Scientific RepoRts 7:42129 DOI: 10.1038srep42129www.nature.comscientificreportsBioproduction, characterization, [600704]

1
Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129www.nature.com/scientificreportsBioproduction, characterization,
anticancer and antioxidant
activities of extracellular melanin
pigment produced by newly
isolated microbial cell factories
Streptomyces glaucescens NEAE-H
Noura El-Ahmady El-Naggar & Sara M. El-Ewasy
In this present study, a newly isolated strain, Streptomyces sp. NEAE-H, capable of producing high
amount of black extracellular melanin pigment on peptone-yeast extract iron agar and identified
as Streptomyces glaucescens NEAE-H. Plackett–Burman statistical design was conducted for initial
screening of 17 independent (assigned) variables for their significances on melanin pigment production by Streptomyces glaucescens NEAE-H. The most significant factors affecting melanin production are
incubation period, protease-peptone and ferric ammonium citrate. The levels of these significant variables and their interaction effects were optimized by using face-centered central composite design.
The maximum melanin production (31.650 μg/0.1 ml) and tyrosinase activity (6089.10 U/ml) were
achieved in the central point runs under the conditions of incubation period (6 days), protease-peptone
(5 g/L) and ferric ammonium citrate (0.5 g/L). Melanin pigment was recovered by acid-treatment. Higher
absorption of the purified melanin pigment was observed in the UV region at 250 nm. It appeared to
have defined small spheres by scanning electron microscopy imaging. The maximum melanin yield was
350 mg dry wt/L of production medium. In vitro anticancer activity of melanin pigment was assayed
against skin cancer cell line using MTT assay. The IC
50 value was 16.34 ± 1.31 μg/ml for melanin and
8.8 ± 0.5 μg/ml for standard 5-fluorouracil.
Melanins are macromolecules formed by oxidative polymerization of phenolic or indolic compounds. Often the
resulting pigments are brown or black in color but many other colors have also been observed. Melanins are also hydrophobic and negatively charged
1. The biosynthesis of melanin is initiated from L-tyrosine via a series of enzy-
matic and nonenzymatic reactions by the enzyme tyrosinase2. First, tyrosinase (monophenol monooxygenase
EC 1.14.18.1) catalyzes oxidation of L-tyrosine to L-3, 4- dihydroxyphenyl alanine (L-DOPA), which is further converted into dopachrome. Dopachrome is converted to melanin by a series of nonenzymatic oxidoreduction reactions
3. Tyrosinases from different biological sources have been utilized for the synthesis of L-DOPA and the
removal of phenolic compounds from wastewaters4.
There are three types of melanins i.e. eumelanins, pheomelanins and allomelanins. Eumelanins are black to
brown color pigments produced by oxidative polymerization of tyrosine (and/or phenylalanine) to L-DOPA, which is further converted into dopachrome and then to melanin
1. Eumelanin is the predominant pigment syn-
thesized in humans and microorganisms5. Pheomelanins are red or yellow color pigments which are initially
synthesized just like eumelanins, but DOPA undergoes cysteinylation (Supplementary Figure S1) (incorporation of cysteine in the polymer)
6 and contain sulphur. The allomelanins forming the third class are heterogeneous
Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City for
Scientific Research and Technological Applications, Alexandria, Egypt. Correspondence and requests for materials should be addressed to N.E.E. (email: nouraelahmady@yahoo.com)Received: 27 September 2016
Accepted: 06 January 2017
Published: 14 February 2017OPEN

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129pigments include nitrogen free heterogeneous group of polymers formed from a variety of sources like dihydro-
folate, homogentisic acid, catechols, etc.7.
Melanins play important roles in microorganisms against thermal, chemical (heavy metal and oxidizing
agent) and biochemical stresses (reactive oxygen generated by the exposure of solar UV radiation)8. The melanin
synthesized by microbes shows metal ions chelating ability9. It has also been shown to provide structural rigidity
to cell walls and to help to store water and ions1. Melanins may also play a role in protecting against antimicrobial
drugs10 whereas in plants melanin is incorporated in their cell walls as strengtheners11. In humans, melanin not
only determines the skin color, but also plays an important role in protecting against UV radiation12 and its lack
leads to several abnormalities and diseases. Due to their chemical composition, melanins have physicochemical properties that allow them to act as ultraviolet absorbers, cation exchangers, drug carriers, amorphous semicon-ductors, X-ray and γ -ray absorbers
13. Water soluble melanins are used in sun-screens, solid plastic films, lenses,
paints, varnishes, and other surface protection formulations to provide greater UV protections14. Melanins have
important biological activities, including antimicrobial15, antitumor16, antivenin activity17 and liver protecting
activity18. There is report of bacterial melanins with anti inflammatory activity19. Hoogduijn et al.20 observed that
melanin protects melanocytes and keratinocytes from the induction of DNA strand breaks by hydrogen peroxide, indicating that this pigment has an important antioxidant role in the skin
21. AIDS treatment reveals the selective
antiviral activity of synthetic soluble melanin against human immunodeficiency virus22.
Recently, melanin production by microorganisms has attracted attention as an environmentally friendly and
economic alternative to chemical production23. Actinomycetes are the biotechnologically valuable bacteria which
are well exploited for secondary metabolites24. Naturally they are able to synthesize and excrete dark soluble pig-
ments, the melanins or melanoid pigments25.
The objectives of this study is to isolate and identify efficient extracellular melanin producing Streptomyces sp.
for pharmaceutical needs, to identify a suitable promotional medium for enhancing melanin production and for characterization of the extracted microbial pigment.
Results and Discussion
The total of one hundred and thirty morphologically different actinomycete strains were isolated from different localities in Egypt and Saudi Arabia. All these isolates were purified and screened for the extracellular synthesis of melanin on peptone yeast extract iron agar and tyrosine agar using plate method. The formation of brown or black zone around the colonies of the tested isolates on peptone-yeast extract iron and/or tyrosine agar signifi-cantly reveals the synthesis of melanin. Melanin production by Streptomyces sp. strain NEAE-H in peptone yeast
extract iron agar is shown in Fig. 1A. Also the isolates were screened for the extracellular synthesis of melanin in peptone yeast extract iron broth. Melanin production by Streptomyces sp. strain NEAE-H in peptone yeast extract
iron broth after 2 days of incubation is shown in Fig. 1B and at different elapsed times is shown in Fig. 1C.
Out of the 130 isolates screened, only 9% of the isolates had the ability to produce melanin in the recom-
mended melanin production media. The results correlated with previous findings where streptomycetes from various sources were screened and only less than 10% were found to produce melanin pigments
26. Only very few
actinomycetes from different ecological habitats exhibited the ability to produce melanin. It was noticed that out of 30 isolates from Egyptian soil, only a single strain had the ability to produce melanin
27. Nine strains among 180
(5%) Streptomyces isolates from soil samples of Gulbarga region produced a diffusible dark brown pigment on
both peptone-yeast extract iron agar and synthetic tyrosine agar25. The most promising isolate, Streptomyces sp.
strain NEAE-H, was selected as potential isolate for the synthesis of melanin and identified on the basis of mor –
phological, cultural, physiological and chemotaxonomic properties, together with 16 S rRNA sequence.
Morphology and cultural characteristics of the strain no. NEAE-H. Strain NEAE-H had morpho-
logical characteristics that were consistent with members of the genus Streptomyces . Strain NEAE-H develops
abundant and well-developed substrate and aerial mycelium. It grew well on all tested media (tryptone-yeast extract agar, yeast extract -malt extract agar, oatmeal agar, inorganic salt-starch agar, glycerol–asparagine agar, peptone-yeast extract iron agar and on tyrosine agar) (Supplementary Table S1). The color of the mature spor –
ulating aerial mycelium was green on several standard tested media (Supplementary Figure S2). Reverse side of colony is yellowish brown on tryptone-yeast extract agar; brownish orange on yeast extract -malt extract agar; yel-lowish green on oatmeal agar and brown on inorganic salts-starch agar, glycerol asparagines agar, peptone-yeast extract iron agar and tyrosine agar; substrate pigment is not pH indicator. Y ellow pigments produced in oat-meal agar medium and brown pigments produced in inorganic salts-starch agar, glycerol asparagines agar, peptone-yeast extract iron agar and tyrosine agar media (Supplementary Table S1). Verticils are not present and the mycelium does not fragment. From electron microscopic observations, it was found that strain NEAE-H had spirals-type spore chains, mature spore chains are short. Spore surface is hairy; hairs are coarse, showing some tendency towards spines. Spore shape is globose to oval, this morphology is seen on starch nitrate agar medium
(Fig. 2).
Physiological and chemotaxonomic characteristics. The physiological characteristics of strain
NEAE-H are shown in Table 1. Melanin pigments are formed in peptone-yeast-iron agar and tyrosine agar.
Lecithinase activity, α –amylase (starch hydrolysis), protease (degradation of casein), cellulase (growth on cel-
lulose), uricase, gelatinase (gelatin liquification) and asparaginase of strain NEAE-H were produced while chitosanase was not produced. Coagulation and peptonization of milk were positive while hydrogen sulphide
production and reduction of nitrate to nitrite were negative. The optimal growth temperature was 30 °C and
optimal pH was 7.0. The isolate exhibited NaCl tolerance up to 5% (w/v). D-fructose, D-xylose, D-galactose,
D-glucose, L-arabinose, ribose, D-mannose, sucrose, maltose, rhamnose and cellulose are utilized as sole carbon

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129sources, while raffinose is weakly utilized as sole carbon source. It exhibited no antimicrobial activities against
Staphylococcus aureus, Alternaria solani, Bipolaris oryzae, Sacchromyces cerevisiae, Candida albicans, Bacillus sub-
tilis, Escherichia coli, Pseudomonas aeruginosa, Rhizoctonia solani, Fusarium oxysporum, Aspergillus niger and Klebsiella pneumoniae. Strain NEAE-H is aerobic, mesophilic and Gram-positive actinomycete.
