3.1 Isolation and identification 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 confirmed as melanin through standard protocols. The melanin positive organism MAPPPL 017A was identified as Prauserella sp. based on the morphological, physiological, biochemical, molecular and chemotaxonomic identification methods. The resultant nucleotide sequence from the above strain was deposited in the gene bank with the accession no MH341971.
3.2 Melanin Confirmation 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 confirmed 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 Purification of melanin pigments produced by Prauserella sp.
The purification of melanin produced by Prauserella sp. was done using acid precipitation method which is the familiar and sp.ecific method preferred by most of the researchers [12, 14, 18-34] and it was characterized using several state-of-the-art techniques which are described below.
3.4 Physical properties of purified melanin
The purified melanin has been found to be insoluble in water, organic solvents and partially soluble in DMSO. However, the pigment was completely soluble in ammonia water, NaOH and KOH solutions. The purified melanin has been found to be in characteristic black color, viscous in nature and susceptibility to oxidizing agents [35] as has been observed with melanin pigments from various bacterial, fungal and plant sources (Bell) [39] such as seeds of O. fragrans [14], Klebsiella sp. GSK [21], Asp.ergillus nidulans [22], Asp.ergillus bridgeri ICTF-201 [24], Pseudomonas stutzeri [28] Aeromonas media [37], Vibrio cholerae [38], Klebsiella pneumoniae [39], Tea leaves [18, 40], Escherichia coli [41-43], Frankia strain [44], Pleurotus cystidiosus [45] and Black-bone silky fowl [42, 43, 46] as well as synthetic melanin.
3.5 UV-Vis Sp.ectroscopic analysis of purified 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” [47-49]. 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 purified 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.
3.6 FT-IR sp.ectroscopic analysis of purified melanin
The functional groups present in the purified melanin have been characterized with FT-IR sp.ectroscopic analysis. The broad absorption band centered around 3450 and 3445 cm-1 with EM and SM, resp.ectively has been found to be characteristic of O-H or N-H stretching vibrations as has been observed by many investigators [24, 27, 53-57]. The strong characteristic band observed at 1637 and 1636 cm-1 in resp.ect of EM and SM have been attributed to the bending vibrations modes of aromatic ring C=C and C=N bond of aromatic system in addition to C=O double bond (COOH) of carboxylic function involved in the bond formation with the metal ions [2, 24, 27, 53, 54, 57-60]. This may be due to the presence of carbonyl groups conjugated with a benzene ring and a similar situation has been found with quinone structure of DOPA- melanin [61]. Finally, the weak bands observed below 700 cm-1 have been ascribed to alkenes C-H substitution and out-of-plane bending of the aromatic carbon-hydrogen bond in the melanin pigment. The signals present at the region of 600-500 cm-1 (below 700 cm-1) have been attributed to the out-of-plane bending of the oxygen hydrogen bonds [24, 57, 60, 62, 63] and 720 -590 cm-1 areas indicate the presence of aliphatic iodo group (Fig. 3C). The above observation made in the present study has been found to be similar to the eumelanin obtained from Sepia officinalis [55, 64-66].
3.7 Raman sp.ectroscopy analysis of purified melanin
The molecular vibration and crystal structures of the purified 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 purified melanin (EPR)
The EPR sp.ectrum obtained with melanin pigments from Prauserella sp. appeared as a singlet without any hyperfine structure as found in synthetic melanin and DPPH, a well-known antioxidant compound (Fig. 4). EPR Sp.ectroscopy analysis of purified melanin pigment produced signals at the magnetic fields 337.918 [mT], 337.919[mT], 337.881[mT], and their G- values were calculated as 1.99776, 1.99743, 1.99751 for extracellular melanin, synthetic melanin and DPPH, resp.ectively. Similar observations were found in Cryptococcus neoformans [72], Paracoccidioides brasiliensis [73], Histoplasma capsulatum [74], Sp.orothrix schenckii [75], Sepia officinalis [75], Klesiella sp. [21]. The G-values obtained with purified 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].
3.9 1H Nuclear magnetic resonance (NMR) sp.ectral analysis of purified melanin
The NMR sp.ectral analysis performed with purified extracellular melanin from Prauserella sp. has shown signals in both aliphatic and aromatic regions (Fig. 5A & B). The peaks centered in the region of 0.2-2.0 ppm could be assigned to the CH3 group of alkyl fragments such as CH2CH3, CH (CH3)2 as has been observed with melanin pigments obtained from different organisms [30, 31, 54, 60, 78-79]. The peaks centered between 2.2-2.8 ppm indicated the presence of characteristic methylene group [54, 60, 78] while the peak centered at 3.5 ppm has been assigned to the presence of methyl N-CH3 [78] which might be attached to either the pyrrole or indole ring [80]. The peaks centered in the region of 3.5-4.2 ppm have been assigned to the carbon attached to nitrogen or carbon (-CH2 or -CH3 group connected with N/O) as has been found in pigments of other [30, 54, 60]. The peaks observed in the region of 4.2-5.4 ppm are due to the presence of C=C-H structures in the aromatic nucleus [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].
