Microbial Production and Characterization of 3, 4-dihydroxy-L Phenylalanine Mediated an Extracellular Heterogenic Eumelanin Pigment From Marine Actinomycetes Prauserella Sp. MAPPL 017A

Melanin is one of the natural biological pigment and it is derived from the enzyme tyrosinase by the oxidation of an amino acid tyrosine. The enzyme tyrosinase [EC 1.14.18.1] is a type-3 copper protein family found large amount in microorganisms and human beings involved in the biosynthesis of melanin and other polyphenolic compounds. Melanin plays an important role in all living creatures such as photo protectants against UV radiation, antioxidant, metal adsorption, electrical conductivity, camouage, anti-aging properties, virulence and fungal pathogenesis. The above properties of melanin can be used in various application purposes which make the melanin expensive and great demand in the industry. To overcome the above situation, there is a necessity to nd out the new biological sources for the maximum production of melanin. Numerous reports are available on fungal and bacterial melanin but only a few of them from actinomycetes. Based on the above view, the present investigation was aimed to purify the DOPA melanin from the novel marine actinomycetes Prauserella sp. (MAPPL 017A). Puried melanin was characterized using several state-of-the-art techniques and it’s showed the broadband absorbance, presence of carboxylic and indolic groups, high amount of carbon, nitrogen and low level of sulfur, presence of other metal ions such as calcium, sodium and magnesium with ecient thermal properties.


Introduction
Melanin pigments are highly insoluble natural biological macromolecules [1] which occurs in all the animal kingdom. Based on the color and substrate sp.eci city these macromolecules are classi ed into ve major types such as eumelanin, pheomelanin, pyomelanin, allomelanin and neuromelanin. Among these, eumelanin is the predominant pigment synthesized in humans and microorganisms [2] which are hydrophobic, negatively charged and high molecular weight polymer [3].
In microorganisms, the melanin pigments are metabolically derived from the enzyme tyrosinase which catalyzed by the oxidation of an amino acid tyrosine [4][5] and the resultant melanin pigments are black or brown in color [3]. It is also been proved that the microbial tyrosinases are resemblance to the type-3 copper metalloprotein [6].
Melanin pigments are polymers of phenolic compounds [7] possess antioxidant and radioprotective properties which can be used as a protective shield from the harmful radiations and it is evident that the low level of melanin in the human skin has high risk of tumor [8][9]. It also plays an important role in the virulence of a broad range of phytopathogenic fungi [3,10]. In addition, melanin has an a nity towards the metal ions and is naturally associated with them. The above characteristics made the researchers make use of it in different technologies such as metal adsorption and electric conductivity (semiconductors) and bioelectronics. Due to the above information, the present study was aimed to purify the extracellular melanin from the novel marine Actinomycetes Prauserella sp. and this is the rst report on melanin production from the genus Prauserella.

Isolation and identi cation of melanin producing marine actinomycetes
Melanin producing marine actinobacterium was isolated from water collected in the marina beech, (Lat. 13° 3' 20" N and Long. 80° 17' 1" E) a region of Bay of Bengal, Chennai, Tamil Nadu, India and screened for the melanin production. The melanin positive strain was identi ed through polyphasic, molecular and chemotaxonomic characterization.

Test for melanin formation and tyrosinase activity
The test for melanin formation and tyrosinase activity were carried out by a simple, rapid and sensitive method proposed by Mikarni et al., [11].

2.3
Extraction and puri cation of melanin pigment The extraction and puri cation of extracellular melanin produced by Prauserella sp. (MAPPL 017A) was performed by the method [12].

Puri cation of melanin from Prauserella sp. (MAPPL 017A)
For the puri cation of melanin pigments, different solvents and extraction procedures were carried out to remove the associated components such as protein carbohydrate in the melanin pigment. The removal of carbohydrates and proteins were carried out by acid hydrolysis which was followed by lipids extraction with the solvent chloroform and the metabolites were sequentially removed by the extraction of different solvents such as hexane, ethyl acetate, acetone and ethanol.

