Abstract

Polyphenol oxidases (PPOs) oxidize phenolic compounds forming quinones, which undergo non-enzymatic reactions resulting in colored compounds. We studied ferulic acid and catechol oxidation by Myceliophthora thermophila laccases using oxidoreduction potential (ORP or Eh) as a novel method to determine PPO activity compared to the traditional spectrophotometric method. Eh varied time-dependently on ferulic acid and catechol concentrations. Eh increased to a maximum value (Eh_max), then decreased irreversibly proportional with substrate concentration. The ΔEh curves of ferulic acid and catechol oxidation at different concentrations by laccase exhibited two steps substrate oxidation: enzymatic and non-enzymatic; linear segments with increasing and decreasing slopes (rate of ΔEh change). The Km values determined spectrophotometrically and ORP were 0.55, 0.75 and 0.39, 0.32mM for catechol and ferulic acid, respectively. The kinetic parameters (K_m, V_max) of enzymatic reaction were not significantly different between the spectrophotometry and ORP methods. The ORP method is simple, low-cost and fast enabling the differentiation between the enzymatic and nonenzymatic phenol oxidation reactions.

Keywords: Myceliophthora thermophila laccases; Polyphenol oxidase; Enzyme kinetic; Oxidoreduction potential; Phenolic compound

Abbreviations: ADA: 4 Amino N, N-Diethyl Aniline Sulphate; KM: Michaels Menten Kinetics Constants; L-DOPA: L-3,4 Dihydroxyphenylalanine; LACs: Laccase; ORP: Oxidoreduction Potential; PPOs: Polyphenol Oxidizes; S: Substrate; V: Reaction velocity; Vmax: Maximum velocity