Biotechnological Production and Characterization of Extracellular Melanin by Streptomyces nashvillensis
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ABSTRACT:
SUBMITTER: Restaino O
PROVIDER: S-EPMC10892051 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:A novel metal ion-independent phospholipase A1 of Streptomyces albidoflavus isolated from Japanese soil has been purified and characterized. The enzyme consists of a 33-residue N-terminal signal secretion sequence and a 269-residue mature protein with a deduced molecular weight of 27,199. Efficient and extracellular production of the recombinant enzyme was successfully achieved using Streptomyces lividans cells and an expression vector. A large amount (25 mg protein, 14.7 kU) of recombinant enzyme with high specific activity (588 U/mg protein) was purified by simple purification steps. The maximum activity was found at pH 7.2 and 50 °C. At pH 7.2, the enzyme preferably hydrolyzed phosphatidic acid and phosphatidylserine; however, the substrate specificity was dependent on the reaction pH. The enzyme hydrolyzed lysophosphatidylcholine and not triglyceride and the p-nitrophenyl ester of fatty acids. At the reaction equilibrium, the molar ratio of released free fatty acids (sn-1:sn-2) was 63:37. The hydrolysis of phosphatidic acid at 50 °C and pH 7.2 gave apparent V max and k cat values of 1389 μmol min(-1) mg protein(-1) and 630 s(-1), respectively. The apparent K m and k cat/K m values were 2.38 mM and 265 mM(-1) s(-1), respectively. Mutagenesis analysis showed that Ser11 is essential for the catalytic function of the enzyme and the active site may include residues Ser216 and His218.
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Project description:Pigments are compounds of importance to several industries, for instance, the food industry, where they can be used as additives, color intensifiers, and antioxidants. As the current trend around the world is shifting to the use of eco-friendly commodities, demand for natural dyes is increasing. Melanins are pigments that are produced by several microorganisms. Pseudomonas putida ESACB 191, isolated from goat cheese rind, was described as a brown pigment producer. This strain produces a brown pigment via the synthetic Müeller-Hinton Broth. This brown compound was extracted, purified, analyzed by FTIR and mass spectrometry, and identified as eumelanin. The maximum productivity was 1.57 mg/L/h. The bioactivity of eumelanin was evaluated as the capacity for scavenging free radicals (antioxidant activity), EC50 74.0 ± 0.2 μg/mL, and as an acetylcholinesterase inhibitor, with IC50 575 ± 4 μg/mL. This bacterial eumelanin did not show cytotoxicity towards A375, HeLa Kyoto, HepG2, or Caco2 cell lines. The effect of melanin on cholesterol absorption and drug interaction was evaluated in order to understand the interaction of melanin present in the cheese rind when ingested by consumers. However, it had no effect either on cholesterol absorption through an intestinal simulated barrier formed by the Caco2 cell line or with the drug ezetimibe.
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Project description:Extracellular vesicle(EV) secretion and capture is an important method of communication between cells. Melanocytes and their surrounding keratinocytes form epidermal melanin units in the thin, outermost layer of the skin to protect the skin from ultraviolet radiation damage. It is important to understand the mechanisms by which changes in melanocyte EV signal transduction influence pigmentation. Mouse melanocytes (melan-α cell) overexpressing MITF and CD63-GFP were used to to investigate the effect on vesicle formation by WB and SEM. Coculturing of melanocyte with keratinocyte(PAM212) was used to explore the polarization of vesicles and transfer between cells by immunofluorescence, flow cytometry. EVs from melanocytes were extracted and used to investigate changes in the miRNA profile of EVs, and the effect of exosomal miR-28 on keratinocyte growth and transcriptomics was analyzed by flow cytometry, TEM, particle size analysis, WB, the iCELLigence system and RNA-seq analysis.Our results indicate that melanocytes in the skin affected vesicle synthesis under the condition of enhanced melanin synthesis function: the expression of CD81, VPS37, VPS36B and Rab27b (melanosome and vesicle transport protein during ESCRT machinery) increased. Keratinocytes can cause the polarization of vesicles in melanocytes and capture EVs from melanocytes via plasmalemma fusion. Analysis of the miRNA profile derived from melanocyte EVs showed that the expression levels of miR-125, miR-130, miR-211, miR-127, miR-692 and miR-709 were increased, while the expression levels of miR-28, miR-744, let-7g and miR-6240 were decreased. The content of miR-28 in EVs may be reduced to induce keratinocytes to prevent carcinogenesis, resist infection and prevent premature apoptosis.
2022-03-23 | GSE199035 | GEO
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