Acid hydrolysis
The crude melanin obtained from the above procedure was hydrolyzed with 10 ml of 6 M HCl for 2 h at 100ºC to remove carbohydrates and proteins. The non-hydrolysable residue was collected by centrifugation at 10,000 rpm for 10 min and washed with distilled water for several times and dried under desiccation and stored in airtight containers for further use.

Washing with solvents
The melanin obtained after acid hydrolysis was re-dissolved in 2 M NaOH and centrifuged at 10,000 rpm for 15min. The supernatant was collected and subjected to precipitation with 1 M HCl and centrifuged to obtain the pellet. The pellet was washed several times with distilled water and dried well. The nonhydrolysable melanin was re-dissolved in 5 ml of 1M NaOH to which 2 mL of chloroform and 0.2 mL of 1butanol were added. The mixture was shaken for 30 min and centrifuged at 10,000 rpm for 10 min. The chloroform and butanol phase was discarded and the procedure was repeated twice. The solution containing melanin was treated with organic solvent such as, hexane, ethyl acetate, acetone for the removable of secondary metabolites and nally with ethanol for the removable of water molecules bound with the melanin pigment. The resulting content was lyophilized and stored as 'pure melanin' for further characterization.
2.5 Characterization of melanin pigment 2.5.1 Physio-chemical analysis of puri ed melanin The chemical analysis of puri ed melanin pigment was carried out by the modi ed method of [13][14].
Commercially available synthetic melanin was purchased from Sigma (Catalog No. M 8631) and used as the standard for all the characterization directly without any further treatment.

FT-IR analysis of puri ed melanin
For FT-IR analysis, approximately 5 mg of melanin and 700 mg of KBr were thoroughly mixed and made into pellets. The pellet was used for recording the IR sp.ectra using an FT-IR sp.ectrometer (Perkin Elmer, USA).

Raman sp.ectroscopic analysis of puri ed melanin
The puri ed melanin was subjected to Raman sp.ectroscopic analysis (Raman 11i, Nano photon, Japan) at the range of 1000-2500 cm -1 . The sp.ectrometer has back scattering geometry for the detection of Raman sp.ectrum with the resolution of 1 cm and the excitation source was 532 nm. The percentage of elements such as carbon, hydrogen, nitrogen and sulfur present in the melanin pigment was determined over a wide range of sample matrices and concentrations with Trusp.ec micro analyzer at Sophisticated Analytical Instruments Facility, IIT Madras, Chennai, India.

Inductively Coupled Plasma Optical Emission Sp.ectrometry (ICP-OES) analysis of puri ed melanin
The atomic sp.ectrum emitted by puri ed melanin is used to determine its elemental composition by ICP-OES analysis (Perkin Elmer optima 5300 DV, USA). The wavelength at which emission occurs identi ed the element while the intensity of the emitted radiation quanti es its concentration.

Procedure for Melanin digestion for ICP-OES
Thirty milligrams of melanin was dissolved in 10 mL of deionized water which was added with 1 mL each of nitric acid (65 %, w/v) and perchloric acid (70%, w/v). The mixture was heated at 100 o C for 5 min and the volume was made up to 100 mL with deionized water.

X-ray diffraction analysis of puri ed melanin
The puri ed melanin obtained from Prauserella sp. was ground to ne powder with mortar and pestle and subjected to analysis by X-ray diffraction with the following parameters: CuKa radiation (λ-1.5406 Å), voltage 40 kV, current 40 mA, 2θ range 3º-80º.

Thermal degradation analysis of puri ed melanin
The puri ed melanin obtained from Prauserella sp. was weighed (3.343 mg) and analyzed with a thermal analyzer (SDTQ600v 8.0 build 95). The TGA and DSC data were recorded under nitrogen atmosp.here at the ow rate of 20 mL/min. The analysis was carried out with ambient temperature to 1000ºC at a heating rate of 10ºC/min.

