A
Melanin, a polyphenolic polymer formed by the oxidative polymerization of phenolic and/or indolic compounds, was generally produced from the oxidation of L-tyrosine by tyrosinase or laccase to L-DOPA and dopaquinones, finally to dihydroxyindole carboxylic acid and their reduced forms [
Melanin is commercially extracted from cuttlefish and depends on irregular supply of natural material and also is expensive [
Melanin plays an important role in humans and its lack leads to several abnormalities and diseases. The reduced melanin in neurons causes Parkinson’s disease [
Efficient isolate for melanin synthesis was characterized by 16S rRNA analysis [
Submerged bioprocess in tyrosine broth was standardized for the production of melanin using
Response Surface Method (RSM) with central composite design (CCD) was employed [
The extraction and purification of the melanin were carried out as per the standard protocols described by Fava et al. [
An indigenous method of slant culture system was standardized and operated for the synthesis and extraction of soluble pigment. Potential isolate of
The pigments obtained by both submerged bioprocess and slant culture were confirmed as insoluble and soluble melanin by following chemical method, UV-vis spectroscopy, FT-IR spectroscopy, and thin layer chromatographic techniques. The solubility of pigments in deionized water, 1 N HCl, 1 N NaOH, 1 N KOH, 1 N NH4OH, ethanol, acetone, chloroform, and benzene was assessed [
The cytotoxic activities of insoluble and soluble melanins were determined by following the standard protocol of Meyer et al. [
Seven isolates of
Screening of isolates for the synthesis of melanin on starch tyrosine (a), tyrosine agar (b), and phylogenetic tree (c) of
Actinobacteria can be analyzed at various molecular levels to gain information suitable for constructing databases and effective identification. Sequence analysis of various genes provides a stable classification and accurate identification, which has become the cornerstone of modern phylogenetic taxonomy [
Operation of process variables using manually one at a time and keeping others constant is a precondition to detect critical variables for the production of melanin employing response surface methodology. 4.2 g/L melanin was produced by
In the recent past, Response Surface Method, a software based statistical design, is the most validated method for the production of biomolecules, especially in the submerged fermentation. In the present investigation, an attempt was made to achieve the higher production of melanin by
Optimization of critical process variables employing response surface method for the production of insoluble melanin by
Run | Critical process variables | Melanin production (g/L) | ||||
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Actual value | Predicted value | |
1 | 42.50 | 7.75 | 1.25 | 1.25 | 4.35 | 4.35 |
2 | 42.50 | 7.75 | 1.25 | 1.25 | 4.35 | 4.35 |
3 | 35.00 | 7.00 | 0.50 | 2.00 | 1.08 | 2.12 |
4 | 35.00 | 8.50 | 2.00 | 2.00 | 3.05 | 3.51 |
5 | 27.50 | 7.75 | 1.25 | 1.25 | 2.12 | 1.90 |
6 | 35.00 | 8.50 | 2.00 | 0.50 | 4.21 | 3.73 |
7 | 42.50 | 7.75 | 1.25 | 1.25 | 4.35 | 4.35 |
8 | 42.50 | 7.75 | 2.75 | 1.25 | 4.05 | 3.91 |
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10 | 42.50 | 9.25 | 1.25 | 1.25 | 3.15 | 3.35 |
11 | 35.00 | 7.00 | 2.00 | 2.00 | 3.11 | 2.99 |
12 | 50.00 | 7.00 | 0.50 | 0.50 | 1.63 | 1.57 |
13 | 35.00 | 7.00 | 2.00 | 0.50 | 2.41 | 2.91 |
14 | 50.00 | 8.50 | 2.00 | 2.00 | 3.92 | 4.53 |
15 | 57.50 | 7.75 | 1.25 | 1.25 | 2.16 | 2.27 |
16 | 42.50 | 7.75 | 1.25 | 1.25 | 4.35 | 4.35 |
17 | 50.00 | 8.50 | 0.50 | 2.00 | 1.55 | 1.44 |
18 | 35.00 | 8.50 | 0.50 | 2.00 | 1.53 | 1.38 |
19 | 42.50 | 7.75 | 1.25 | 2.75 | 4.48 | 3.47 |
20 | 42.50 | 7.75 | 1.25 | 1.25 | 4.35 | 4.35 |
21 | 42.50 | 7.75 | 1.25 | −0.25 | 2.78 | 3.67 |
22 | 50.00 | 8.50 | 0.50 | 0.50 | 1.89 | 1.72 |
23 | 35.00 | 8.50 | 0.50 | 0.50 | 1.55 | 1.75 |
24 | 50.00 | 7.00 | 2.00 | 2.00 | 3.20 | 3.40 |
25 | 42.50 | 7.75 | −0.25 | 1.25 | 0.090 | 0.12 |
26 | 42.50 | 6.25 | 1.25 | 1.25 | 2.98 | 2.67 |
27 | 42.50 | 7.75 | 1.25 | 1.25 | 4.35 | 4.35 |
28 | 50.00 | 7.00 | 2.00 | 0.50 | 3.35 | 3.22 |
29 | 50.00 | 7.00 | 0.50 | 2.00 | 1.38 | 1.58 |
30 | 35.00 | 7.00 | 0.50 | 0.50 | 3.10 | 2.21 |
Analysis of variance (ANOVA) of model response data.
