV-induced damages: Melanin is an excellent UV-absorbing compound and it protects organisms from UV-induced DNA damages and environmental stress. Therefore, melanin has potential applications in cosmetics, pharmaceuticals, optical lenses, paints and varnishes and other technologies (Gallas, 1991; Gaskin,1993, Andrean et al., 1995; Pawelek; 1995; Gallas and Eisner, 2006).
• Bio-electricity: Melanin could be useful in bio-electronics as semiconductors (Mostert et al., 2012).
• Protection against gamma radiation: Melanin has a very effective radical-scavenging capacity and possesses a high ability to accept and give back electrons. The availability of radioprotective materials suitable for internal administration which would protect normal organs without protecting the tumor would greatly enhance the efficacy of radiation therapy by permitting higher tumoricidal doses while protecting normal organs. Radioprotective materials could also be extremely useful for protection against terrorist actions using radiological devices and for protecting astronauts in space (Dadachova et al., 2007a, b; Dadachova and Casadevall, 2012).
• Studies on radiation protection properties of Myko Tech’s melanin have shown that the melanin exhibited both prophylactic and mitigative activities, increasing the 30-day survival of mice by 100% and 60%, respectively, after exposure to radiation (7 Gy, whole body irradiation). The protective activity was due to inhibition of radiation-induced hematopoietic damages. The melanin also reversed the radiation-induced decrease in ERK phosphorylation in splenic tissue, which may be the key feature in its radioprotective action. The melanin prevented apoptosis in splenic tissue and reduced the oxidative stress in hepatic tissue and abrogated immune imbalance (Kunwar et al., 2012).

2. Sources of melanin, the market and the price: Commercial melanin is presently available from the following sources.
• The marine cephalopods, the cuttle fish or Sepia eject an ink made of melanin as a defence mechanism. Sepia melanin is the best studied melanin so far (Adhyaru et al., 2003; Magarelli et al., 2010) and has been used as a standard in many research studies on the compound.
• Soluble synthetic melanins: Pawelek et al. (1993) described a method to produce synthetic melanin by combining dopachrome and an appropriate enzyme, or by incubating 5,6-dihydroxyindole-2-carboxylic acid alone or with 5,6-dihydroxyindole, or with 3-amino-tyrosine.
• Vegetable melanins: Kerestes and Kerestes (2003) provided a process to make plant melanin, whereby a vegetable raw material containing polymers or monomeric units of the flavonoids, especially of catechins and leucoanthocyanidins is chemically treated to obtain melanin. Yao et al. (2012) provided a method for the production of melanin-like pigments with different solubilities from chestnut shells.
Eumelanin from cuttlefish and synthetic melanin each cost about US $ 100.- per gram.

3. Status of present research: Several studies have been carried out to develop more economical ways to produce melanin
• Microbial melanin: Many species of Streptomyces produce small amounts of extracellular melanin by oxidization of tyrosine by tyrosinase (Dastager et al., 2006). Many other bacteria synthesize melanin similarly. Many fungi produce DHN allomelanin. The production of extracellular melanin and tyrosine-derived eumelanin by fungi has been doubted by Butler and Day (1998).
• A recombinant approach has been adopted by many to increase tyrosinase or eumelanin from bacteria (Della-Cioppa et al. 1998a, b; Lin et al., 2005; Cabrarera-Valladares et al., 2006; Lagunas-Munoz et al. 2006; ) .
• Mani et al. (2001) prepared protein- and/or peptide-bound melanin, soluble in an aqueous solution at pH 2 to 11 and temperature of 0 to 50 degree 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 pl of 3-6. The soluble protein- and/or peptide-bound melanin of this invention is useful as a sunscreen and as a coloring and/or flavoring for food.