16S rRNA gene sequence comparisons and phylogenetic analysis. A BLAST search of the
GenBank database using 1487 bp 16 S rRNA gene sequence of strain NEAE-H showed its similarity to that of
many members of the genus Streptomyces. 16 S rRNA gene sequence similarities between the strain NEAE-H
and these type strains of the genus Streptomyces were between 98 and 100%. A phylogenetic tree (Fig. 3) based
on 16 S rRNA gene sequences of members of the genus Streptomyces was constructed according to the bootstrap
test of neighbor-joining algorithm method of Saitou and Nei28 with MEGA429. This tree shows the close phy-
logenetic association of strain NEAE-H with certain other Streptomyces species. Phylogenetic analysis indicated
that the strain NEAE-H consistently falls into a clade together with Streptomyces glaucescens strain NRRL B-2706
(GenBank/EMBL/DDBJ accession No. NR_115773.1, similarity 100%), Streptomyces althioticus strain NBRC
12740 (GenBank/EMBL/DDBJ accession No. NR_112254.1, similarity 99%), Streptomyces thinghirensis strain S10
(GenBank/EMBL/DDBJ accession No. NR_116901.1, similarity 99%), Streptomyces griseoflavus strain 13668 A
(GenBank/EMBL/DDBJ accession No. EU741218.1, similarity 99%).
On the basis of the previously collected data and in view of the comparative study of the morphological, cul-
tural and physiological characteristics of isolate No. NEAE-H in relation to its closest phylogenetic neighbours
of the genus Streptomyces (Table 1), it is most closely related to the type strain of Streptomyces glaucescens strain
NRRL B-2706 (GenBank/EMBL/DDBJ accession No. NR_115773.1, the highest degree of similarity 100%)30.
Therefore, this strain was identified as Streptomyces glaucescens strain NEAE-H.
Evaluation of the factors affecting the extracellular synthesis of melanin using Plackett-Burman
design. The production of a diffusible dark brown pigment on complex organic media is so significant that it
has long been regarded as a key characteristic for the identification and classification of Streptomyce s. The method
of testing melanin formation by L-DOPA as substrate is used to confirm whether the diffusible pigments produced
are melanoid (dark brown) or merely a brown substance, especially when complex organic media are employed25.
The experiment was conducted in 20 runs to study the effect of the selected variables on the production of melanin. Plackett-Burman experiments showed a markedly wide variation of melanin production from 5.72 to 17.96 μ g/0.1 ml
of medium (Table 2); this variation reflected the importance of medium optimization to attain higher
Figure 1. (A) Melanin production by Streptomyces glaucescens strain NEAE-H in peptone yeast extract iron
agar; (B) culture broth after 2 days of incubation; (C) tubes containing culture samples at different elapsed times. Number below each vial indicates incubation time in hours.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129melanin production. The maximum melanin production (17.96 μ g/0.1 ml of medium) and tyrosinase activity
(5454.27 U/ml) were achieved in the run number 17, while the minimum melanin production (5.72 μ g/0.1 ml
of medium) and tyrosinase activity (996.05 U/ml) were observed in the run number 8 (Table 2).
The relationship between a set of independent variables and melanin production is determined by a math-
ematical model called multiple-regression model. The data revealed that, medium volume (E) and potassium
nitrate (J) are insignificant variables with zero effect (0.0) and zero percent of contribution (0.0). Lower % of con –
tribution indicated higher p -value. Thus instead of starting with the maximum model effects, backward regression
Figure 2. Scanning electron micrograph showing the spore-chain morphology and spore-surface
ornamentation of strain NEAE -H grown on starch nitrate agar medium for 14 days at 30 °C at different
magnification.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129at alpha 0.15 was applied to eliminate the effect of medium volume and potassium nitrate. Then, the model fitted
for the test of significance. Statistical analysis of the response was performed which is represented in Table 3, Supplementary Table S2.
Supplementary Table S2 and Fig. 4A show the main effect of each variable on melanin production. With
respect to the main effect of each variables, we can see that eight variables from the seventeen different inde-
pendent variable named incubation period, L-tyrosine, peptone, protease-peptone, yeast extract, K
2HPO4, ferric
ammonium citrate and sodium thiosulfate affect positively melanin production, where the seven variables named
glycerol, MgSO4, NaCl, pH, temperature, agitation speed and starch affect negatively melanin production. The
significant variables with positive effect were fixed at high level and the variables which exerted a negative effect on melanin production were maintained at low level for further optimization by a face-centered central compos-ite design. Glycerol, KNO
3, MgSO4, NaCl and starch were excluded from the production medium due to the low
levels of these factors is “0” . Medium volume was maintained at low level for further optimization.
The Pareto chart illustrates the order of significance of the variables affecting melanin production in
Plackett-Burman experimental design (Fig. 4B). It displays the absolute values of the effects, and draws a reference line on the chart. Any effect that extends past this reference line is potentially important. Pareto chart in design Characteristic Streptomyces sp. strain NEAE-H Streptomyces glaucescens Streptomyces griseoflavus Streptomyces thinghirensis Streptomyces althioticus
Aerial mass color on ISP
mdeium 2Green with white margins Green or blue Gray White–grey Gray
Reverse side of colony on ISP medium 2Brownish orange red Y ellow to orange-yellow Ye l l ow Blue-violet or red
Production of diffusible pigmentNone None None Ye l l ow Blue-violet or red
Spore chain morphology Spirals* Spirals* Spirals, long Spirals Spirals or flexuous, long
Spore surface Hairy** Hairy** Spiny Smooth Spiny or warts
Spore shape Globose to oval Oval
Melanin production on
Peptone-yeast extract
iron agar + + − − −
Tyrosine agar + + − − −
Tryptone-yeast extract
broth− + − −
Maximum NaCl tolerance
(%, w/v)5 7
Coagulation of milk + +
Peptonization of milk + +
Nitrate reduction – +
H
2S production − −
Gelatin liquification + −
Utilization of carbon sources (1%,w/v) D(− ) fructose + + + + +
D(+ ) xylose + + + − +
D(+ ) galactose + +
D(+ ) glucose + + + + +
L-arabinose + + + − +
Ribose +
D(+ ) mannose + +
Sucrose + ± ± ±
Maltose + ±
Rhamnose + + + + +
Raffinose ± ± ± − ±
Cellulose + ±
Table 1. Phenotypic properties that separate strain Streptomyces sp. strain NEAE-H from related
Streptomyces species. Data for reference species were taken from Bergey’s Manual of Systematic
Bacteriology -volume five the actinobacteria30. Abbreviations: + , Positive; −, Negative; ± , Doubtful; Blank
cells, no data available. The optimal growth temperature was 30 °C and optimal pH was 7.0. It exhibited
no antimicrobial activities against Staphylococcus aureus, Alternaria solani, Bipolaris oryzae, Sacchromyces
cerevisiae, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Rhizoctonia solani, Fusarium oxysporum, Aspergillus niger and Klebsiella pneumoniae. Lecithinase activity, α –amylase (starch
hydrolysis), protease (degradation of casein), cellulase (growth on cellulose), uricase, gelatinase and asparaginase of strain NEAE-H were produced while chitosanase was not produced.
*Mature spore chains are
short. **Hairs are coarse, showing some tendency towards spines.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129expert version 7.0 reproduce the relation between t -value (effect) vs. ranks. Among the tested variables, ferric ammo-
nium citrate showed the highest positive effect by 13.282%. Next to ferric ammonium citrate, protease-peptone
showed positive effect by 11.782%, then incubation period by 11.160% (Supplementary Table S2). Starch showed the highest negative significance by 11.965%. Also, predicted versus actual melanin production plot indicated that, there is a close agreement between the experimental results and theoretical values predicted by the model equation
as shown in Fig. 4C, which confirms the adequacy of the model.
The value of the determination coefficient (R
2 = 0.9998) indicates that 99.98% of the variability in the response
was attributed to the given independent variables and only 0.02% of the total variations are not explained by the
independent variables. In addition, the value of the adjusted determination coefficient (Adj. R2 = 0.9979) is also
very high which indicates a high significance of the model31. The “Pred R-Squared” of 0.9778 is in reasonable
agreement with the “ Adj R-Squared” of 0.9979.This indicated a good adjustment between the observed and pre-dicted values. “ Adeq Precision” measures the signal to noise ratio. A ratio greater than 4 is desirable. Our ratio of 67.877 indicates an adequate signal (Table 3).
The analysis of variance (ANOV A) of the experimental design was calculated, and the sum of square, mean
square, F-value, P-value and confidence level are given in Table 3. The significance of each coefficient was deter –
mined by p -values, which are listed in Table 3. The Model F -value of 529.93 implies the model is significant.