Isolation and identi cation of melanin producing marine actinomycete
Pigment producing isolate MAPPL 017A was isolated using the sea water collected from the sea shore of marina beech, a region of Bay of Bengal, Chennai, Tamil Nadu, India. And produced the diffusible purplish black color pigments on Czapek tyrosine (Fig. 1). The resultant pigment was con rmed as melanin through standard protocols. The melanin positive organism MAPPPL 017A was identi ed as Prauserella sp. based on the morphological, physiological, biochemical, molecular and chemotaxonomic identi cation methods. The resultant nucleotide sequence from the above strain was deposited in the gene bank with the accession no MH341971.

Melanin Con rmation test
The qualitative and quantitative determination of melanin production by the isolate MAPPL 017A was carried out by the addition of L-DOPA to the culture supernatant. The color changes into brown con rmed the presence of enzyme tyrosinase (Fig. 2) and the amount of tyrosinase enzyme produced by the melanin positive isolate was quantitatively determined and the maximum enzyme production occurred on 15 day (Fig. 2). Similar experiments were carried out in Streptomyces griseus and Streptomyces phaeochromogenes [11].
3.3 Puri cation of melanin pigments produced by Prauserella sp.

UV-Vis Sp.ectroscopic analysis of puri ed melanin
Melanin is an amorphous pigment which naturally exhibit scattering phenomenon (broadband absorption) in the UV-Vis sp.ectrum (scattering phenomenon) and it believed to be formed by the superposition of the peaked sp.ectra which are termed as "chemical disorder model" [47][48][49]. The melanin pigments show strong UV absorption in the region of 200-300 nm which could be attributed to the π =>π* and n=>π* of the amino, carboxylic and aromatic moieties [50]. In the present study, the puri ed melanin pigment obtained from Prauserella sp. and synthetic melanin showed strong absorbance in the shorter wavelengths such as 211 and 213 nm, resp.ectively (Fig. 3A) and the absorption decreased rapidly when the wavelengths increased to 400-800 nm. The absorbance of the melanin pigments were monotonically increased towards the higher energy region (200-300 nm) as it can be observed only in the range of 210-240 nm and decreased rapidly with increasing wavelength towards the visible region (400-800). The above characteristics have been considered as general features of the melanin pigments [51] which increases the protection against the most damaging high energy from harmful radiations. This phenomenon is due to the superposition of absorption of individuals, distinct chemical constituents [52] and the presence of complex conjugated structure of the melanin pigments.

Raman sp.ectroscopy analysis of puri ed melanin
The molecular vibration and crystal structures of the puri ed melanin was analyzed using Raman sp.ectroscopic analysis. The extra cellular melanin (EM) from Prauserella sp. and Synthetic melanin (SM) exhibited two prominent peaks in the ranges between 1300 cm -1 -1600 cm -1 (Fig. 3B). The presence of lower wave number centered at 1345 cm -1 , 1340 cm -1 (EM and SM) were related to the C-N stretching mode of the indole structure and peaks centered at 1557 cm -1 , 1576 cm -1 (EM and SM) were assigned to aromatic C=C stretching modes of the basic indole structure and C=N stretching/N-H bending (Matsunuma, Capozzi, 2005, Nagano, Priti Vairale) [67 -70]. Similar peak were observed in cutaneous melanin [71] 3.8 Electro paramagnetic resonance sp.ectroscopic analysis of puri ed melanin (EPR) The EPR sp.ectrum obtained with melanin pigments from Prauserella sp. appeared as a singlet without any hyper ne structure as found in synthetic melanin and DPPH, a well-known antioxidant compound (Fig. 4) [75], Sepia o cinalis [75], Klesiella sp. [21]. The G-values obtained with puri ed melanin were found to be nearer to the value of standard antioxidant compound which indicated the presence of stable free radicals in the melanin pigments [77].

