Homogentisic Acid and Gentisic Acid Biosynthesized Pyomelanin Mimics: Structural Characterization and Antioxidant Activity

Abstract

Pyomelanin mimics from homogentisic acid (HGA) and gentisic acid (GA) were biosynthesized by the oxidative enzyme T. versicolor laccase at physiological pH to obtain water soluble melanins. The pigments show brown-black color, broad band visible light absorption, a persistent paramagnetism and high antioxidant activity. The EPR approach shows that at least two different radical species are present in both cases, contributing to the paramagnetism of the samples. This achievement can also shed light on the composition of the ochronotic pigment in the Alkaptonuria disease. On the other hand, these soluble pyomelanin mimics, sharing physico-chemical properties with eumelanin, can represent a suitable alternative to replace the insoluble melanin pigment in biotechnological applications.

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

“…In some pyomelanin samples, a clear semiquinone signal was reported in the literature. [ 31 ] The absence of this signal in our samples can be explained by the small contribution of the semiquinone radical to the overall EPR signal. The spectra recorded at high power, however, suggested the presence of some EPR‐active metals in all enzyme‐derived samples, possibly originating from the traces of residual enzymes (supported by EDX data; see Figures S16–S23 in the Supporting Information).…”

“…Peaks at ≈1200–1210 cm –1 corresponding to phenolic OH and at ≈1560–1570 cm –1 corresponding to the C═O bond of the carboxylic acid functional groups are present in all spectra, albeit broader. [ 31 ]…”

“…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).…”

“…Peaks at ≈1200-1210 cm -1 corresponding to phenolic OH and at ≈1560-1570 cm -1 corresponding to the C═O bond of the carboxylic acid functional groups are present in all spectra, albeit broader. [31] EPR spectroscopy was used to study the powders. X-band EPR spectra of all polyHGA powders showed nearly identical spectra (Figure 7).…”

“…Here we report the results of an investigation of the polymerization of HGA to form polyHGA (a simplified version of pyomelanin, Figure ) in the absence or presence of enzymes (a laccase (LACC), [ 31 ] peroxidase (horseradish peroxidase (HRP)), or tyrosinase (TYR)) at either pH 5.0 or 7.4. A selection of different analytical techniques was applied to study the resultant pyomelanins, including UV–vis spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, X‐ray photoelectron spectroscopy (XPS), size exclusion chromatography (SEC), dynamic light scattering (DLS), scanning electron microscopy (SEM), energy‐dispersive X‐ray (EDX) spectroscopy, Fourier‐transform infrared (FTIR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, cyclic voltammetry (CV), and conductive probe atomic force microscopy (C‐AFM).…”