Values of “Prob > F” (P-value) less than 0.05 indicate model terms are significant. In this case A, B, C, D, F, G,
H, K, L, M, N, O, P , Q and R are significant model terms. The analysis showed that, ferric ammonium citrate
Figure 3. Neighbour-joining phylogenetic tree based on 16 S rRNA gene sequences, showing the
relationships between strain NEAE-H and related species of the genus Streptomyces . Only bootstrap values
above 50%, expressed as percentages of 500 replications, are shown at the branch points. GenBank sequence accession numbers are indicated in parentheses after the strain names. Phylogenetic analyses were conducted in the software package MEGA4. Bar, 0.1 substitution per nucleotide position.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129(O) with a probability value of 0.0006 was determined to be the most significant factor affecting melanin pro-
duction by Streptomyces glaucescens strain NEAE-H at 99.940% confidence followed by protease-peptone (L)
(P-value = 0.0007) at 99.930% confidence, and starch (F) (P -value = 0.0007) at 99.930% confidence, then incuba-
tion period (A) (P-value = 0.0008) at 99.920% confidence.
The coefficient of variation % (C.V .%) is a measure of residual variation of the data relative to the size of the
mean. Here a lower value of C.V . (1.690%) indicates a greater reliability of the experimental performance. The predicted residual sum of squares (PRESS) statistic is used as an indication of the predictive power of a model. A model with a small value of PRESS statistic indicates better prediction. Our value of PRESS is 7.29. The model shows standard deviation and mean value of 0.191and 11.294, respectively.
By neglecting the terms that were insignificant (P > 0.05) (Supplementary Table S2), the first order polynomial
equation was derived representing melanin production as a function of the independent variables:
=. +. −. −. −. −.
−. +. +. +. +. +.
+. +. −. −. OY1 1294 1523A 066B 0612C 1118D 1637F
0468G 0647H 0965K 1611L 0618M 0208N
1813 0691P 0271Q 0818R (1)(melaninproduction)
Where Y is the response (melanin production) and A, B, C, D, F, G, H, K, L, M, N, O, P , Q, R are incubation period, pH, temperature, agitation speed, starch, glycerol, L-tyrosine, peptone, protease-peptone, yeast extract, K
2HPO4, ferric ammonium citrate, sodium thiosulfate, MgSO4 and NaCl.
In a confirmatory experiment, to evaluate the accuracy of Plackett-Burman, a medium, which expected to
be optimum of the following composition:(g/L: L-tyrosine 3, peptone 15, protease-peptone 4, yeast extract 1, K
2HPO4 1, ferric ammonium citrate 0.4, sodium thiosulfate 0.08), incubation period 5 days, pH 7, temperature
30 °C, agitation speed 100 rpm and medium volume (50 ml/250 ml flask) gives 19.17 μ g melanin/0.1 ml of medium
which is higher than result obtained from the basal medium before applying Plackett-Burman by about 2.24 times (8.57 μ g/0.1 ml of medium).
Optimization by face-centered central composite design. The face-centered central composite
design was employed to study the interactions among the significant variables and also determine their optimal Run
no.Coded levels of independent variablesMelanin production
(μg/0.1 ml of medium)
ResidualsTyrosinase activity
(U/ml) A B C D E F G H J K L M N O P Q R D1D2Actual
valuePredicted
value
1 − 1− 1 1 1 1 1− 1 1− 1 1− 1− 1− 1− 1 1 1− 1 1 1 5.91 5.991 − 0.081 1202.06
2 − 1 1 1− 1 1 1− 1− 1 1 1 1 1− 1 1− 1 1− 1− 1− 1 12.57 12.597 − 0.027 2284.11
3 1 1 1 1− 1 1− 1 1− 1− 1− 1− 1 1 1− 1 1 1− 1− 1 7.12 7.093 0.027 1666.08
4 1− 1 1 1− 1− 1 1 1 1 1− 1 1− 1 1− 1− 1− 1− 1 1 16.33 16.249 0.081 4148.21
5 1− 1− 1 1 1 1 1− 1 1− 1 1− 1− 1− 1− 1 1 1− 1 1 7.13 7.157 − 0.027 1272.06
6 − 1 1− 1 1− 1− 1− 1− 1 1 1− 1 1 1− 1− 1 1 1 1 1 6.59 6.509 0.081 1180.06
7 1− 1 1− 1− 1− 1− 1 1 1− 1 1 1− 1− 1 1 1 1 1− 1 14.95 14.869 0.081 1158.06
8 − 1 1 1 1 1− 1 1− 1 1− 1− 1− 1− 1 1 1− 1 1 1− 1 5.72 5.747 − 0.027 996.05
9 − 1 1− 1− 1− 1− 1 1 1− 1 1 1− 1− 1 1 1 1 1− 1 1 16.56 16.533 0.027 4220.21
10 − 1− 1 1 1− 1 1 1− 1− 1 1 1 1 1− 1 1− 1 1− 1− 1 7.85 7.823 0.027 1510.08
11 − 1− 1− 1− 1− 1− 1− 1− 1− 1− 1− 1− 1− 1− 1− 1− 1− 1− 1− 1 8.97 8.943 0.027 1998.10
12 1− 1− 1− 1− 1 1 1− 1 1 1− 1− 1 1 1 1 1− 1 1− 1 13.82 13.739 0.081 2686.13
13 1 1− 1− 1 1 1 1 1− 1 1− 1 1− 1− 1− 1− 1 1 1− 1 8.35 8.431 − 0.081 1680.08
14 1 1− 1 1− 1 1− 1− 1− 1− 1 1 1− 1 1 1− 1− 1 1 1 14.37 14.343 0.027 3052.15
15 − 1 1 1− 1− 1 1 1 1 1− 1 1− 1 1− 1− 1− 1− 1 1 1 6.92 6.839 0.081 1838.09
16 − 1− 1− 1− 1 1 1− 1 1 1− 1− 1 1 1 1 1− 1 1− 1 1 12.53 12.557 − 0.027 2762.14
17 1− 1 1− 1 1− 1− 1− 1− 1 1 1− 1 1 1− 1− 1 1 1 1 17.96 18.041 − 0.081 5454.27
18 − 1− 1− 1 1 1− 1 1 1− 1− 1 1 1 1 1− 1 1− 1 1− 1 14.09 14.171 − 0.081 2934.15
19 1 1− 1 1 1− 1− 1 1 1 1 1− 1 1− 1 1− 1− 1− 1− 1 16.65 16.677 − 0.027 1608.08
20 1 1 1− 1 1− 1 1− 1− 1− 1− 1 1 1− 1 1 1− 1− 1 1 11.49 11.571 − 0.081 1866.09
Level daysοCrpm ml g/L g/L g/L g/L g/L g/L g/L g/L g/L g/L g/L g/L
− 1 2 7 30 100 50 0 0 1 0 7 2 0.5 0.5 0.1 0.01 0 0
1 5 8.5 37 200 100 5 5 3 2 15 4 1 1 0.4 0.08 0.5 0.5
Table 2. Twenty-trial Plackett–Burman experimental design for evaluation of nineteen independent
variables with coded values along with the melanin production and tyrosinase activity. A (incubation
period); B (pH); C (temperature); D (agitation speed); E (medium volume, ml/250 ml flask); F (starch); G
(glycerol); H (L-tyrosine); J (potassium nitrate); K (peptone); L (protease-peptone); M (yeast extract); N (K
2HPO4); O (ferric ammonium citrate); P (sodium thiosulfate); Q (MgSO4) and R (NaCl).

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129levels. Results of Placket-Burman design revealed that incubation period, protease-peptone and ferric ammo-
nium citrate were the most significant positive independent variables affecting melanin production, thus they were selected for further optimization using face-centered central composite design.
In this study, a total of 20 experiments with different combination of incubation period (X
1), protease-peptone
(X2) and ferric ammonium citrate (X3) were performed and the results of experiments are presented along with
predicted response and residuals in Table 4. Concentrations of three independent variables at different coded and actual levels of the variables also presented in Table 4. The central point was repeated six times (run order: 1, 3, 10, 13, 15 and 20). The maximum melanin production (31.650 μ g/0.1 ml of medium) and tyrosinase activity (6089.10
U/ml) were achieved in central point runs number 1, 3, 10, 13, 15 and 20 under the conditions of incubation
period (6 days ), protease-peptone (5 g/L) and ferric ammonium citrate (0.5 g/L), while the minimum melanin
production (3.006 μ g/0.1 ml of medium) and tyrosinase activity (2174.51 U/ml) was observed in run number
17 under the conditions of incubation period (8 days ), protease-peptone (3 g/L) and ferric ammonium citrate
(0.3 g/L) (Table 4).
Multiple regression analysis and ANOVA. The data were analyzed using Design Expert ® 7.0 for
Windows to perform statistical analysis. The positive coefficients for X2, X3, X1 X2, X1 X3, X2 X3 (Table 5) indicate
that linear effect of X2, X3 and interaction effects for X1 X2, X1 X3, X2 X3 increase melanin production, while other
negative coefficients indicate decrease in melanin production.
The adequacy of the model was checked using analysis of variance (ANOV A) which was tested using Fisher’s
statistical analysis and the results are shown in Table 5. The Model F -value of 66.903 with a very low probability
value ( P model > F 0.0001) implies the model is significant. It can be seen from the degree of significance that the
quadratic effect of incubation period (X1), protease-peptone (X2) and ferric ammonium citrate (X3) are signifi-
cant model terms (P value > 0.05). Linear coefficients and interaction between three variables are not significant
(P value > 0.05) indicating that there is no significant correlation between three variables and that they did not
help much in increasing the production of melanin (Table 5).