1 H Nuclear magnetic resonance (NMR) sp.ectral analysis of puri ed melanin
The NMR sp.ectral analysis performed with puri ed extracellular melanin from Prauserella sp. has shown signals in both aliphatic and aromatic regions (Fig. 5A & B). The peaks centered in the region of 0.  [54,60]. The peaks centered between 6.5-9.0 ppm have been assigned to the proton attached to the substituted aromatic and hetero-aromatic regions [30,54,60,79].

Solid state 13 C NMR analysis of puri ed melanin
The solid state NMR analysis performed with puri ed melanin obtained from Prauserella sp. has shown distinctive sp.ectral regions (Fig. 6) such as aliphatic , aromatic (110-160) and carbonyl groups (160-225) which corroborates the observation made with Sepia melanin, acid free Sepia melanin and melanin from human hair [81]. The peaks observed in the lower region (10-95ppm) are characteristics of aliphatic carbons, which might have arisen from the proteinaceous substance as sociated with pigment as has been observed in Catharsius molossus [79] and Auricularia auricula [82]. The aliphatic carbon in the lower regions centered at 10-40 ppm could be due to the presence of the alkyl group such as (-CH 2

Elemental analysis of puri ed melanin
Melanin pigments naturally have the tendency to bind to various metals and therefore the CHNS analysis has been routinely performed to identify the type of melanin and to determine the purity of the pigment. The monomeric units of eumelanin and pheomelanin resp.ectively should have 6-9 and 8-11% nitrogen content with a sulfur content of 9-12% in the case of benzothiazine monomer [91][92]. The elemental analysis performed with the puri ed melanin has shown the presence of relatively higher content of carbon (41.04%) when compared to other elements such as hydrogen (8.18%) and nitrogen (7.15%; Table. 1). However, the melanin obtained from different sources have exhibited varied elemental compositions [18,22,31,60,79,[93][94][95][96][97][98][99]. The sulfur content found in the EM and SM in the present study has been 1.56 and 0.66%, resp.ectively. Naturally, the subunits of eumelanin and pheomelanin are formed through similar pathways and exist in a mixed form however it can be differentiated based on the percentage of sulfur content. The sulfur content of eumelanin obtained from different sources has been found to be in the range of 0.001 to 14.83% [31,63,91,[98][99]. It has been suggested that the sulfur content in the melanin samples might be due to the addition of some thiol containing compounds or presence of sulfur containing aminophenol in the melano proteins during the polymerization of eumelanin. The melanin pigments isolated from a sp.eci c environment has been linked with numerous metal ions including Mg (II), Ca (II), Na (I), K (I) and almost all the rst transition metals have been shown to contain Fe (III) in abundant quantity. Therefore, melanin pigments could serve as a reservoir of metal ions such as Ca (II) Cu (II) and Fe (III). In the present investigation, the quantitative determination of the abundant metal ions in the puri ed melanin pigments has been carried out with ICP-OES analysis which indicated the presence of Na + , K + , Mg2 + ,Ca2 + and Fe3 + . Both EM and SM have been found to contain the same set of metal ion with varied concentration (  In the present study the morphological characteristics and quantitative determination of the elements present in the melanin pigments have been carried out with SEM-EDAX analysis ( Fig.7 & 8). The obtained biopolymer appeared to be in amorphous nature as has been observed in synthetic melanin as well as the melanin obtained from a mushroom, pigments obtained from Jurassic period have been found to be sp.herical in shape [89]. The elemental composition in resp.ect of EM and SM has been found in the following orders C, O, N, S, Cl, Fe, K, Na and C, O, N, Fe, Cl, S, K, Na. It has been observed that carbon, oxygen and nitrogen have been found to be the most abundant elements while sulfur, chlorine, iron, potassium and sodium are the least elements (Table. 3). A higher quantity of carbon, nitrogen and lower quantity of oxygen was observed in the puri ed melanin when compared to the synthetic melanin which indicated the density of the melanin layers. A traces of sulfur content was also found in the puri ed melanin which could be due the presence of copolymer of eumelanin and pheomelanin or aminophenol in the melanoprotein. Table 3. Quantitative elemental analysis of extracellular melanin from Prauserella sp.. (MAPPL 017A) through EDAX analysis 3.14 X-Ray Diffraction Analysis (XRD) of puri ed melanin The XRD sp.ectra of melanin pigments obtained from Prauserella sp. has shown broad peak on the 2θ scale of 20°C in all the diffractograms (Fig. 9). The broad band peaks observed are characteristic of amorphous materials like melanin polymers appeared in the region of 20°C which might be due to the parallel planar layers found in melanin structure [100]. Melanin pigments obtained from Catharsius molossus [79] and Pseudomonas stutzeri [28] have exhibited similar peaks.

