Extracellular Pyomelanin Pigment Production, Purication and Characterization with Streptomyces griseus MPPS2

Melanins are natural pigments widely distributed in nature from bacteria to humans. These complex, negatively charged, amorphous, high molecular mass natural biopolymers have many different bioactive properties such as antimicrobial, antiviral, antioxidant, liver protective effects etc. In this study, some chemical and physical properties of the extracellular pyomelanin pigment puried from Streptomyces griseus MPPS2 was investigated via XRD (X-Ray diffraction), FT-IR (Fourier transform infra-red) and 1H NMR (Nuclear magnetic resonance). Additionally, the melanin pigment-producing Streptomyces griseus MPPS2 strain was identied at species level by matrix-assisted laser desorption/ionization time-of-ight mass spectrometry (MALDI-TOF/TOF MS) and 16S rDNA sequence analysis. 16S rDNA sequence analysis result was deposited in NCBI under accession number MT825616.


Introduction
Melanins are ubiquitous natural pigments which are formed with polymerized phenolic and/or indolic compounds. These complex dark colored biopolymers are hydrophobic, negatively charged and high molecular weight. These amorphous bio-macromolecules polymerize in acidic solutions, but are insoluble in water and organic solvents. They are known to have a maximum absorbance capacity at 200-400 nm wavelengths and also, these natural pigments are characterized by its sensitivity to oxidizing agents such as H 2 O 2 and its loss of color (Tarangini and Mishra, 2014;Banerjee et al. 2014).
Under adverse conditions, many microorganisms synthesize melanin pigment and this pigment protects microorganisms against external threats such as desiccation, UV radiation, temperature uctuations, heavy metals, hydrolytic enzymes and digestion. In this way, microorganisms increase their chances of survival (Venil et al. 2013; Ye et al. 2014).
Melanin pigment has many different bioactive properties such as antioxidant, antiviral, antimicrobial, antivenin, anti-in ammatory, anti-proliferative effects. In addition to these properties, liver protective, drug carrier and UV absorbing properties of melanin pigment were also determined in different studies (El- In a study performed by Dadachova and Cassadevall (2008), it was stated that the melanized fungi species found around the Chernobyl reactor responded to ionizing radiation with increased growth (Dadachova and Cassadevall 2008). These data support the radioprotective effect of the melanin pigment and show that the melanin pigment has the potential to be used as a protective agent for patients with cancer undergoing radiation therapy. After this study, in a study conducted by Schweitzer et al. (2010), melanin coated nanoparticles were used to protect the bone marrow against radiotoxicity during radioimmunotherapy. The obtained results in this study showed that melanin coated nanoparticles reduced hematological toxicity in mice treated with radioimmunotherapy. Furthermore these nanoparticles did not have a protective effect against tumor tissues (Schweitzer et al. 2010).
In another study, conducted by Ozlu et al. (2019) for breast cancer therapy, controlled release of doxorubicin from polyethylene glycol functionalized melanin nanoparticles was investigated. Results from in vitro cytotoxicity assays showed that melanin nanoparticles (MNP) and polyethylene glycol (PEG) conjugated melanin nanoparticles (PEG-MNPs) did not show any toxic effects on mouse broblast cells.
In contrast to these results, it has been observed that doxorubicin loaded PEG-MNPs (DOX-PEG-MNP) can inhibit the proliferation of human breast cancer cells. These results support the potential for the use of melanin nanoparticles in prolonged and controlled drug release for different cancer therapeutics (Ozlu et al. 2019).
Melanin pigment has strong absorption at near-infrared region and higher photothermal conversion e ciency. Taking  In recent studies, it is seen that melanin pigment has an important potential in elds such as medical imaging, drug delivery, optoelectronics, cosmetics (Ozlu et  These results provide an increasing interest in melanin pigment day by day. In this study, pyomelanin pigment production potentials of different Streptomyces strains were investigated and the puri ed extracellular pyomelanin pigment from Streptomyces griseus MPPS2 strain was characterized by using XRD, FT-IR and 1 H NMR and UV-Vis spectrometry. In addition to these characterization process, pyomelanin pigment producer Streptomyces strain identi ed at species level via MALDI TOF/TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometer) and 16S rDNA sequence analysis.

Materials And Methods
Determination of Pigment Producer Strains.
In order to determine pigment producer micoorganisms, strains were seeded (about 1 cm 2 ) to petri dishes containing ISP2 medium and incubated at 30 °C for 96-120 h. At the end of this incubation period, colonies that form dark colored extracellular pigment around it, were selected for pyomelanin pigment production (El-Naggar and El-Ewasy, 2017).