The determination coefficient (R2) of the model was 0.9837 (Table 5). Therefore, the present R2-value reflected
a very good fit between the observed and predicted responses, and implied that the model is reliable for mela-
nin production in the present study. The “Pred R-Squared” of 0.9025 is in reasonable agreement with the “ Adj R-Squared” of 0.9690. This indicated a good adjustment between the observed and predicted values. “ Adeq Precision” ratio of 19.2714 indicates an adequate signal to noise ratio. A lower value of C.V . (11.715) indicated a
better precision and reliability of the experimental performance
32. Value of PRESS is 257.139. The model shows
standard deviation and mean value of 2.076 and 17.720, respectively (Table 5).Source SS MS F-valuep-value
Prob > FConfidence
Level (%)
Model 328.37 19.32 529.93 0.0019* 99.810
Incubation period (A) 46.39 46.39 1272.72 0.0008* 99.920
pH (B) 8.71 8.71 239.01 0.0042* 99.580
Temperature (C) 7.49 7.49 205.51 0.0048* 99.520
Agitation speed (D) 25.00 25.00 685.83 0.0015* 99.850
Starch (F) 53.60 53.60 1470.38 0.0007* 99.930
Glycerol (G) 4.38 4.38 120.18 0.0082* 99.180
L-tyrosine (H) 8.37 8.37 229.69 0.0043* 99.570
Peptone (K) 18.62 18.62 510.96 0.0020* 99.800
Protease-peptone (L) 51.91 51.91 1424.04 0.0007* 99.930
Y east extract (M) 7.64 7.64 209.56 0.0047* 99.530
K2HPO4 (N) 0.87 0.87 23.74 0.0396* 96.040
Ferric ammonium citrate (O) 65.74 65.74 1803.55 0.0006* 99.940
Sodium thiosulfate (P) 9.55 9.55 261.99 0.0038* 99.620
MgSO4 (Q) 1.47 1.47 40.30 0.0239* 97.610
(R) NaCl 13.38 13.38 367.15 0.0027* 99.730
Residual 0.07 0.04
Cor Total 328.44
Std. Dev. 0.191 R-Squared 0.9998
Mean 11.294 Adj R-Squared 0.9979
C.V .% 1.690 Pred R-Squared 0.9778
PRESS 7.29 Adeq Precision 67.8771
Table 3. Regression statistics and analysis of variance (ANOV A) for the experimental results of Plackett-
Burman design used for melanin production by Streptomyces glaucescens strain NEAE-H. *Significant
values, SS – sum of squares, MS- mean square, F: Fishers’s function, P: Level of significance, PRESS -the
predicted residual sum of squares, CV %-the coefficient of variation%.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129In order to evaluate the relationship between dependent and independent variables and to determine the
maximum melanin production corresponding to the optimum levels of incubation period (X1), protease-peptone
(X2) and ferric ammonium citrate (X3), a second-order polynomial model (Equation 2) was proposed to calculate
the optimum levels of these variables and defines predicted response (Y) in terms of the independent variables
(X1, X2 and X3):
=. −. +. +. +.
+. +. −. −. −.Y3 0592 0484X 0191X 0454X 1323X X
0338X X0 249X X5 166X 5072X 15508X (2)(melaninproduction)1 23 12
13 23 12
22
32
Where Y is the response (melanin production) and X1, X2 and X3 are incubation period, protease-peptone and
ferric ammonium citrate, respectively.
The fit summary results are presented in Supplementary Table S3. The aim of sequential model sum of squares
is to select the highest order polynomial where terms are significant; quadratic model type was selected to be the proper model that fit the FCCD of melanin production by Streptomyces glaucescens strain NEAE-H, where fit
summary results showed that, the quadratic model is a highly significant model with a very low probability value [(P
model > F) < 0.0001]. The model summary statistics focus on the models that have lower standard deviation and
Figure 4. (A) The main effects of the factors affecting melanin production, eight variables affect positively melanin production, where seven variables affect negatively melanin production, (B) The Pareto chart shows the amount of influence of each factor on melanin production, C) Correlation between the experimented and predicted values for melanin production by Streptomyces glau cescens strain NEAE-H according to the Plackett–
Burman experimental results.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129Std RunVariablesMelanin production (μg/0.1 ml of
medium)
ResidualsTyrosinase activity
(U/ml) X1 X2 X3 Experimental Predicted
17 1 0 0 0 31.650 30.592 1.058 6089.10
14 2 0 0 1 13.993 15.539 − 1.546 3981.80
20 3 0 0 0 31.650 30.592 1.058 6089.10
10 4 1 0 0 24.059 24.942 − 0.883 4087.40
7 5− 1 1 1 4.658 4.564 0.093 2503.33
4 6 1 1− 1 4.597 4.835 − 0.238 2496.12
5 7− 1− 1 1 7.362 6.331 1.031 3331.37
1 8− 1− 1− 1 8.067 6.596 1.471 3367.37
13 9 0 0− 1 13.003 14.630 − 1.628 3784.99
19 10 0 0 0 31.650 30.592 1.058 6089.10
3 11− 1 1− 1 3.529 3.834 − 0.305 2251.31
12 12 0 1 0 26.838 25.711 1.127 4497.82
18 13 0 0 0 31.650 30.592 1.058 6089.10
9 14− 1 0 0 23.620 25.911 − 2.290 4051.40
15 15 0 0 0 31.650 30.592 1.058 6089.10
8 16 1 1 1 6.240 6.917 − 0.677 3062.55
2 17 1− 1− 1 3.006 2.306 0.700 2174.51
6 18 1− 1 1 4.491 3.392 1.099 2462.52
11 19 0− 1 0 21.029 25.330 − 4.301 4008.20
16 20 0 0 0 31.650 30.592 1.058 6089.10
LevelIncubation period
(day)Protease-peptone (g/L)Ferric ammonium
citrate (g/L)
−1 4 3 0.3
0 6 5 0.5
1 8 7 0.7
Table 4. Face-centered central composite design representing the melanin production by Streptomyces
glau cescens strain NEAE-H as influenced by incubation period (X1), protease-peptone (X2) and ferric
ammonium citrate (X3) along with the predicted melanin production and residuals and the levels of
variables with actual factor levels corresponding to coded factor levels.
SourceCoefficient
estimate Sum of Squares dfMean
Square F-valueP-value
Prob > F
Model 2594.424 9 288.269 66.903 < 0.0001*
X1 − 0.484 2.345 1 2.345 0.544 0.4776
X2 0.191 0.364 1 0.364 0.084 0.7774
X3 0.454 2.063 1 2.063 0.479 0.5047
X1 X2 1.323 13.998 1 13.998 3.249 0.1016
X1 X3 0.338 0.914 1 0.914 0.212 0.6550
X2 X3 0.249 0.496 1 0.496 0.115 0.7415
X12− 5.166 73.378 1 73.378 17.030 0.0021*
X22− 5.072 70.742 1 70.742 16.418 0.0023*
X32− 15.508 661.349 1 661.349 153.490 < 0.0001*
Residual 43.088 10 4.309
Lack of Fit 43.088 5 8.618
Pure Error 0.000 5 0.000
Cor Total 2637.511 19
Std. Dev. 2.076 R-Squared 0.9837
Mean 17.720 Adj R-Squared 0.9690
C.V .% 11.715 Pred R-Squared 0.9025
PRESS 257.139 Adeq Precision 19.2714
Table 5. Regression statistics, analysis of variance (ANOV A) for FCCD results used for optimizing
melanin production by Streptomyces glaucescens strain NEAE-H. *Significant values, df : Degree of freedom,
F: Fishers’s function, P: Level of significance, C.V: Coefficient of variation, intercept coefficient estimate: 30.592.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129higher adjusted and predicted R-squared; the model summary statistics of the quadratic model showed the small-
est standard deviation of 2.076 and the largest adjusted and predicted R-squared of 0.9690 and 0.9025 respectively.
Three dimensional plots. The three dimensional response surface curves were plotted by statistically sig-
nificant model to understand the interaction of the variables and the optimal levels of each variable required for the optimal melanin production. Three dimensional plots for the significant pair-wise combinations of the three variables (X
1 X2, X1 X3, and X2 X3) were generated by plotting the response (melanin production) on Z-axis against
two independent variables while keeping the other variable at its center point (zero levels) (shown in Fig. 5A–C). Figure 5A represents the three dimensional plot as function of incubation period (X
1), protease-peptone (X2) on
the production of melanin. Maximum melanin production was clearly situated close to the central point of the incubation period and protease-peptone. Further increase or decrease led to the decrease in the production of melanin.
The maximum pigment production was observed on 6
th day of incubation. This result was in agreement with
the finding of Rani et al.33 who extracted the highest amount of crude melanin at 6th day from halophilic black
yeast Hortaea werneckii. In contrast, Amal et al.34 and Vasanthabharathi et al.35 reported that the maximum level
of pigment formation by Streptomyces was observed on 10th and 7th day of incubation period respectively after
which it slowly declined. The maximum amount of melanin pigments was synthesized by the fungus Aspergillus carbonicus at 15
th–25th day’s incubation period36.
Quadri and Agsar37 reported that simple nitrogen source tyrosine gave the maximum production of melanin
by thermo-alkaliphilic Streptomyces followed by phenylalanine. Tyrosine has given the maximum production
when compared to complex nitrogen sources. Potassium nitrate was reported as the best nitrogen source for the experimental actinomycete isolate to produce melanin
27. The nitrogen source utilized varies among different
species of Streptomyce s. The formation of brown color for Streptomyces isolates on peptone-yeast extract iron agar
was observed by Vasanthabharathi et al.35. Twenty-one cultures produced a diffusible dark brown pigment on
peptone-yeast extract-iron-agar, but failed to do so on synthetic tyrosine-agar. In these cases, the growth or the production of the enzyme is not enough to be detected on synthetic tyrosine-agar
38. Proteose peptone is enzy-
matic digests of protein. It is rich in peptides with the higher molecular weight.