Adhyaru B.B., Akhmedov, N.G., Katritzky A.R. and Bowers, C.R. 2003. Magn. Reson. Chem. 2003; 41: 466–474
Andrean, H. et al. 1995. US Patent 5451254.
Babitskaya, V.G., V. V. Shcherba, T. V. Filimonova, and E. A. Grigorchuk. 2000. Applied Biochemistry and Microbiology, Vol. 36, No. 2, 2000, pp. 128-133.
Butler M.J. and A.W. Day. 1998. Can. J. Microbiol. 44: 1115–1136
Butler, M.J. R.B. Gardiner and A.W. Day. 2005. Mycologia 97: 312-319.
Cabrarera-Valladares N et al., 2006. Enzyme Microbial Technol. 38: 772–779
Dadachova, E.,, R.A. Bryan, X. Huang, T. Moadel, A. D. Schweitze, P. Aisen, J. D. Nosanchuk and A. Casadevall. 2007a. PLoS ONE 2(5): e457. doi:10.1371/journal.pone.0000457.
Dadachova, E., R.A. Bryan, R.C. Howell, A.D. Schweitzer, P. Aisen , J.D. Nosanchuk and A. Casadevall. 2007b. Pigment Cell Melanoma Res. 21; 192–199.
Dadachova E. and A. Casadevall. 2012. WIPO Patent Application WO/2012/129047
Dastager S.G. et al. 2006. African Journal of Biotechnology Vol. 5: 1131-1134.
Della-Cioppa, G. et al., 1998a. US Patent 5814595
Della-Cioppa, G. et al., 1998b. US Patent 5837505.
Gallas, M. 1991. US Patent 5,047,447.
Gallas J and M. Eisner. 2007. US Patent 7029758
Gaskin, F.C. 1993. US Patent 5256403.
Kerestes J. Jr. and J. Kerestes. 2003. US Patent 6,576,258 B2.
Kunwar, A., B. Adhikary, S. Jayakumar, A. Barik, S. Chattopadhyay, S. Raghukumar and K.I. Priyadarsini. 2012. Toxicology and Applied Pharmacology 264 (2012) 202–211.
Lagunas-Munoz VH et al., 2006. Journal of Applied Microbiology 101: 1002-1008
Lin, W-P. et al. 2005. J. Microbiol. Immunol. Infect. 38: 320-326.
Magarelli M, Passamonti P, Renieri C. 2010. Rev CES Med Vet Zootec; Vol 5 (2): 18-28.
Mani, I. et al. 2001. US Patent 6315988.
Mostert AB, Powell BJ, Pratt FL, Hanson GR, Sarna T, Gentle IR, Meredith P.. 2012. Proc Natl Acad Sci U S A. 2012 Jun 5;109(23):8943-7. doi: 10.1073/pnas.1119948109. Epub 2012 May 21.
Nosanchuk JD and A Casadevall. 2006. 50: 3519-3528.
Pawelek J.M. 1993. US Patent 5225435.
Pawelek J.M. et al. 1995. US Patent 5384116.
Plonka PM and M Grabacka. 2006. Acta Biochimica Polonica 53: 429–443.
Yao, Z., Qi, J. and Wang, L. 2012. Isolation, Fractionation and Characterization of Melanin-like Pigments from Chestnut (Castanea mollissima) Shells.
Zhong, J., S. Frases,§ H. Wang, A. Casadevall and R.E. Stark. 2008. Biochemistry 47: 4701–4710

4. Myko Tech’s process to produce melanin and advantages

Myko Tech’s melanin overcomes the following constraints in presently available melanins.
• Natural melanins, including those derived from cuttlefish and fungi are insoluble in water, thus restricting their use. Melanins, such as human hair and fungal melanin are extracted under harsh conditions, such as boiling using NaOH.
• Few microbes produce extracellular, soluble (Aghajanyan et al., 2005).. However, the yield of such melanin is low.
• Soluble, microbial melanin has been produced using recombinant Escherichia coli (della-Cioppa et al., 1997; 1998 a and b). However, this is not commercially available. Besides, use of recombinant organisms has restrictions based on regulations.
• Synthetic, soluble melanins described by Pawelek (1993) and Pawelek et al. (1995) are produced enzymatically by converting melanin precursors to the pigment and to use these in cosmetics. However, these precursors, DOPA and DHI are themselves expensive, resulting in high cost of synthetic melanin.
• Vegetable melanins are different from the true, or eumelanins and are also expensive.

Myko Tech Pvt. Ltd., has filed a patent for a process to produce extracellular, soluble melanin from the fungus Gliocephalotrichum sp.

Myko Tech’s melanin has the following advantages.

