Biotechnological Production and Characterization of Extracellular Melanin by Streptomyces nashvillensis

Abstract

Melanins are pigments employed in food, cosmetic, and textile industries, manufactured by extraction from cuttlefishes. Their biotechnological production by Streptomycetes, instead, has been poorly investigated so far. In this paper, for the first time, the strain Streptomyces nashvillensis DSM 40314 was tested as an extracellular melanin producer by investigating the influence of diverse temperatures (26, 28, and 30 °C) and pH values (6.0 and 7.0) on bacterial growth, melanin production, and on the activity of the secreted tyrosinase, the first enzyme of the pigment biosynthetic pathway. In physiological 96-h shake flask experiments, the optimal growth parameters resulted to be 28 °C and pH 7.0, at which a maximum biomass of 8.4 ± 0.5 gcdw/L, a melanin concentration of 0.74 ± 0.01 g/L (yield on biomass of 0.09 ± 0.01 g/gcdw and productivity of 0.008 ± 0.001 g/L/h), and a final tyrosinase activity of 10.1 ± 0.1 U/mL were reached. The produced pigment was purified from the broth supernatant with a two-step purification process (75.0 ± 2.0% of purity with 65.0 ± 5.0% of recovery) and tested for its chemical, antioxidant, and photoprotective properties. Finally, characterization by UV-visible and FT-IR spectroscopy, elemental analyses, and mono- and bi-dimensional NMR suggested the eumelanin-like nature of the pigment.

Streptomycetes as Microbial Cell Factories for the Biotechnological Production of Melanin

“…A simpler downstream procedure, consisting of two steps of precipitation and washing with acidic solutions, was used to obtain a purified form of the melanin produced by Streptomyces nashvillensis DSM 40314. The melanin was found to have a purity of 75%, and the protein fraction associated with the pigment was almost completely removed, as evident from the low number of protons signals in the 2.6–4 ppm region of the 1 H NMR spectrum [ 47 ]. By analyzing the purified pigment also by 1 H, 1 H COSY, the signals of the catechol ring system at 6.64, 7.01, and 7.22 ppm were also found to be correlated with each other, while the signals at 7.17, 7.30, and 7.41 ppm of the 5,6-dihydroxyindole ring system were also clearly visible [ 47 ].…”

“…Streptomyces lusitanus DMZ-3 was able to synthesize both insoluble and soluble melanins [ 31 ], while Streptomyces bellus MSA1, isolated from marine sediments, also demonstrated the production of high contents of the pigment as an intracellular product [ 29 ]. In other papers, strains belonging to microbial cell banks, not newly isolated ones, like Streptomyces roseochromogenes ATCC 13400 and Streptomyces nashvillensis DSM 40314, were reported as good melanin producers as well [ 23 , 47 ]. Engineered Streptomyces strains have also been built to overproduce melanin by overexpressing the genes encoding tyrosinase and by inserting them in a replicative plasmid vector under the control of an inducible promoter [ 31 ].…”

“…Optimization studies of parameters, like pH, temperature, carbon and nitrogen sources, the salinity of the medium, inoculum size, time of growth (2–30 days), agitation speed (100–250 rpm), and the ratio between the medium volume and total air volume, have been performed in the literature to increase the melanin production [ 22 , 23 , 28 , 29 , 30 , 31 , 40 , 51 , 52 , 53 ]. Although pH and temperature conditions are key factors for secondary metabolite synthesis in Streptomycetes [ 50 ], only very few papers have performed a systematic study of the best pH and temperature values for each strain, as in the case of Streptomyces cavourensis RD8 [ 40 ], Streptomyces nashvillensis DSM 40314 [ 47 ], and Streptomyces sp. ZL-24 [ 32 ], for which the best growth conditions resulted to be pH 6.0 and 30 °C, pH 7.0 and 28 °C, and pH 7.0 and 30 °C, respectively.…”

“…Assays of the secreted tyrosinase were performed in several cases to correlate the enzymatic activity with the quantity of melanin produced in different conditions, for example, in the cases of Streptomyces sp. ZL-24 [ 32 ], Streptomyces roseochromogenes ATCC 13400 [ 23 ], and Streptomyces nashvillensis DSM 40314 [ 47 ]. The determination of tyrosinase activity might also be useful in correlating the enhancement in melanin production with supplementation to the growth medium of some metal ions that might influence both the gene transcription of tyrosinase synthesis as well as its activation from an apo-tyrosinase form [ 26 , 27 , 53 , 54 , 55 ].…”

“…Melanins produced by Streptomyces strains were demonstrated to be soluble in aqueous alkali (i.e., 0.1 M NaOH) and DMSO but insoluble in acid solutions with a pH lower than 2.0. Some of these melanins were completely or slowly soluble in water, as in the case of the pigments produced by S. roseochromogenes ATCC 13400 and S. nashvillensis DSM 40314 [ 23 , 47 ], while in the case of Streptomyces sp. ZL-24 [ 32 ], Streptomyces lusitanus DMZ-3 [ 52 ], Streptomyces sp.…”