Direct Spectrophotometric Assay of Laccase Using Diazo Derivatives of Guaiacol

Abstract

The effects of 12 common metal ionic compounds on the laccase catalytic activity in reactions using guaiacol as the substrate was determined using spectrophotometry. Furthermore, the influence of several metal ionic compounds on the generation of reactive oxygen species (ROS) by oxidation of lignin in jute fiber under laccase catalysis was studied by electron paramagnetic resonance (EPR) spectroscopy using N-tert-butyl-alpha-phenylnitrone (PBN) as the spin-trapping agent. Common metal cations, such as K + , Na + , Mg 2+ , Ca 2+ , and Cu 2+ and the anion SO4 2-had almost no effect on laccase activity during the initial stage of the catalytic reactions. High concentrations of the Mn 2+ ion exhibited weak inhibition of laccase; Ag + and NO3-showed a moderate inhibitory effect on laccase activity during the initial stage of the catalytic reactions. Fe 2+ had no direct effect on the binding of laccase to its substrate, but strongly retarded the progress of the catalytic reaction by reducing the intermediate free radicals. The ions Cl-, Fe 3+ , and Ag + exhibited either strong inhibitory effects on the catalysis of the substrate or a destructive effect on the structure of laccase itself. Furthermore, the results showed that an appropriate concentration of Cu 2+ helped to promote the thermal stability of laccase during the enzymatic reaction. This study could help researchers to avoid the use of inhibitory exogenous metal ions and anions in the application of laccase and to maximize the value of laccase.

Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities

“…Previous studies showed that molecules carrying substituents at ortho M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 position to the binding terminal were poor inhibitors [10,20]. We also showed that guaiacols are not oxidized by MT, assumingly due to the spatial hindrance created by the methoxy group [31]. PA and CA have both benzene ring and CAFG in their structures, but neither serves as a substrate for MT.…”

New enzymatic time–temperature integrator (TTI) that uses laccase

“…To date, the kinetics of laccase with guaiacol as a substrate have been studied by measuring the increase in absorbance at a wavelength of 470 nm, at which the oxidized form of the substrate shows the maximum absorption (Moshtaghioun et al, 2011;Manole et al, 2008). Considering that TTI monitoring is designed to provide a history of time-temperature along the food distribution chain for perishable products such as vegetables, meats, and dairy products, the color change should be easily measurable so that the amount of temperature abuse the product has been subjected to can be easily determined.…”

“…In some cases, laccases enhance the color formation by reaction with syringaldazine, hydroquinone, vanillic acid, tannic acid (Manole et al, 2008) and Diazo derivatives of guaiacol (Moshtaghioun et al, 2011). In the food industry, laccase is also used to modify, enhance, or change colors for foods and beverages to indicate safety (Minussi et al, 2002).…”

Laccases in the Context of Potentially Cooperating Enzymes

“…Since the structural skeleton of the examined substrates were the same and the enzymatic reactions were monitored directly through the consumption of the substrates, from the decrease in the substrate optical density at its λ max and not the formation of products, the changes in the kinetics could be solely ascribed to the effect of the substituents. Results of this study clearly showed the instant rates of the reactions (ÀdA/min) had the trend of -OCH 3 > -CH 3 % -H > -SO 2 NH 2 > -Cl, while the V max(s) of these substrates were in the same range (Moshtaghioun et al 2011). This result could be explained based on the radical nature of laccase reaction as the electron demand of organic radicals varies dramatically with the structure and induction effect of a substituent.…”

“…To examine the electronic effect of a substituent, Moshtaghioun et al (2011) studied oxidation of some synthetic diazo derivatives of guaiacol (S1, Fig. 2) by the extracellular Neurospora crassa laccase.…”