Bioproduction, characterization, anticancer and antioxidant activities of extracellular melanin pigment produced by newly isolated microbial cell factories Streptomyces glaucescens NEAE-H

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 IC50 value was 16.34 ± 1.31 μg/ml for melanin and 8.8 ± 0.5 μg/ml for standard 5-fluorouracil.


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 significantly 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 recommended 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 agar 25 . 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 morphological, 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 morphological 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 sporulating 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; yellowish 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. Yellow pigments produced in oatmeal 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 cellulose), 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 sources, while raffinose is weakly utilized as sole carbon source. It  On the basis of the previously collected data and in view of the comparative study of the morphological, cultural 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 Streptomyces. 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 employed 25 . 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 melanin 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 mathematical 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 contribution indicated higher p-value. Thus instead of starting with the maximum model effects, backward regression at 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 independent variable named incubation period, L-tyrosine, peptone, protease-peptone, yeast extract, K 2 HPO 4 , ferric ammonium citrate and sodium thiosulfate affect positively melanin production, where the seven variables named glycerol, MgSO 4 , 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 composite design. Glycerol, KNO 3 , MgSO 4 , 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  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. R 2 = 0.9979) is also very high which indicates a high significance of the model 31 . 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 predicted 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 (ANOVA) 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 determined by p-values, which are listed in Table 3  (O) with a probability value of 0.0006 was determined to be the most significant factor affecting melanin production 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 incubation 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: 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 2 HPO 4 , ferric ammonium citrate, sodium thiosulfate, MgSO 4 and NaCl.
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 variables
Melanin production (μg/0.1 ml of medium)

Actual value
Predicted value .093 0.027 1666.08 13.739 0.081 2686.13 Level days ο C rpm 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 levels. Results of Placket-Burman design revealed that incubation period, protease-peptone and ferric ammonium 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 (X 2 ) and ferric ammonium citrate (X 3 ) 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 X 2 , X 3 , X 1 X 2 , X 1 X 3 , X 2 X 3 ( Table 5) indicate that linear effect of X 2 , X 3 and interaction effects for X 1 X 2 , X 1 X 3 , X 2 X 3 increase melanin production, while other negative coefficients indicate decrease in melanin production.
The adequacy of the model was checked using analysis of variance (ANOVA) 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 (X 1 ), protease-peptone (X 2 ) and ferric ammonium citrate (X 3 ) are significant 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 (R 2 ) of the model was 0.9837 (Table 5). Therefore, the present R 2 -value reflected a very good fit between the observed and predicted responses, and implied that the model is reliable for melanin 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).  In 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 (X 1 ), protease-peptone (X 2 ) and ferric ammonium citrate (X 3 ), 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 (X 1 , X 2 and X 3 ): (melanin production) 1 2 3 1 2 Where Y is the response (melanin production) and X 1 , X 2 and X 3 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   higher adjusted and predicted R-squared; the model summary statistics of the quadratic model showed the smallest standard deviation of 2.076 and the largest adjusted and predicted R-squared of 0.9690 and 0.9025 respectively.

Table 4. Face-centered central composite design representing the melanin production by Streptomyces glaucescens strain NEAE-H as influenced by incubation period (X 1 ), protease-peptone (X 2 ) and ferric ammonium citrate (X 3 ) along with the predicted melanin production and residuals and the levels of variables with actual factor levels corresponding to coded factor levels.
Three dimensional plots. The three dimensional response surface curves were plotted by statistically significant 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 X 2 , X 1 X 3, and X 2 X 3 ) 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 (X 2 ) 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 6 th 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 10 th and 7 th 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 -25 th day's incubation period 36 .
Quadri and Agsar 37 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 Streptomyces. 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 enzymatic digests of protein. It is rich in peptides with the higher molecular weight. Figure 5B depicts the incubation period (X 1 ) and ferric ammonium citrate (X 3 ) interactions. At moderate levels 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 Fe 2+ 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 reduction 39 . A study reports that iron levels can modulate the transcriptional control of melanin biosynthesis in C. neoformans 40 . Fe 2+ ion is required as cofactor for the activity of several aromatic hydroxylases, such as phenylalanine, tryptophan and tyrosine hydroxylases 41 . 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 (X 2 ) and ferric ammonium citrate (X 3 ) 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 predicted 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 production 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 granules 43 . 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. GSK 46 .
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 of melanin. The absorption peak at 250 nm was similar to the absorption peak for melanin pigment extracted from Phyllosticta capitalensis 47 . 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 nm 48 and Actinoalloteichus sp. MA-32 melanin at 300 nm 15 . 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 the characteristic property of melanin 44 . 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 spectra 48 .
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 group 49 . 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 carboxylic acid, phenolic and aromatic amino functions present in the indolic and pyrrolic systems 50 . 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 COOgroups. 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 pigment 46 . The peak centered at 1423.51 cm −1 (CH 2 -CH 3 bending) characteristic of melanin pigment. Phenolic COH stretching 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 pigments 51 . 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 substitution in the melanin pigment 46 . The spectroscopic properties of the pigment extracted from Streptomyces glaucescens strain NEAE-H correlated with those of melanin produced by various microorganisms as reported previously 46 . On the basis of the above results, it was concluded that the pigment was eumelanin. Scanning electron micrograph (SEM). SEM was used to examine the structure of melanin and the natural melanin appears to be small spheres 5 . 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 natural Sepia melanin sample has a significant structural order with subunits that have a lateral dimension of ~15 nm 53 . 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 (IC 50 ) 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 cytotoxic activity against HFB4 skin cancer cell line. After 24 h, the total mortality was 81.3% in the highest concentration (100 μ g/ml) of melanin comparable to standard 5-fluorouracil which showing 92.2% mortality ( Table 6). The IC 50 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 normal 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 inhibited growth of L929 cell line, cytotoxic concentration of melanin was found to be 105.4 μ g/ml (IC 50 ). 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 an 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  character. Melanin is a polymer able to donate or accept an electron. Melanin pigment interacts with free radicals 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 evaluate 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. Screening for melanin producers. During the primary screening, isolates were spot inoculated on peptone yeast extract iron agar (ISP medium 6) plates containing g/L: peptone 15; protease peptone 5; ferric ammonium citrate 0.5; K 2 HPO 4 1; sodium thiosulfate 0.08; yeast extract 1 and distilled water 1 L; agar 20 g; pH 7-7. 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.

Methods
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-dihydroxyphenyl 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 absorbance 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 2.1 29 was used for multiple alignment and phylogenetic analysis. The phylogenetic tree was constructed via the bootstrap test of neighbor-joining algorithm method 28 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 variables 61 . 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 D 2 ) are used to estimate experimental errors in data analysis. Table 2 shows the seventeen different independent variables including incubation period, pH, temperature, agitation speed, medium Scientific RepoRts | 7:42129 | DOI: 10.1038/srep42129 Conclusion 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, expensive 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 commercial potential in biotechnological applications in the pharmaceutical and cosmetic industries.