Figure 5B depicts the incubation period (X1) and ferric ammonium citrate (X3) interactions. At moderate lev-
els of incubation period and ferric ammonium citrate, the production of melanin was high. The graph pointed a decline in production level when the interaction was carried beyond high and low levels of incubation period and ferric ammonium citrate. The organism reduced ferric citrate by ferric reductase activity, converting Fe
3+ to Fe2+
which might facilitate iron acquisition/assimilation by providing a ferrous iron source to growing Streptomyces .
Increases in the levels of ferric reductase activity in culture supernatants of Legionella pneumophila correlated
with increased pigmentation. Legionella pneumophila is one of only a small number of microorganisms in which
melanin secretion has been linked to ferric reduction39. A study reports that iron levels can modulate the tran-
scriptional control of melanin biosynthesis in C. neoformans40. Fe2+ ion is required as cofactor for the activity of
several aromatic hydroxylases, such as phenylalanine, tryptophan and tyrosine hydroxylases41. With regard to a
possible mechanism for the observed effect of iron, that the expression of the hydroxylase activity of tyrosinase is dependent on a pre-reduction site of the enzyme
42.
Figure 5C plot reveals that lower and higher levels of the protease-peptone (X2) and ferric ammonium citrate
(X3) support relatively low levels of melanin production. On the other hand, the maximum melanin production
clearly situated close to the central point of the protease-peptone and ferric ammonium citrate. In addition, the interaction terms between these variables were not significant, indicating that there is no significant correlation between each two variables and that they did not help much in increasing the production of melanin.
Model verification. In order to determine the accuracy of the model and to verify the result, an experiment
under the new conditions which obtained from face-centered central composite design was preformed. The pre-dicted optimal levels of the process variables for melanin production by Streptomyces glaucescens strain NEAE-H
were incubation period (6 days), protease-peptone (5 g/L) and ferric ammonium citrate (0.5 g/L). Melanin pro-
duction by Streptomyces glaucescens strain NEAE-H (31.650 μ g/0.1 ml of medium) obtained from the experiment
was very close to the response (30.607 μ g/0.1 ml of medium) predicted by the regression model, which proved the
validity of the model. The verification revealed a high degree of accuracy of the model of 96.70%, indicating the model validation under the tested conditions.
Extraction of melanin. It was suggested that melanin polymers constitute the building blocks of melanin
granules43. The process of granules formation and their dimension are strongly pH dependent, where a low pH
promotes the aggregate growth and a high pH induces the breakup of the granules to small particles-oligomers with a lower degree of polymerization. This process is a consequence of the polyelectrolyte nature of melanin, and it is dependent on the ionization state of melanin groups like carboxylic, phenolic, and aminic groups as well as on the ionic strength of the environment. The physical appearance of the purified melanin is shown in Fig. 6A with a
true black color typical of melanins in general.
In the present study, the maximum yield of melanin per liter of peptone yeast extract iron broth was 350 mg
dry wt/L of production medium, which is comparable with that of the maximum yield of M8 melanin extracted
from peptone yeast extract iron broth culture of S. bikiniensis which was 166 mg/L
44; yeast melanin synthesized by
Yarrowia lipolytica (160 mg/L)45 and tyrosine-mediated melanin production (130 mg/L) by Klebsiella sp. GSK46.
UV–visible spectrophotometeric analysis of the purified melanin pigment. The UV-visible
absorbance spectrum (200–700 nm) of the purified melanin is shown in Fig. 6B. Higher absorption was observed
in the UV region at 250 nm which then decreased towards the visible region, which is the characteristic property

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129of melanin. The absorption peak at 250 nm was similar to the absorption peak for melanin pigment extracted
from Phyllosticta capitalensis47. The melanins of different sources had various maximum UV-Vis absorption
peaks, such as the purified Chroogomphus rutilus melanin which had maximum absorption peak at 212 nm48
and Actinoalloteichus sp. MA-32 melanin at 300 nm15. Whereas, wavelength scan of melanin synthesized by
Streptomyces bikiniensis M8 exhibited an absorbance in the UV region with highest absorption peak at 230 nm,
but decreased towards the visible region due to the presence of the very complex conjugated structure, which is
Figure 5. Three-dimensional response surface plots for melanin production showing the interactive
effects of incubation period (X1), protease-peptone (X2) and ferric ammonium citrate (X3) when one of the
variables is fixed at optimum value and the other two are allowed to vary.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129the characteristic property of melanin44. The purified Chroogomphus rutilus melanin did not contain nucleic acid
or protein because of no obvious absorption peak between 260–280 nm in the UV spectra48.
FTIR analysis of melanin. One of the main tests for identifying melanin is the FTIR spectrum. The FTIR
spectrum of the extracted melanin (Fig. 7A) shows a peak around 3421.83 cm−1, correspond to the OH group,
small band at 2947.33 cm−1 can be assigned to stretching vibration of aliphatic C-H group49. The signals in the
3600–2800 cm−1 area are attributed to the stretching vibrations (O-H and N-H) of the amine, amide, or carbox-
ylic acid, phenolic and aromatic amino functions present in the indolic and pyrrolic systems50. Peak observed
around 1647.26 cm−1 is attributed to bending of secondary NH group. The characteristic strong band at between
1650–1620 cm−1(1647.26 cm−1) attributed to vibrations of aromatic ring C = C of amide I C = O and/or of COO-
groups. The N-H bending vibration peak at 1539.25 cm−1, indicates that the pigment had typical indole structure
of melanin. Bands at ~1400 to 1500 cm−1 can be due to aliphatic C-H groups in the melanin pigment46. The
peak centered at 1423.51 cm−1 (CH2-CH3 bending) characteristic of melanin pigment. Phenolic COH stretch-
ing at 1240.27 cm−1 relates to phenolic compounds. It was proposed that peaks at 1243 to 1305 cm−1 relates to
the anhydride group (C-O) in synthetic melanin and all extracted microbial pigments51. The peak centered at
1058.96 cm−1 is the indication of CH in-plane of aliphatic structure characteristic of melanin pigment. The peak
observed at 864.14 cm−1 due to aromatics C–H group. Weak bands below 700 cm−1 ascribed to alkene C-H sub-
stitution in the melanin pigment46. The spectroscopic properties of the pigment extracted from Streptomyces
glaucescens strain NEAE-H correlated with those of melanin produced by various microorganisms as reported
previously46. On the basis of the above results, it was concluded that the pigment was eumelanin.
NMR spectrum. NMR spectrum of the purified melanin pigment synthesized by Streptomyces glaucescens
strain NEAE-H (Fig. 7B) shows resonances between 8.0 and 6.0 ppm and between 0.813 and 4.504 ppm.
Resonances between 8.0 and 6.0 ppm in NMR spectra are assigned to aromatic functionality, between 3.2 and
about 4.3 ppm are assigned to protons attached to N and/or O, based upon similar assignments in human hair and
Sepia melanin52.The four broad aromatic resonances at 7.60, 7.35, 7.00, and 6.60 ppm are assigned to indole and/
or pyrrole repeat units of the melanin polymer52. Resonances between 1.0 and 3.2 ppm are assigned to residual
protein.
Scanning electron micrograph (SEM). SEM was used to examine the structure of melanin and the nat-
ural melanin appears to be small spheres5. In the present study, the purified melanin pigment synthesized by
Streptomyces glaucescens strain NEAE-H appears to have defined small spheres by SEM imaging (Fig. 8). The
Figure 6. (A) Granules of extracted lyophilized melanin; (B) UV-visible absorbance spectrum (200–700 nm) of
the purified melanin pigment of Streptomyces glaucescens strain NEAE-H.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129natural Sepia melanin sample has a significant structural order with subunits that have a lateral dimension of
~15 nm53. Structural order is lacking in the case of melanin produced by S. bikiniensis.
In vitro anticancer activity. The safety pattern of the purified melanin pigment of Streptomyces glaucescens
strain NEAE-H was assayed on human lung fibroblast (WI-38) and human amnion (WISH). The results revealed that, the treatment IC
50 on all cells ranged from 37.05 ± 2.40 to 48.07 ± 2.76 μ g/ml (Table 6). The anti-proliferative
activity of the purified melanin pigment of Streptomyces glaucescens strain NEAE-H was assayed for its anticancer
activity in vitro against skin cancer cell line (HFB4) using MTT assay. The obtained results were expressed as
growth inhibitory concentration (IC50) values, which represent the melanin pigment concentration required to
produce a 50% inhibition of cell growth after 24 h of incubation, compared to untreated controls (Table 6). MTT
assay revealed that melanin pigment produced by Streptomyces glaucescens strain NEAE-H showed potent cyto-
toxic activity against HFB4 skin cancer cell line. After 24 h, the total mortality was 81.3% in the highest concentra-
tion (100 μ g/ml) of melanin comparable to standard 5-fluorouracil which showing 92.2% mortality (Table 6). The
IC50 value was 16.34 ± 1.31 μ g/ml for melanin and 8.8 ± 0.5 μ g/ml for standard 5-fluorouracil. From the obtained
results, it was obvious that the melanin pigment displayed strong anticancer activity against the tested cell line. Melanin at 50 μ g/ml inhibited cell viability by 70.9%.