Identi cation of strains by MALDI TOF/TOF MS
Species identi cation of pyomelanin pigment producer Streptomyces strain was carried out with MALDI TOF/TOF MS (Bruker Daltonics, Auto ex Speed). For this purpose, mass signals from most abundant and conserved ribosomal protein fractions that are speci c at genus, species or sub-group levels was detected. MALDI-TOF MS log (score) values range between 2.3-3.000 was interpreted as highly probable species-level identi cation and values range between 2.000-2.299 was interpreted as secure genus identi cation and probable species identi cation. For the extraction of ribosomal protein, formic acid method which was applied by Bizzini et al. (2010) was used and samples were prepared for MS analysis.
The samples taken from a single bacterial colony using a sterile wood applicator were transferred to the steel target plate (Ground Steel Target, Bruker Daltonics). Transferred microorganisms were overlaid with 1.0 μL of a saturated HCCA matrix (a solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile -2.5% tri uoroacetic acid -CAS Number 28166-41-8). To allow co-crystallization, the sample was kept for drying at room temperature (Bizzini et
Pyomelanin particles was scanned at 4000-400 cm -1 and spectrum recorded. FT-IR spectra is shown in In this study, puri ed extracellular pyomelanin pigment from Streptomyces griseus MPPS2 strain was analyzed by X ray diffractometer (Fig. 1). Additionally, Fourier-transform infrared spectroscopy (Fig. 2), 1 H Nuclear magnetic resonance (Fig. 3) and UV-vis spectrometry (Fig. 4) spectrums interpreted by comparing it with previously published data. In addition to these analyzes, the pyomelanin pigment producer Streptomyces strain was characterized at the species level by 16S rDNA sequence analysis and Matrixassisted laser desorption ionization time-of-ight (MALDI-TOF) mass spectrometry techniques. As a result of the analyzes, the pyomelanin-producing bacterium was identi ed as Streptomyces griseus at the species level and 16S rDNA sequence analysis result was deposited in NCBI under accession number MT825616.
Initially, dark colored pyomelanin pigment was scanned by using the Cu Kα radiation (λ=1.5406 Å). XRD Secondly we used FT-IR spectroscopy to further identify the structure of the puri ed pyomelanin polymer. When the obtained FT-IR spectra in this study were compared with the previously performed studies, it was observed that IR spectra were highly consistent and compatible (Tarangini and Mishra, 2014; El-Naggar and El-Ewasy, 2017; Li et al. 2017). Bacterial pyomelanin pigment showed strong absorption at 3276.37 cm -1 and this stretching vibration band is attributed to O\\H stretch. After that, a weak signal at 2918.75 cm −1 can be assigned as the C-H stretching band. The spectrum shows an absorption peaks at 1614.14 cm -1 was attributed to the NH bending. The broad infra-red absorption peak in the region between 3600 cm -1 and 2400 cm -1 attribute to the stretching vibrations of phenolic, carboxylic and aromatic amino functional groups present in the indole and pyrrole core structures. The band at around 1614.14 cm -1 in Fig. 2 is attributed to bending of secondary N-H group. Additionaly, IR band near 1521 cm -1 indicates the the presence of indole structure in the pyomelanin pigment. Another infra-red absorption peak about 1443 cm -1 seen in Figure 2 is also characteristic of melanin pigments (CH 2 -CH 3 bending). Another characteristic IR absorption peak around 1210 cm -1 and this IR peak is generally attributed to the phenolic COH stretch in previously performed studies. In this study, obtained FT-IR spectra re ects the characteristic absorption peaks and band stretching vibrations of melanins. As a result of the interpretation of this data and comparison with the previous literature, we can state that the puri ed pigment is pyomelanin pigment. (El-Naggar and El-Ewasy, 2017; Bayram et al. 2020;Li et al. 2017).
In addition to these data, 1 H NMR analysis of the puri ed pyomelanin pigment was performed and spectrum of the puri ed pyomelanin pigment in DMSO-d6 was measured using a 400 MHz NMR Spectrometer (Bruker AVANCE III) at 25 °C (9 T). The puri ed pyomelanin pigment is sparingly soluble in DMSO-d6. Since melanin pigment is a heterogeneous polymer, di culties have been experienced in obtaining NMR spectra but it has been observed that NMR spectra are substantially similar to the The absorbance values obtained from UV-vis spectrophotometric measurement show that the puri ed pyomelanin pigment has high absorbance values in the UV region and reaches the maximum level, especially between 250-280 nm wavelengths. (Fig. 4). These obtained data overlap with previously published studies (Li et al. 2017).
In conclusion, when XRD diffractograms and FT-IR, 1 H NMR, UV-vis spectra are examined, it is observed that the pyomelanin pigment has a high similarity with the data published previously in the literature and Based on these results, it can be stated that the puri ed pyomelanin pigment has the potential to be used in cosmetic products due to its high level of UV absorption. In addition to these results, the chemical and physical properties of melanin pigments should be investigated more deeply and thus the structure of this enigmatic pigment should be further clari ed.

Conclusion
The obtained analysis results show that the pyomelanin pigment puri ed from Streptomyces griseus MPPS2 strain has a highly similar structure to eumelanin pigment. It was observed that all obtained analysis results are compatible with the literature however, due to the heterogeneous polymer structure of pyomelanin pigments, it could not be obtained regular and clear spectra in repeated 1 H NMR analyzes.
Also, in the research results published before, it is seen that the 1 H NMR spectra are not clear in the literature. In order to strengthen and support these results, it was planned to characterize melanin pigments using pyrolysis GC-MS in the future researchs.
As a result, based on these analysis results, it can be stated that puri ed pyomelanin pigment has the potential of use in different areas such as medicine, pharmacology and cosmetics. In addition to these properties, the amorphous semiconductor, X-ray and γ-ray absorbing properties of pyomelanin polymers should be studied in depth and their potential for use in nanocomposite and biocomposite material production should be investigated in detail.
Con ict of interest disclosure: The author declare no con ict of interest.