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

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-Mn chemical autoxidation (Pyo yield 0.317 g/g substrate), (2) an induced bacterial culture of Halomonas titanicae through the 4-hydroxyphenylacetic acid-1-hydroxylase route (Pyo, 0.55 g/L), and (3) a process using a recombinant laccase extract with the highest level produced (Pyo, 1.25 g/g substrate) and all the criteria for a large-scale prototype. The chemical structures had been investigated by C solid-state NMR (CP-MAS) and FTIR. C-C bindings predominated in the three polymers, C-O-C (ether) linkages being absent, proposing mainly C-C (α-bindings) and C-C (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, Pyo exhibited an M of 5,400 Da, was hyperthermostable, non-cytotoxic even after irradiation, scavenged ROS induced by keratinocytes, and had a highly DPPH-antioxidant and Fe-reducing activity. As a representative pigment of living cells and an available standard, Pyo might also be useful for applications in extreme conditions and skin protection.