It can be observed that the purified melanin pigment of Streptomyces glaucescens strain NEAE-H showed
less cytotoxicity even at high concentrations with an IC
50 value 37.05 ± 2.40 and 48.07 ± 2.76 against nor –
mal non-cancerous, human lung fibroblast and human amnion cells; respectively as compared with standard 5-fluorouracil which showing IC
50 value 6.68 ± 0.57 and 5.07 ± 0.38; respectively. The potent cytotoxic activity of
melanin against HFB4 skin cancer cell line and low cytotoxicity of against normal non-cancerous cells shows that melanin pigment can be used as potential natural anticancer. Arun et al.
54 reported that the in vitro inhibition of
cell proliferation in HEP 2 carcinoma cell line was concentration dependent. Melanin at 60 μ g inhibited the cell
viability by 53%. On the other hand, Kurian et al.19 found that MTT assay revealed that BTCZ31 melanin inhib-
ited growth of L929 cell line, cytotoxic concentration of melanin was found to be 105.4 μ g/ml (IC50).
ABTS+ radical scavenging (antioxidant) activity. The ability of the melanin pigment to scavenge
free radicals was evaluated by inhibition of the oxidation of 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The results showed that the purified melanin pigment of Streptomyces glaucescens strain NEAE-H
showed good antioxidant activity. The results revealed that 100 μ g/ml melanin exhibited percentage inhibition
57.2% radical scavenging activity, which was comparable to that of standard antioxidant ascorbic acid showing
Figure 7. (A) FT-IR Spectroscopic analysis of the extracted melanin pigment; (B) NMR spectrum of the extracted melanin pigment.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129an activity of 89.6%. ABTS radical was quickly and effectively scavenged by the melanin pigment. Interaction of
melanin pigment (antioxidant) with ABTS+ transfers hydrogen atoms to ABTS+ thus neutralizing its free radical
Figure 8. Scanning electron microscope micrographs of the extracted melanin pigment granules at
different magnifications showing small spheres.
Concentration ( μg/ml)Cytotoxicity (%)
Skin cancer
cell line
(HFB4)Human lung
fibroblast
(WI-38)Human
amnion
(WISH)
5-fluorouracil
1.56 5.9 12.7 24.8
3.125 21.8 35 37.1
6.25 45.1 49.4 54.9
12.5 61.7 66.9 72.4
25 75.6 78.6 81.7
50 83.8 85.8 90.3
100 92.2 92.5 94.2
Cytotoxicity IC50 (μ g/ml) 8.85 ± 0.52 6.68 ± 0.57 5.07 ± 0.38
Melanin pigment 1.56 0 0 0
3.125 7.7 0 0
6.25 28.2 9.8 3.5
12.5 49.4 32.9 25.9
25 66 43.7 37.8
50 70.9 57.3 51
100 81.3 68.1 64.4
Cytotoxicity IC
50 (μ g/ml) 16.34 ± 1.31 37.05 ± 2.40 48.07 ± 2.76
Table 6. Cytotoxicity and anticancer activities of various concentrations of the purified melanin pigment
of Streptomyces glaucescens strain NEAE-H on both cancerous and non-cancerous cells. 5-fluorouracil was
used as a standard anticancer drug for comparison. IC50 (μ g/ml): 1–10 (very strong). 11–20 (strong). 21–50
(moderate). 51–100 (weak) and above 100 (non-cytotoxic).

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129character. Melanin is a polymer able to donate or accept an electron. Melanin pigment interacts with free radi-
cals and other reactive species readily due to the presence of unpaired electrons in its molecules and acts as an antioxidant, suggesting its use as a raw cosmetic material to minimize toxin-induced tissue destruction. Melanin interacts with free radicals via the simple one electron transfer processes
55.
Anti-haemolytic activity. In vitro anti-haemolytic assay using spectroscopic method was used to eval-
uate the effect of melanin pigment on the erythrocytes. The results revealed that, melanin pigment exhibited considerable anti-hemolytic activity. Melanin exhibited percentage erythrocyte hemolysis of 11.9%, which was comparable to that of standard ascorbic acid showing percentage erythrocyte hemolysis of 4.4%. The effective anti-hemolytic activity of melanin pigment is because of the ability of phenolic compounds in neutralizing the
free radicals and thereby protecting the erythrocytes membrane from destruction and lysis.
Methods
Microorganisms and cultural conditions. Streptomyces spp. used in this study were isolated from various
soil samples collected from different localities of Egypt and Saudi Arabia. Using standard dilution plate method on
Petri plates containing starch nitrate agar medium of the following composition (g/L): Starch, 20; KNO3, 2; K2HPO4, 1;
MgSO4.7H2O, 0.5; NaCl, 0.5; CaCO3, 3; FeSO4.7H2O, 0.01; agar, 20 and distilled water up to 1 L; then plates were incu-
bated for a period of 7 days at 30 °C. Actinomycetes isolates were maintained as spore suspension in 20% (v/v) glycerol
at − 20 °C for subsequent investigation.
Screening for melanin producers. During the primary screening, isolates were spot inoculated on pep-
tone yeast extract iron agar (ISP medium 6) plates containing g/L: peptone 15; protease peptone 5; ferric ammo-
nium citrate 0.5; K2HPO4 1; sodium thiosulfate 0.08; yeast extract 1 and distilled water 1 L; agar 20 g; pH 7–7.2
and tyrosine agar (ISP medium 7) plates containing g/L: glycerol 15.0; L-tyrosine 0.5; L-asparagine 0.5; K2HPO4
0.5; MgSO4.7H2O 0.5; FeSO4.7H2O 0.01 and distilled water 1 L; agar 20 g; pH 7.2. Loopful of spores were inocu-
lated on to the agar plates and incubated at 30 °C for 2–6 days. Brown to back zone of diffusible pigment around
the colonies in the medium was scored as positive. Melanin production was quantitatively analyzed by seeding
the isolates selected after the primary screening into the melanin production medium (peptone yeast extract iron broth). Melanin pigment production was evaluated by measuring O.D. of the filtrate spectrophotometrically at
280 nm.
Production conditions. 100 ml of fermentation medium (peptone yeast extract iron broth) were dispensed in
250 ml Erlenmeyer conical flasks, inoculated with six disks of 9 mm diameter taken from the 7 days old stock culture
grown starch nitrate agar medium. The inoculated flasks were incubated for 3–6 days on a rotatory incubator shaker
at 100–200 rpm and 30–37 °C. After incubation time, Streptomyces cells were collected by centrifugation at 10000 g
for 10 min. The cell free supernatant was used for assay of tyrosinase activity and melanin formation.
Assay of tyrosinase activity. The tyrosinase activity test was done to confirm whether the diffusible black/
brown pigment formed in peptone yeast extract iron agar and synthetic tyrosine agar is melanin (not melanoid pigments). Tyrosinase activity was assayed as described by the modified method of Robb
56 by using L-DOPA as a
substrate, measuring conversion of L-DOPA to red colored oxidation product dopachrome. 0.5 ml of the enzyme
solution was added to freshly prepared 2 ml of 0.1 M potassium phosphate buffer (pH 7) containing L-3, 4- dihy-
droxyphenyl alanine (L-DOPA, 4 mg/ml of phosphate buffer) as substrate. The reaction mixture was incubated at
37 °C for 15 min. Red coloration resulting from dopachrome formation was monitored by measuring the absorb-
ance spectrophotometrically at 480 nm. One unit of tyrosinase activity was defined as the amount of enzyme that
catalyzes the formation of 1 μ mol dopachrome per minute at 37 °C.
Morphology and cultural characteristics. Detailed information is reported in the Supplementary Information.
Physiological characteristics. Detailed information is reported in the Supplementary Information.
16 S rRNA sequencing, sequence alignment and phylogenetic analysis. The preparation of
genomic DNA of the strain was conducted in accordance with the methods described by Sambrook et al.57. The
PCR amplification reaction was performed in accordance with the methods described by El-Naggar et al.58.
Sequencing product was deposited in the GenBank database under accession number KJ467537.
The partial 16 S rRNA gene sequence of strain NEAE-H was aligned with the corresponding 16 S rRNA
sequences of the type strains of representative members of the genus Streptomyces retrieved from the GenBank,
EMBL, DDBJ and PDB databases by using BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_
TYPE= BlastSearch)59 and the software package MEGA4 version 2.129 was used for multiple alignment and phy-
logenetic analysis. The phylogenetic tree was constructed via the bootstrap test of neighbor-joining algorithm
method28 based on the 16 S rRNA gene sequences of strain NEAE- H and related organisms.
Screening of main factors influences melanin production by Plackett–Burman design. Plackett–
Burman experimental design is a two factorial design, which identifies the critical environmental and nutritional variables required for elevated melanin production and is very useful for screening the most important factors with respect to their main effects
60. The total number of experiments to be carried out according to Plackett–
Burman is n + 1, where n is the number of variables61. A total of 17 independent (assigned) and two unassigned
variables (commonly referred as dummy variables) were screened in Plackett–Burman experimental design. Dummy variables (D
1 and D2) are used to estimate experimental errors in data analysis. Table 2 shows the sev-
enteen different independent variables including incubation period, pH, temperature, agitation speed, medium

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129volume, starch, glycerol, L-tyrosine, potassium nitrate, peptone, protease-peptone, yeast extract, K2HPO4, ferric
ammonium citrate, sodium thiosulfate, MgSO4 and NaCl which chosen to be screened by Plackett Burman exper –
iment. Each variable is represented at two levels, high and low denoted by ( + ) and (− ), respectively. The experi-
ment was conducted in 20 runs to study the effect of the selected variables on the production of melanin. All trials
were performed in duplicate and the average of melanin production was treated as response. Plackett–Burman experimental design is based on the first order model:
∑β β =+YX (3) ii 0
Where, Y is the response or dependent variable (melanin production); it will always be the variable we aim to predict, β
0 is the model intercept and βi is the linear coefficient, and Xi is the level of the independent variable; it
is the variable that will help us explain melanin production.