Electron Sp.ray Ionization Mass Sp.ectroscopic (ESI-MS) analysis of puri ed melanin
Due to the remarkable structural diversity and amorphous nature, melanin pigments fail to dissolve in organic solvents. Therefore, investigations to study their structural property and functions have become di cult task however are considered essential for application purposes. Therefore, the characterization of melanin pigments has been performed with Mass Sp.ectrometry (MS) such as ESI-MS and MALDI-MS [101][102][103][104]. In the present study, the ESI-MS analysis has revealed the molecular masses of 341.0523 m/z and the molecular formula was derived as C 18 H 10 N 2 O 4 Na, which fell in the lower molecular mass range ( Fig. 10) as has been observed earlier in LEM404 [31].
3.16 Thermal properties of puri ed melanin 3.16.1 Thermal gravimetric analysis The extracellular melanin from Prauserella sp. underwent two stages of thermal degradation. The initial degradation occurred between 75.40 -97.47ºC which was treated as the glass transition temperature where the compound lost a weight of 0.394 mg (11.79 %) as a result of loss of free and bound water molecules present in the melanin pigment. The second degradation occurred at 236.53 -369.80ºC which resulted in the loss of 1.252 mg (37.44 %) due to the destruction of advanced and sub-structure of melanin pigments, non-covalent bonds present between layers of the structural units, covalent bonds of the monomer units such as indole and pyrrole group. The major weight loses occurred at 88.21 and 302.01ºC and their derived weight were 0.1458 mg/ 3.74 min and 0.1946 mg/ 14.31 min, resp.ectively. The complete weight loss occurred at 979.00 ºC (Fig. 11A and

Conclusion
Melanin the natural biopolymer which plays an important role in most living organisms. Based on the physical and chemical composition it has been isolated and puri ed from different sources and the exact structure of the melanin was not yet identi ed. Among the different types of melanin, eumelanin is the predominant one. Sepia melanin is well-known eumelanin puri ed from cuttle sh (Sepia o cinalis) which was completely characterized by many of the researchers and it occupies a foremost position in melanin industries. The second position was occupied by fungi followed by bacterial melanin but only a few reports are available on actinomycetes. In the present study, the melanin pigment puri ed and characterized from the novel actinomycetes Prauserella sp. and it was con rmed as eumelanin based on the color and chemical nature. For the additional support, the enhanced production of melanin by the isolate Prauserella sp. was observed only in the presence of tyrosine through DOPA pathway thus con rmed that the resultant eumelanin was DOPA type. The various biological activities of the melanin from Prauserella sp. can be used various elds like cosmetics, bioelectronics, semiconductors, anticancer drugs, and protective equipment.

Declarations Con ict of Interest
We know of no con icts of interest with this publication and there has been no signi cant nance support for this work that could have in uenced its outcome.  Qualitative and quantitative determination of tyrosinase activity by Prauserella sp. at different time intervals