Source | Sum of squares | Df | Mean square |
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Model | 42.50 | 14 | 3.04 | 8.10 | 0.0001 |
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0.21 | 1 | 0.21 | 0.56 | 0.4647 |
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0.69 | 1 | 0.69 | 1.84 | 0.1948 |
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21.57 | 1 | 21.57 | 57.55 | <0.0001 |
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0.061 | 1 | 0.61 | 0.16 | 0.6923 |
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0.38 | 1 | 0.38 | 1.00 | 0.3329 |
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0.90 | 1 | 0.90 | 2.40 | 0.1425 |
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1 |
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0.025 | 0.8756 |
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1.61 | 1 | 1.61 | 4.29 | 0.0561 |
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0.086 | 1 | 0.086 | 0.23 | 0.6397 |
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0.026 | 1 | 0.026 | 0.070 | 0.7943 |
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8.81 | 1 | 8.81 | 23.51 | 0.0002 |
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3.09 | 1 | 3.09 | 8.24 | 0.0117 |
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9.36 | 1 | 9.36 | 24.99 | 0.0002 |
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1.04 | 1 | 1.04 | 2.76 | 0.1173 |
Residual | 5.62 | 15 | 0.37 | ||
Lack of fit | 5.62 | 10 | 0.56 | ||
Pure error | 0.000 | 5 | 0.000 | ||
Cor total | 48.12 | 29 | |||
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Contour plots and response surface curves of the production of insoluble melanin by
Temperature, pH, tyrosine, and beef extract were the most critical factors to produce enhanced level (5.29 g/L) melanin by
The preserved culture of
Formation of dark brown pigment droplets on the surface of the slant culture of
The pigment obtained under submerged bioprocess was purified by chromatographic technique using silica gel columns. Thus purified pigment (pigment 1) and the pigment directly extracted from the slant culture system (pigment 2) were analyzed for their characteristic chemical features (Table
Chemical analysis of the pigments.
Color and reactions | Observations | |
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Pigment 1 | Pigment 2 | |
Color | Blackish brown | Blackish brown |
Solubility in water | Insoluble | Soluble |
Solubility in 0.1 N NH4OH | Readily soluble | Readily soluble |
Solubility in 0.1 N NaOH | Soluble | Soluble |
Solubility in 0.1 N KOH | Soluble | Soluble |
Solubility in organic solvents |
Insoluble | Insoluble |
Reaction with sodium dithionite and with potassium ferricyanide | Decolorized and turned to brown | Decolorized and turned to brown |
Reaction with H2S | Reduced | Reduced |
Both pigments gave maximum UV absorption between 200 and 300 nm but decreased towards visible range in the similar line of standard melanin under UV-Vis spectrophotometer (Figure
UV-visible spectrophotometric analysis of insoluble and soluble melanins.
FT-IR Spectroscopic analysis of pigments 1 and 2.
Thin layer chromatograph of insoluble and soluble melanins.
Assessment of cytotoxicity of chemicals using cell lines is not an uncommon procedure and is accurately correlated [
Cytotoxic activity of the insoluble and soluble melanins.
Concentration of melanin |
Log of concentration of melanin | Survived brine shrimps | Percent mortality |
Lc50 ( |
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6 h | 12 h | 24 h | 6 h | 12 h | 24 h | |||
Insoluble melanin | ||||||||
01.0 | 0 | 19 | 11 | 06 | 10 | 45 | 70 | 0.80 |
02.0 | 0.301 | 17 | 11 | 07 | 15 | 45 | 65 | |
04.0 | 0.602 | 17 | 10 | 06 | 15 | 50 | 70 | |
08.0 | 0.903 | 17 | 09 | 04 | 15 | 55 | 80 | |
16.0 | 1.204 | 16 | 09 | 04 | 20 | 55 | 80 | |
32.0 | 1.505 | 16 | 09 | 03 | 20 | 55 | 85 | |
64.0 | 1.806 | 16 | 07 | 01 | 20 | 65 | 95 | |
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Soluble melanin | ||||||||
01.0 | 0 | 17 | 05 | 04 | 15 | 75 | 80 | 0.40 |
02.0 | 0.301 | 17 | 05 | 04 | 15 | 75 | 80 | |
04.0 | 0.602 | 16 | 05 | 04 | 20 | 75 | 80 | |
08.0 | 0.903 | 16 | 05 | 04 | 20 | 75 | 80 | |
16.0 | 1.204 | 15 | 05 | 03 | 25 | 75 | 85 | |
32.0 | 1.505 | 15 | 04 | 02 | 25 | 80 | 90 | |
64.0 | 1.806 | 15 | 03 | 00 | 25 | 85 | 100 |
A successful production of both insoluble and soluble melanins from a single isolate of
The authors have no conflict of interests to declare.
The research Grant provided by Department of Biotechnology (BT/PR13864/PID/06/580/2010, Dt. 05.08.2011), Government of India, New Delhi, is sincerely acknowledged.