Face-centered central composite design. The levels and the interaction effects between various significant
variables which exerted a positive effect on the melanin production were analyzed and optimized by using face-centered central composite design (FCCD). In this study, the experimental plan consisted of 20 trials and the independent var –
iables were studied at three different levels, low (− 1), middle (0) and high ( + 1). All the experiments were done in
duplicate and the average of melanin production obtained was taken as the response (Y). The experimental results of
FCCD were fitted via the response surface regression procedure using the following second order polynomial equation:
∑∑ ∑ ββ ββ =+ ++ YX XX X
(4)ii i
jijij
ii
iii
i02
In which Y is the predicted response, β 0 is the regression coefficients, β i is the linear coefficient, β ii is the quad-
ratic coefficients, β ij is the interaction coefficients and Xi is the coded levels of independent variables.
Statistical analysis. Design Expert ® 7.0 software version 7 (Stat-Ease Inc., USA) for Windows was used for
the experimental designs and statistical analysis. The statistical software package, STATISTICA software (Version
8.0, StatSoft Inc., Tulsa, USA) was used to plot the three-dimensional surface plots, in order to illustrate the rela-tionship between the responses and the experimental levels of each of the variables utilized in this study.
Purification of melanin. Melanin was purified by centrifuging the fermentation broth for 15 min at 5000 g
to remove cells and debris. To precipitate the melanin, the pH of the supernatant was adjusted to 2.0 using 6 M
HCl and was allowed to stand for 4 h. The precipitate was then collected by centrifugation at 9000 g for 15 min.
Melanin pellets were washed with distilled water four times and centrifuged at 9000 g for 15 minutes to obtain the
purified pigment. Purified pigment was lyophilized and stored at − 20 °C until further use.
UV–visible spectrophotometeric analysis of the purified melanin pigment. The purified melanin
powder was first dissolved in 0.5 M NaOH solution and then scanned in a UV–visible spectrophotometer (Optizen
pop) at UV , visible and near-infrared wavelengths (200–700 nm). The blank control was 0.5 M NaOH solution62.
Scanning electron microscope. Surface topography of melanin was performed on gold coated samples that
had been previously lyophilized using an analytical Scanning Electron Microscope (SEM) (JEOL JSM-6390LV).
Fourier transform infra red spectroscopy. Fourier transform infrared spectroscopy (FTIR) is most useful
for identifying the functional groups and interpretation of structure of unknown compounds. The melanin powder and KBr powder were mixed in an agate mortar and ground for a few seconds to break up the melanin and KBr lumps. The mixed disc was scanned at 4000–400 cm
1 in an FTIR spectrophotometer (Shimadzu FTIR-8400 S).
1H NMR. The 1H NMR spectrum was obtained by JEOL DELTA2 Nuclear Magnetic Resonance Spectrometer
in 5-mm NMR tubes at 25 °C. Operating parameters were: Freq 500.16 MHz, field strength 11.75 T (500 MHz),
Resolution 0.57 Hz and acquisition time 1.75 s.
In vitro cytotoxicity and anticancer activities using microculture tetrazolium assay (MTT assay).
Both safety and the anticancer activities of the purified melanin pigment of Streptomyces glaucescens strain
NEAE-H were measured in vitro on both cancerous (skin cancer cell line (HFB4)) and non-cancerous cells
(human lung fibroblast (WI-38) and human amnion (WISH)) which obtained from ATCC via holding com-pany for biological products and vaccines (V ACSERA), Cairo, Egypt. HFB4 cell line was used to determine the
inhibitory effects of melanin pigment on cell growth using standard 3-(4, 5 dimethythiazol-2-yl)-2, 5-diphenyl
tetrazolium bromide (MTT) assay
63. This colorimetric assay is based on the conversion of the yellow tetrazolium
bromide (MTT) to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells. The cells were cultured in RPMI-1640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/ml penicillin and 100 μ g/ml streptomycin at 37 °C in a 5% CO
2 incubator. The cells were seeded in a 96-well plate at a
density of 1.0 × 104cells/well at 37ᵒ C for 48 h under 5% CO2. After incubation, the cells were treated with different
concentration of melanin pigment (1.56, 3.125, 6.25, 12.5, 25, 50 and 100 μ g/ml) and incubated for 24 h. After
24 h of drug treatment, 20 μ l of MTT solution at 5 mg/ml was added and incubated for 4 h. Dimethyl sulfoxide
(DMSO) in volume of 100 μ l is added into each well to dissolve the purple formazan formed. The colorimetric
assay is measured and recorded at absorbance of 570 nm using a plate reader (EXL 800, USA). The relative cell
viability in percentage was calculated as (A570 of treated samples/A570 of untreated sample) X 100. 5-fluorouracil was used as a standard anticancer drug for comparison.

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Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129Conclusion
A melanin producer Streptomyces glaucescens strain NEAE-H was isolated from soil sample collected from
Al-Taif, Saudi Arabia. The purified melanin exhibited the physical and chemical properties of typical melanin.
Maximum melanin yield was obtained using a simple culture process, avoids the use of purified tyrosinase, expen-sive chemical methods or the cumbersome extraction of this polymer from animal or plant tissues. Streptomyces glaucescens strain NEAE-H can be viewed as a promising source of melanin. Melanin pigment produced by
Streptomyces glaucescens NEAE-H is soluble in water, it is critical for melanin to be water soluble for a better com-
mercial potential in biotechnological applications in the pharmaceutical and cosmetic industries.
References
1. Langfelder, K., Streibel, M., Jahn, B., Haase, G. & Brakhage, A. A. Biosynthesis of fungal melanins and their importance for human
pathogenic fungi. Fungal Genet Biol 38, 143–158 (2003).
2. Ikeda, K. et al. Construction of a new cloning vector utilizing a cryptic plasmid and the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus . FEMS Microbiol Lett 168, 195–199 (1998).
3. Marino, S. M. et al. Investigation of Streptomyces antibioticus tyrosinase reactivity toward chlorophenols. Arch Biochem Biophys 505,
67–74 (2011).
4. Seetharam, G. B. & Saville, B. A. Degradation of phenol using tyrosinase immobilized on siliceous supports. Water Res 37, 436–440 (2003).
5. Tarangini, K. & Mishra, S. Production, characterization and analysis of melanin from isolated marine Pseudomonas sp. using
vegetable waste. Res J Eng Sci 2(5), 40–46 ISSN 2278: 9472 (2013).
6. Nappi, A. J. & Ottaviani, E. Cytotoxicity and cytotoxic molecules in invertebrates. Bioessays 22, 469–480 (2000).
7. Plonka, P . M. & Grabacka, M. Melanin synthesis in microorganisms-biotechnological and medical aspects. Acta Biochim Pol 53,
429–443 (2006).
8. Allam, N. G. & El-Zaher, E. A. Protective role of Aspergillus fumigatus melanin against ultraviolet (UV) irradiation and Bjerkandera adusta melanin as a candidate vaccine against systemic candidiasis. Afr J Biotechnol 11, 6566–6577 (2012).
9. Gadd, G. M. & de Rome, L. Biosorption of copper by fungal melanin. Appl Microbiol Biot 29, 610–617 (1988).
10. Casadevall. A., Rosas, A. L. & Nosanchuk, J. D. Melanin and virulence in Cryptococcus neoformans . Curr Opin Microbiol 3, 354–358
(2000).
11. Riley, P . Melanin. Int j biochem cell boil 29, 1235–1239 (1997).
12. Huang, H. C. & Chang, T. M. Antioxidative properties and inhibitory effect of Bifidobacterium adolescentis on melanogenesis. World
J Microb Biot 28, 2903–2912 (2012).
13. Y e, M. et al. Purification, structure and anti-radiation activity of melanin from Lachnum YM404. Int J Biol Macromol 63, 170–176
(2014).
14. Gallas, J. & Eisner, M. Melanin polyvinyl alcohol plastic laminates for optical applications. US Patent 7029758 (2006).
15. Manivasagan, P ., Venkatesan, J., Senthilkumar, K., Sivakumar, K. & Kim, S. K. Isolation and characterization of biologically active melanin from Actinoalloteichus sp. MA-32. Int J Biol Macromol 58, 263–274 (2013).
16. El-Obeid, A., Al-Harbi, S., Al-Jomah, N. & Hassib, A. Herbal melanin modulates tumor necrosis factor alpha (TNF-α ), interleukin
6 (IL-6) and vascular endothelial growth factor (VEGF) production. Phytomed 13, 324–333 (2006).
17. Hung, Y . C., Sava V ., Hong, M. Y . & Huang, G. S. Inhibitory effects on phospholipase A2 and antivenin activity of melanin extracted
from Thea sinensis Linn. Life Sci 74, 2037–2047 (2004).
18. Sava, V ., Hung, Y ., Blagodarsky, V ., Hong, M. Y . & Huang, G. The liver-protecting activity of melanin-like pigment derived from black tea. Food Res Int 36, 505–511 (2003).
19. Kurian, N. K., Nair, H. P . & Bhat, S. G. Evaluation of anti-inflammatory property of melanin from marine Bacillus sp. BTCZ31. Asian
J Pharm Clin Res 8, 251–255 (2015).
