Production and properties of non-cytotoxic pyomelanin by laccase and comparison to bacterial and synthetic pigments

Abstract

Pyomelanin is a polymer of homogentisic acid synthesized by microorganisms. This work aimed to develop a production process and evaluate the quality of the pigment. Three procedures have been elaborated and optimized, (1) an HGA-Mn2+ chemical autoxidation (PyoCHEM yield 0.317 g/g substrate), (2) an induced bacterial culture of Halomonas titanicae through the 4-hydroxyphenylacetic acid-1-hydroxylase route (PyoBACT, 0.55 g/L), and (3) a process using a recombinant laccase extract with the highest level produced (PyoENZ, 1.25 g/g substrate) and all the criteria for a large-scale prototype. The chemical structures had been investigated by 13C solid-state NMR (CP-MAS) and FTIR. Car–Car bindings predominated in the three polymers, Car–O–Car (ether) linkages being absent, proposing mainly C3-C6 (α-bindings) and C4-C6 (β-bindings) configurations. This work highlighted a biological decarboxylation by the laccase or bacterial oxidase(s), leading to the partly formation of gentisyl alcohol and gentisaldehyde that are integral parts of the polymer. By comparison, PyoENZ exhibited an Mw of 5,400 Da, was hyperthermostable, non-cytotoxic even after irradiation, scavenged ROS induced by keratinocytes, and had a highly DPPH-antioxidant and Fe3+-reducing activity. As a representative pigment of living cells and an available standard, PyoENZ might also be useful for applications in extreme conditions and skin protection.

The Polymerization of Homogentisic Acid In Vitro as a Model for Pyomelanin Formation

“…The product of the polymerization of HGA (Figure 1) was studied in the absence (control) or presence of enzymes (a laccase, [31,36] peroxidase, or tyrosinase) at either pH 5 or 7.4 with a view to understand the potential role of enzymes and pH on the formation of ochronotic pigment (Figure 2).…”

New insights and advances on pyomelanin production: from microbial synthesis to applications

“…Accurate identification is achieved by their respective retention time, UV–visible spectra, and m/z mass data, over all standards available and spectra from the databank (NIST or Wiley). Cultures must be induced by appropriate substrates assayed separately at concentrations not exceeding 1–5 mM in the medium: (i) L-Tyr or even tyrosol, a phenolic strongly accumulated in olive-wastewaters, for the global identification of the intermediates at different times, (ii) 4-HPP (route 1 or 2), (iii) 4-HPA (route 2b, 3, or 3’), and (iv) HPA isomers (HPA, 2-HPA, and 3-HPA) for other HGA routes and through the respective hydroxylases involvement (Liebgott et al., 2007–2009; Lorquin et al., 2021 ; Turick et al., 2009 ) (Fig. 1 ).…”

“…1 ). HGA and pyomelanin were shown to increase in the presence of an excess of 4-HPA and 4-tyrosol during the growth of Halomonas titanicae and Halomonas Olivaria , respectively, these two bacteria being unable to grow in the presence of L-Tyr (Liebgott et al., 2008 ; Lorquin et al., 2021 ). Identical strategies had also been used in fungi and yeasts (Almeida-Paes et al., 2017 ; Schmaler-Ripcke et al., 2009 ).…”

“…Indeed, alkaline-H 2 O 2 oxidation of L-Dopa melanin led to the identification of four possible degradation products, pyrrole-2,3-dicarboxylic acid, pyrrole-2,5,5-tricarboxylic acid, thiazole-4,5-dicarboxylic acid, and thiazole-2,3,5-tricarboxylic acid (d'Ischia et al., 2013 ; Wakamatsu et al., 2003 ). Such hydrolysis treatments failed for pyomelanin structure determination (Lorquin et al., 2021 ). A few researchers also tried to characterize pyomelanin by MALDI-ToF and ESI-MS-MS (tandem) mass spectrometry but gave inaccurate molecular weight and unknown fragments (Roberts et al., 2015 ; Singh et al., 2018 ).…”

Legionella pneumophila—Virulence Factors and the Possibility of Infection in Dental Practice

“…The brown pigment, or pyomelanin, is one of the frequently studied secreted factors. It is a polymer formed with homogenetic acid (HGA), produced by bacteria with oxygen [68]. The lly gene is necessary for its formation [69].…”