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Mar Drugs
2022 Jan 06;201:. doi: 10.3390/md20010054.
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Characterization of an Insoluble and Soluble Form of Melanin Produced by Streptomyces cavourensis SV 21, a Sea Cucumber Associated Bacterium.
Wibowo JT
,
Kellermann MY
,
Petersen LE
,
Alfiansah YR
,
Lattyak C
,
Schupp PJ
.
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Melanin is a widely distributed and striking dark-colored pigment produced by countless living organisms. Although a wide range of bioactivities have been recognized, there are still major constraints in using melanin for biotechnological applications such as its fragmentary known chemical structure and its insolubility in inorganic and organic solvents. In this study, a bacterial culture of Streptomyces cavourensis SV 21 produced two distinct forms of melanin: (1) a particulate, insoluble form as well as (2) a rarely observed water-soluble form. The here presented novel, acid-free purification protocol of purified particulate melanin (PPM) and purified dissolved melanin (PDM) represents the basis for an in-depth comparison of their physicochemical and biological properties, which were compared to the traditional acid-based precipitation of melanin (AM) and to a synthetic melanin standard (SM). Our data show that the differences in solubility between PDM and PPM in aqueous solutions may be a result of different adjoining cation species, since the soluble PDM polymer is largely composed of Mg2+ ions and the insoluble PPM is dominated by Ca2+ ions. Furthermore, AM shared most properties with SM, which is likely attributed to a similar, acid-based production protocol. The here presented gentler approach of purifying melanin facilitates a new perspective of an intact form of soluble and insoluble melanin that is less chemical altered and thus closer to its original biological form.
Figure 1. (A) Color of three replicates of Streptomyces cavourensis SV 21 cultures grown in marine broth (MB) at day 0 and after 14 days of incubation. (B) Transmission and fluorescence light microscopy (magnification 10 × 100, oil immersion) pictures of S.
cavourensis SV 21 colonies. Here, colonies were grown in liquid MB media shaken at 22 °C and sampled at 7, 10, and 20 days. (C) Top and bottom view of 5-day old S. cavourensis SV 21 cultures on marine agar (MA). (D) SEM pictures of a 5-day old colony of S. cavourensis SV 21 grown on MA media and cultured at a temperature of 22 °C (single bacterial colony shown); (i) to (iii) represent further magnifications into the bacterial culture down to the cellular, µm-level. In (i) substrate and aerial mycelia are shown, (ii) zooms into the substrate mycelium and the main stem of the aerial mycelia, and (iii) magnifies on the spore-chains of S. cavourensis SV 21.
Figure 2. Physicochemical characteristics of the MB growth media (A) and the purified acid-based melanin (AM), purified particulate melanin (PPM), purified dissolved melanin (PDM) and a commercially available, synthetic melanin standard (SM; B–E). (A) shows the absorption spectra in the UV (230–400 nm) and visible light range (400–700 nm) of a Milli-Q blank (black line, negative control) and the supernatants of four different 13-day old incubations of S. cavourensis SV 21 (i.e., with and w/o light at 22 and 30 °C). In (B) the absorbance spectra (230–700 nm) of AM, PPM, and PDM purified from S. cavourensis SV 21 (cf. Figure 3) were compared to SM. For that, all samples were treated with 1N NH4OH to rise the pH to 12 and thus increasing the solubility properties of the otherwise poorly or insoluble melanin samples PPM, AM, and SM (cf. Table 1). The elemental composition of the different melanin types SM, AM, PDM, and PPM were determined using both, an elemental combustion (C) and energy dispersive X-ray (D) analyzer. In (E) the RAMAN spectra of the dried melanin powder AM, PDM, and PPM were compared to SM. For all samples the laser was set to 488 nm.
Figure 3. A schematic workflow illustrating how the different melanin types of S. cavourensis SV 21 were obtained. (A) shows the first physical separation step by centrifugation to achieve the water-soluble form of melanin (WSM) and the cellular/particulate form of melanin (PM). In (B) a step-by-step purification protocol is shown for WSM and in (C) for PM.
Figure 4. Scanning electron microscopy (SEM) images of SM, AM, PDM, and PPM. AM and PDM were derived and purified from the supernatant of the aqueous media (WSM, cf. Figure 3), while PPM was isolated from cell pellets (PM).
Figure 5. Antioxidant capacity of different melanin types. In (A) the DPPH radical scavenging activity of SM, AM, PDM and PPM were tested at a concentration of 160 µg mL−1. In (B) the DPPH scavenging activities were compared between AM and SM at three different concentrations (i.e., 1.6, 16, 160 µg mL−1). In (C,D) the oxidation potential of PDM was shown using hydrogen peroxide (H2O2) as oxidation reagent. In (C), 5 mL of the dark-brownish aqueous phase of PDM was spiked with different amounts of H2O2 (final conc.: 0, 0.1, 1, 10, 100, and 200 mM). The absorption spectra of melanin show that H2O2 bleached the pigment in a concentration dependent manner. In (D), the decrease of H2O2 concentration (50 mM) in the presence and absence of PDM is shown over time.
Figure 6. In (A) the heat map displays antibacterial activities of the different melanin types SM, AM, and PDM (x-axis) towards a test panel of selected environmental bacteria (y-axis). The heat map color represents percent antibacterial activity compared to the positive control (10 µM chloramphenicol). In (B,C) the heat map shows the different melanin types (y-axis) tested at two concentrations for their (B) antibacterial activity in % and (C) inhibition of quorum sensing activity in A. fischeri. Positive control for (B) 25 µM chloramphenicol and for (C) 10 µM furanone. In (A) tested bacteria: WHV 001: Aurantimonas coralicida; WHV 002: Vibrio mediterranei; WHV 003: Vibrio coralliilyticus; 852: Acinetobacter solii.; 1334: Aliagarivorans marinus; 1348: Vibrio maritimus; 1682: Rhodococcus corynebacterioides; 1668: Ruegeria areniliticus; 1686: Exiguobacterium profundum; 1792: Pseudovibrio denitrificans; 1809: Ruegeria areniliticus; 1810: Pantoea eucrina.
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