Purification and Physiochemical Characterization of Melanin Pigment from Klebsiella sp. GSK
Sajjan, Shrishailnath1, Guruprasad Kulkarni1, Veeranagouda Yaligara2, Lee Kyoung2, and T. B. Karegoudar1*
Department of Biochemistry, Gulbarga University, Gulbarga - 585 106,Karnataka State, India Department of Microbiology, Changwon National University, Changwon-si, Kyongnam 641-773, Korea
Received: February 4, 2010 / Revised: June 2, 2010 / Accepted: July 21, 2010
A bacterium capable of producing melanin pigment in the presence of L-tyrosine was isolated from a crop field soil sample and identified as Klebsiella sp. GSK based on morphological, biochemical,and 16S rDNA sequencing. The polymerization of this pigment occurs outside the cell wall, which has a granular structure as melanin ghosts. Chemical characterization of the pigment particles showed then to be acid resistant, alkali soluble, and insoluble in most of the organic solvents and water. The pigment got bleached when subjected to the action of oxidants as well as reductants. This pigmentwas precipitated with FeCl3, ammoniacal silver nitrate, and potassium ferricynide. The pigment showed high absorbance in the UV region and decreased absorbance when shifted towards the visible region. The melanin pigment was further charecterized by FT-IR and EPR spectroscopies. A key enzyme, 4hydroxyphenylacetic acid hydroxylase, that catalyzes the formation of melanin pigment by hydroxylation ofLtyrosine was detected in this bacterium. Inhibition studies with specific inhibitors, kojic acid and KCN, proved that melanin is synthesized by the DOPA-melanin pathway. Keywords: Klebsiella sp. GSK, L-tyrosine, melanin, pigment, spectroscopy, 4-hydroxyphenylacetate hydroxylase
Melanins form a diverse group of pigments synthesized in living organisms in the course of hydroxylation andpolymerization of organic compounds. Melanin production is observed in all large taxa from both the Prokaryota and Eukaryota . Melanin is nearly a ubiquitous pigment. Animal melanins may be classified as black eumelanins and yellow-to-brown pheomelanins, whereas melanins from plants, fungi, and bacteria are brown-to-black allomelanins . Melanins are negatively charged, hydrophobic ,*Corresponding author Phone: +91-8472-263289; Fax: +91-8472-245632; E-mail: email@example.com
and high-molecular-weight compounds. These pigments are insoluble in both aqueous and organic solvents, and its is consequently difficult to study their structure by conventional biochemical and biophysical techniques . The ability to produce melanin is widespread among microorganisms. From thechemical point of view, the only common feature of microbial melanins is it being a product of oxidative polymerization of various phenolic substances. Melanins form a quite heterogeneous group of biopolymers. As a consequence, melanogenesis can serve as an example of evolutionary convergence, besides mimicry, and signaling, as well as protection against UV and visible light, and extremetemperatures, and maintaining a proper balance of metal ions . Melanin pigments are synthesized by organism representative of all biological kingdoms and have been implicated in a wide variety of physiological and pathological processes, including the pathogenesis of some microbial infections [5, 37]. Production of melanin is one of the most universal (but at the same time enigmatic) adaptations ofliving organisms to the variable conditions of the Earth. The presence of various kinds of melanins in representatives of almost every large taxon suggests an evolutionary importance of melanogenesis . Melanins have great application potentials in the agriculture, cosmetics, and pharmaceutical industries. Research has revealed that melanin produced by Streptomycete showed photoprotection and...