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

Abstract

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.

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.

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.

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.

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 glaucescens strain NEAE-H according to the Plackett–Burman experimental results.

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.

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.

Figure 7

(A) FT-IR Spectroscopic analysis of the extracted melanin pigment; (B) NMR spectrum of the extracted melanin pigment.

Figure 8. Scanning electron microscope micrographs of the extracted melanin pigment granules at different magnifications showing small spheres.