1. It is produced extracellularly, thus making downstream processing easy.
2. The melanin is water soluble and produced as an optically clear, dark solution. Therefore, it is amenable to a wide variety of uses.
3. Melanin produced amounts to levels of up to 6.6 g per liter culture, being the highest known so far for any natural, wild type microorganism.
4. The melanin chemically corresponds to eumelanin, the same as produced in black human hair and skin.
5. Has shown great promise in protection against gamma radiation exposure in studies using mice models.

Myko Tech’s melanin has been characterized by qualitative tests, UV-visible spectrum, IR spectroscopy and NMR.

1. The melanin is bleached by hydrogen peroxide and turns purple in the presence of potassium permanganate.
2. It absorbs both ultraviolet (UV) and visible light. The absorbance typically increases linearly in the range of 720 to 620 nm and then exponentially toward shorter wavelengths (300–600 nm) (Ou-Yang et al., 2004). Myko Tech’s melanin shows the well-known and much reported monotonic, broadband UV-visible absorption spectrum of eumelanin and the spectrum fits closely a single exponential with no distinct chromophoric peaks (Meredith and Sarna, 2006).
3. IR spectroscopy shows features typical of eumelanin, containing quinones, aromatics and amide structures.
4. Electron paramagnetic resonance (EPR) spectrum: One of the most unusual properties of melanin as a biomacromolecule is its persistent electron spin resonance (ESR) signal – a clear indication of free radical centres present in the material (Meredith and Sarna, 2006). The EPR spectrum of Myko Tech’s melanin showed typical, strong resonance absorption, consisting of a single symmetric EPR peak with no hyperfine structure.
5. Detection of pyrrole tricarboxylic acid (PTCA) in acidic KMnO4 oxidized melanin is specific for dihydroxyindole carboxylic acid (DHICA), the major component of eumelanin (Wakamatsu and Ito, 2002). HPLC analyses of Myko Tech’s melanin confirmed this to be the major component.
6. NMR spectroscopy: The 1H NMR spectrum of Myko Tech’s melanin is very similar to that of human hair melanin given by Katritzky et al. (2002). The 13C NMR spectrum of Myko Tech’s melanin remarkably resembles that of human hair melanin (Adhyaru et al., 2003; Ghiani S et al., 2007).
Myko Tech’s Publications on its melanin

1. Jalmi, P., Bodke, P., Wahidullah, S. and Raghukumar, S. 2012. The fungus Gliocephalotrichum simplex as a source of abundant, extracellular melanin for biotechnological applications. World Journal of Microbiology and Biotechnology.28: 505-512. DOI 10: 1007/s11274-011-0841O
2. Kunwar A., Adjhikary B., Jayakumar, S.,Barik, A., Chattopadhyay, S., Raghukumar, S. and K.I. Priyadarsini. 2012. Melanin: a promising radioprotector; Mechanisms of actions in a mice model. Toxicology and Applied Pharmacology 264: 202-211.

Problem this idea/invention addresses:
The present invention provides a natural, biological compound with multiple uses including cosmetics, material sciences and cancer therapy. In cosmetics, it can be used to protect against the harmful effects of UV radiation by incorporation in sun protection compounds. It can also be used as a black, hair dye. In cancer therapy, it can protect patients undergoing radiation therapy against the harmful effects of gamma rays. In material science, it can be incorporated in plastics, resins and lenses as an antioxidant. The compound is melanin, nature’s solution to all the above problems.

Auxiliary products or services for sale:
This technology is linked to the technology on production of L-dihydroxyphenylalanine, also from Myko Tech. All negotiations will be for both the technologies together.

Sale or license. Negotiable.

Technology for sale or license: Patented microbial strain, technical knowhow, patent rights.

Cost: Negotiable.
Upfront payment: US$ 150,000.
Staggered over the next two years: US$ 150,000; or 5% royalty over profit in the next 10 years.
Total: US$ 300,000.

Myko Tech Private Limited,
313, Vainguinnim Valley,
Dona Paula, Goa – 403 004.

Contact person: Dr Seshagiri Raghukumar
E-mail: [email protected]
Phone Number: +91(0832)256070
Mobile: +919890450007

Attached files:
Melanin pigment.docx

IN 25,452,008
US 2,013,056,202

Asking price: [CONTACT SELLER]
Available for consultation? Yes

Invention #11871
Date posted: 2013-09-09

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