Bioproduction and optimization of newly characterized melanin pigment from Streptomyces djakartensis NSS-3 with its anticancer, antimicrobial, and radioprotective properties

Abstract

Background: Melanin is a natural pigment that is considered a promising biomaterial for numerous biotechnological applications across several industries. Melanin has biomedical applications as antimicrobial, anticancer, and antioxidant properties. Additionally, in the pharmaceutical and cosmetic industries, it is used in drug delivery and as a radioprotective agent. Also, melanin has environmental uses in the fields of bioremediation and the food industry. The biosynthesis of melanin pigment is an area of interest for researchers due to its multifunctionality, high compatibility, and biodegradability. Therefore, our present work is the first attempt to characterize and optimize the productivity of melanin pigment from Streptomyces djakartensis NSS-3 concerning its radioprotection and biological properties.

Results: Forty isolates of soil actinobacteria were isolated from the Wadi Allaqui Biosphere Reserve, Egypt. Only one isolate, ACT3, produced a dark brown melanin pigment extracellularly. This isolate was identified according to phenotypic properties and molecular phylogenetic analysis as Streptomyces djakartensis NSS-3 with accession number OP912881. Plackett-Burman experimental design (PBD) and response surface methodology (RSM) using a Box-Behnken design (BBD) were performed for optimum medium and culturing conditions for maximum pigment production, resulting in a 4.19-fold improvement in melanin production (118.73 mg/10 mL). The extracted melanin pigment was purified and characterized as belonging to nitrogen-free pyomelanin based on ultraviolet-visible spectrophotometry (UV-VIS), Fourier transform infrared (FT-IR), Raman spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and NMR studies. Purified melanin demonstrated potent scavenging activity with IC₅₀ values of 18.03 µg/mL and revealed high potency as sunscreens (in vitro SPF = 18.5). Moreover, it showed a nontoxic effect on a normal cell line (WI38), while it had a concentration-dependent anticancer effect on HCT116, HEPG, and MCF7 cell lines with IC₅₀ = 108.9, 43.83, and 81.99 µg/mL, respectively. Also, purified melanin had a detrimental effect on the tested MDR bacterial strains, of which PA-09 and SA-04 were clearly more susceptible to melanin compared with other strains with MICs of 6.25 and 25 µg/mL, respectively.

Conclusion: Our results demonstrated that the newly characterized pyomelanin from Streptomyces djakartensis NSS-3 has valuable biological properties due to its potential photoprotective, antioxidant, anticancer, antimicrobial, and lack of cytotoxic activities, which open up new prospects for using this natural melanin pigment in various biotechnological applications and avoiding chemical-based drugs.

Keywords: Actinobacteria; Antimicrobial; Melanin bioproduction; Radioprotection; Response surface optimization.

Fig. 1

Schematic diagram of melanin extraction from S. djakartensis NSS-3

Fig. 2

Morphological characteristics of ACT3 strain. Dusty aerial mycelium (A); Brown substrate mycelium (B); Scanning electron micrographs showing spore chain shape and spore surface ornamentation of ACT3 strain at different magnifications (1.000, 7.500, 7.500X) (C, D, E)

Fig. 3

Phylogenetic tree based on 16S rDNA gene sequencing of Streptomyces djakartensis NSS-3 strain with GenBank accession no. OP912881 (arrowed) aligned with closely related sequences of bacterial strains accessed from the GenBank

Fig. 4

Pareto chart depicts the degree to which each variable influences melanin production by S. djakartensis NSS-3 (A); Correlation between the experimentally actual and predicted values for melanin production by S. djakartensis NSS-3 according to the Plackett–Burman experimental results (B)

Fig. 5

The 3D surface response and contour plots showing the effect of L-tyrosine (A, A1); incubation period (B, B1); and ferric ammonium citrate (C, C1) and their mutual interaction on melanin production

Fig. 6

Predicted solution for maximum melanin pigment production using BBD numerical optimization

Fig. 7

Melanin production before and after RSM optimization

Fig. 8

Spectroscopic analysis of the purified melanin produced by S. djakartensis NSS-3. Uv–visible spectrum (A); FTIR spectroscopic spectrum (B); Raman spectrum (C)

Fig. 9

¹HNMR (A); and.¹³CNMR (B) of purified melanin produced by S. djakartensis NSS-3

Fig. 10

SEM images of purified melanin produced by S. djakartensis NSS-3 at different magnifications (A, B); Energy dispersive spectroscopy (EDS) and elemental mapping analysis showing elemental composition of purified melanin (C, D, E, F)

Fig. 11

Antioxidant activity of purified melanin and ascorbic acid as standard

Fig. 12

In vitro cytotoxicity and anticancer activities of various concentrations of the purified melanin pigment of S. djakartensis NSS-3