20. Hoogduijn, M., Cemeli, E., Anderson, D., Wood, J. & Thody, A. Melanin protects melanocytes and keratinocytes against H
2O2-
induced DNA strand breaks through its ability to bind Ca2+. Exp Cell Res 294, 60–7, doi: 10.1016/j.yexcr.2003.11.007 (2003).
21. Kalka, K., Mukhtar, H., Turowski-Wanke, A. & Merk, H. Biomelanin antioxidants in cosmetics: assessment based on inhibition of
lipid peroxidation. Skin Pharmacol Phys 13, 143–149 (2000).
22. Montefiori, D. C. & Zhou, J. Selective antiviral activity of synthetic soluble L-tyrosine and L-dopa melanins against human
immunodeficiency virus in vitro . Antiviral Res 15, 11–25 (1991).
23. Wan, X. et al. Isolation of a novel strain of Aeromonas media producing high levels of DOPA‐melanin and assessment of the
photoprotective role of the melanin in bioinsecticide applications. J Appl Microbiol 103, 2533–2541 (2007).
24. Balagurunathan, R. & Radhakrishnan, M. Biotechnological, genetic engineering and nanotechnological potential of actinomycetes.
In Industrial exploitation of microorganisms Maheshwari, D. K., Dubey, R. C., Saravanamuthu, R. (eds) New Delhi, International
Publishing House Pvt Ltd, 302–321 (2010).
25. Dastager, S. et al. Seperation, identification and analysis of pigment (melanin) production in Streptomyces . Afr J Biotechnol 5 (8),
1131–1134 (2006).
26. Suja Devan, V . Environmental impact assessment. Studies on microbial population of Veli Lake. Indian J Exp Biol 10, 46–58 (1999).
27. Shaaban, M. T., El-Sabbagh, S. M. M. & Alam, A. Studies on an actinomycete producing a melanin pigment inhibiting aflatoxin B1 production by Aspergillus flavus . Life Sci J 10, 1437–1448 (2013).
28. Saitou, N. & Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425
(1987).
29. Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol
biol evol 24, 1596–1599 (2007).
30. Goodfellow, M. et al. Bergey’s Manual of Systematic Bacteriology, vol. 5. Springer New Y ork (2012).
31. Akhnazarova, S. L., Kafarov, V . V ., Mackovskij, V . M. & Rep’ ev, A. P . Experiment optimization in chemistry and chemical engineering: Mir Publishers (1982).
32. Box, G. E. P ., Hunter, W . G. & Hunter, J. S. Statistics for experiments. New Y ork, Wiley, 291–334 (1978).
33. Rani, M. H. S., Ramesh, T., Subramanian, J. & Kalaiselvam, M. Production and characterization of melanin pigment from halophilic black yeast Hortaea werneckii . Int J Pharm Res Rev 2, 9–17 (2013).
34. Amal, A. M. et al. Selection of pigment (melanin) production in Streptomyces and their application in printing and dyeing of wool
fabrics. Res J Chem Sci 1, 22–28 (2011).
35. Vasanthabharathi, V ., Lakshminarayanan, R. & Jayalakshmi, S. Melanin production from marine Streptomyces . Afr J Biotechnol 10,
11224 (2011).
36. Babitskaya, V ., Shcherba, V ., Filimonova, T. & Grigorchuk, E. Melanin pigments from the fungi Paecilomyces variotii and Aspergillus
carbonarius . Appl Biochem Microbiol 36, 128–133 (2000).
37. Quadri, S. R. & Agsar, D. Detection of melanin producing thermo-alkaliphilic Streptomyces from limestone quarries of the Deccan
traps. World J Sci Technol 2, 8–12 (2012).
38. Arai, T. & Mikami, Y . Chromogenicity of Streptomyces . Appl Microbiol 23, 402–406 PMCID: PMC380352 (1972).

www.nature.com/scientificreports/19
Scientific RepoRts | 7:42129 | DOI: 10.1038/srep4212939. Chatfield, C. H. & Cianciotto, N. P . The secreted pyomelanin pigment of Legionella pneumophila confers ferric reductase activity.
Infect Immun 75, 4062–4070 (2007).
40. Jung, W . H., Sham, A., White, R. & Kronstad, J. W . Iron regulation of the major virulence factors in the AIDS-associated pathogen
Cryptococcus neoformans . PLOS Biol 4, e410 (2006).
41. Kuhn, D. M., Ruskin, B. & Lovenberg, W . Tryptophan hydroxylase. The role of oxygen, iron, and sulfhydryl groups as determinants
of stability and catalytic activity. J Biol Chem 255, 4137–4143 (1980).
42. Woolery, G., Powers, L., Winkler, M., Solomon, E. & Spiro, T. EXAFS studies of binuclear copper site of oxy-, deoxy-, and metaquo-,
metfluoro-, and metazidohemocyanin from arthropods and molluscs. J American Chem Society 106, 86–92 (1984).
43. Zajac, G. W . et al. The fundamental unit of synthetic melanin: a verification by tunneling microscopy of X-ray scattering results.
Biochem Biophys Acta 1199, 271–278 (1994).
44. Deepthi, A. & Rosamma, P . Actinomycete isolates from Arabian Sea and Bay of Bengal: biochemical, molecular and functional
characterization: Thesis, Cochin University of Science and Technology, Cochin, Kerala 682022, India (2014).
45. Apte, M., Girme, G., Bankar, A., RaviKumar, A. & Zinjarde, S. 3, 4-dihydroxy-L-phenylalanine-derived melanin from Yarrow ia
lipolytica mediates the synthesis of silver and gold nanostructures. J Nanobiotechnol 11, 1 (2013).
46. Sajjan, S., Kulkarni, G., Y aligara, V ., Kyoung, L. & Karegoudar, T. Purification and physiochemical characterization of melanin
pigment from Klebsiella sp. GSK. J Microbiol Biotechnol 20, 1513–1520 (2010).
47. Suryanarayanan, T. S., Ravishankar, J. P ., Venkatesan, G. & Murali, T. S. Characterization of melanin pigment of a cosmopolitan
fungal endophyte. Mycol Res 108, 974–978 (2004).
48. Hu, W . L., Dai, D. H., Huang, G. R. & Zhang, Z. D. Isolation and characterization of extracellular melanin produced by
Chroogomphus rutilus D447. American J Food Technol 10(2), 68–77 (2015).
49. Coates, J. Interpretation of infrared spectra, a practical approach. Encyclopedia Anal Chem (2000).
50. Magarelli, M., Passamonti, P . & Renieri, C. Purification, characterization and analysis of sepia melanin from commercial sepia ink (Sepia Officinalis). CES Medicina Veterinaria y Zootecnia 5, 18–28 (2010).
51. Hewedy, M. & Ashour, S. Production of a melanin like pigment by Kluyveromyces marxianus and Streptomyces chibaensis . Aust J
Basic Appl Sci 3, 920–927 (2009).
52. Katritzky, A. R., Akhmedov, N. G., Denisenko, S. N. & Denisko, O. V . 1H NMR spectroscopic characterization of solutions of Sepia
melanin, Sepia melanin free acid and human hair melanin. Pigment Cell Res 15, 93–97 (2002).
53. Nofsinger J. B., Forest S. E., Eibest L. M., Gold K. A. & Simon J. D. Probing the building blocks of eumelanins using scanning electron
microscopy. Pigm Cell Res 13, 179–184 (2000).
54. Arun, G., Eyini, M. & Gunasekaran, P . Characterization and biological activities of extracellular melanin produced by Schizophyllum commune (Fries). Indian J Exp Biol 53(6), 380–387 (2015).
55. Rozanowska, M., Sarna, T., Land, E. J. & Truscott, T. G. Free radical scavenging properties of melanin interaction of eu- and pheo-
melanin models with reducing and oxidising radicals. Free Rad Biol Med 26, 518–525 (1999).
56. Robb, D. A. Tyrosinase. In Lontie, R. (Ed.), Copper proteins and copper enzymes (Lontie,. R. ed.), FL: CRC Press, Boca Raton Vol 2,
207–240 (1984).
57. Sambrook, J. & Fritsch, E. F. Maniatis, T. Molecular cloning: Cold spring harbor laboratory press New Y ork (1989).
58. El-Naggar, N. E., Sherief, A. & Hamza, S. S. Streptomyces aegyptia NEAE 102, a novel cellulolytic streptomycete isolated from soil in
Egypt. Afr J Microbiol Res 5, 5308–5315 (2011).
59. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25,
3389–3402 (1997).
60. El-Naggar, N. E. Extracellular production of the oncolytic enzyme, L-asparaginase by newly isolated Streptomyces sp. strain NEAE-
95 as potential microbial cell factories: Optimization of culture conditions using response surface methodology. Curr Pharm
Biotechnol 16, 162–178 (2015).
61. Plackett, R. L. & Burman, J. P . The design of optimum multifactorial experiments. Biometrika 33, 305–325 (1946).
62. Guo, J. et al. High-level production of melanin by a novel isolate of Streptomyces kathirae . FEMS Microbiol Lett 357, 85–91 (2014).
63. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55–63 (1983).
Author Contributions
N.E.E. proposed the research concept, designed the experiments, providing necessary tools for experiments,
experimental instructions, conducted most of the experiments, analyzed and interpreted the data and wrote the manuscript. S.M.E. carried out the experiments. All authors read and approved the final manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.How to cite this article: El-Naggar, N. E. and El-Ewasy, S. M. Bioproduction, characterization, anticancer and
antioxidant activities of extracellular melanin pigment produced by newly isolated microbial cell factories
Streptomyces glaucescens NEAE-H. Sci. Rep. 7, 42129; doi: 10.1038/srep42129 (2017).
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