3.10 Solid state 13C NMR analysis of purified melanin
The solid state NMR analysis performed with purified melanin obtained from Prauserella sp. has shown distinctive sp.ectral regions (Fig. 6) such as aliphatic (10-110), 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 (-CH2CH3), methyl or (-CH2-) methylene and the peaks centered in the region of 40-60 ppm are due to the presence of α-carbon or carbon in CH-N/CH-S. The results obtained in the present study corroborates with the observation made earlier by [60, 82-85]. In addition, the peaks observed in the region of 35.2 and 55.7 ppm has been found to show the presence of methyl (N-CH3) group [86]. The peaks observed in the region 95-160 and 110-160 ppm is characteristic of aromatic carbons which indicate the presence of indole or pyrrole types of carbons in the melanin polymers. The presence of broad signals in the aromatic region (110-160ppm) is characteristic of melanin signal [83] as has been observed with natural and synthetic melanin [60, 79, 81-83, 86-89]. The peaks centered in the region 165–225 ppm are as a result of carbonyl carbon atoms derived from peptide bonds, carboxylates, amides and quinones which might be coupled with the melanin polymers or proteins. A peak observed in the region 175 ppm is characteristic of quinine moieties of the carbonyl group of the melanin pigments which has been found in other melanin pigments [60, 79, 81-83, 89-90].
3.11 Elemental analysis of purified 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 purified 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, 93-99]. 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.
Table 1. Quantitative elemental analysis of melanin pigments from Prauserella sp.. (MAPPL 017A) through CHNS analysis
S. No.
|
Elements
|
Extracellular melanin
|
Synthetic melanin
|
Sepia melanin (Magarelli,
et al.,2010)
|
1.
|
Carbon
|
41.0350
|
46.105
|
33.859
|
2.
|
Hydrogen
|
8.1800
|
8.6280
|
3.362
|
3.
|
Nitrogen
|
7.1470
|
5.3290
|
6.068
|
4.
|
Sulfur
|
1.5585
|
0.660
|
< 0.001
|
3.12 Inductively Coupled Plasma Optical Emission sp.ectrometry (ICP-OES) analysis of purified melanin
The melanin pigments isolated from a sp.ecific environment has been linked with numerous metal ions including Mg (II), Ca (II), Na (I), K (I) and almost all the first 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 purified 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 (Table. 2). Similar observation has been made with melanin of Sepia officinalis [66] and Pseudomonas stutzeri [28].
Table 2. Quantitative elemental analysis of melanin pigments from Prauserella sp.. (MAPPL 017A) through ICP-OES analysis
S. No.
|
Elements
|
Wavelength (nm)
|
Extra
cellular melanin
|
Synthetic melanin
|
Sepia melanin
(Magarelli
et al., 2010)
|
Concentration (mg/g)
|
1.
|
Ca
|
317.933
|
1.393
|
3.789
|
47.302
|
2.
|
Fe
|
238.204
|
0.189
|
1.569
|
0.100
|
3.
|
K
|
766.490
|
0.090
|
0.140
|
1.300
|
4.
|
Mg
|
285.213
|
0.648
|
1.714
|
17.310
|
5.
|
Na
|
589.592
|
1.948
|
5.144
|
5.500
|
3.13 HRSEM–EDAX analysis of purified melanin
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, Auricularia auricula ([82], silky fowl [99] however the melanin 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 purified 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 purified 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
S. No.
|
Element
|
Extracellular melanin
|
Synthetic melanin
|
Wt%
|
At%
|
Wt%
|
At%
|
1.
|
CK
|
61.47
|
69.47
|
60.66
|
68.58
|
2.
|
NK
|
09.12
|
08.84
|
07.19
|
06.97
|
3.
|
OK
|
22.28
|
18.90
|
26.76
|
22.71
|
4.
|
NaK
|
00.08
|
00.05
|
00.22
|
00.13
|
5.
|
SK
|
03.46
|
01.47
|
00.58
|
00.25
|
6.
|
ClK
|
02.81
|
01.07
|
01.64
|
00.63
|
7.
|
KK
|
00.11
|
00.04
|
00.22
|
00.08
|
8.
|
FeK
|
00.68
|
00.17
|
02.73
|
00.66
|
3.14 X-Ray Diffraction Analysis (XRD) of purified 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.
3.15 Electron Sp.ray Ionization Mass Sp.ectroscopic (ESI-MS) analysis of purified 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 difficult 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 [101-104]. 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 C18H10N2O4Na, which fell in the lower molecular mass range (Fig. 10) as has been observed earlier in LEM404 [31].
3.16 Thermal properties of purified 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 Table. 4). Similar observations have been made for the melanin obtained from Catharsius molossus [79].
Table 4. Thermal gravimetric analysis of extracellular melanin from Prauserella sp.. (MAPPL 017A)
S. No.
|
Temperature
|
Weight
|
Weight
|
Degree Celsius
|
Mg
|
%
|
Step transition
|
1.
|
75.40 - 109.86
|
0.3941
|
11.79
|
2.
|
236.53 - 369.80
|
1.252
|
37.44
|
Residue
|
3.
|
979.00
|
0.8506
|
25.44
|
Point value
|
4.
|
88.21
|
3.74
|
0.1458
|
5.
|
302.01
|
14.31
|
0.1946
|
3.16.2 Differential Scanning Calorimetric analysis of purified melanin
It was observed that there was a difference in the heat flow at three different temperature ranges such as 85.04 - 111.22, 210.61 - 247.57 and 272.79 - 309.80oC and the maximum heat energy absorption were at 91.47/0.06059 area j/g, 229.61/86.44 area j/g and 282.65 /23.62 area j/g, resp.ectively (Fig. 11B and Table. 5). Similar results were obtained for the melanin obtained from Catharsius molossus [79].
Table 5. Differential Scanning calorimetric analysis for extracellular melanin from Prauserellasp.. (MAPPL 017A)
S. No.
|
Start and end temperature
|
Maximum
|
Area
|
Degree Celsius
|
Degree Celsius
|
J/g
|
1.
|
85.04 - 111.22
|
91.47
|
0.06059
|
2.
|
210.61 - 247.57
|
229.61
|
86.44
|
3.
|
272.79 - 309.80
|
282.65
|
23.62
|