Graphical abstract

Various aromatic compounds that are structurally analogous to lignin were tested as possible/preferred substrates for purified laccase from newly isolated white rote fungi, Trametes polyzona WRF03. The pH optima were tested using different substrates and kinetic studies were conducted at these pH optima. The pH optima in the presence of ABTS, α-naphthol, o-dianisidine, and catechol were 4.5 but 5.0 and 5.5 in the presence of guaiacol and pyrogallol, respectively. The initial velocities obtained from the kinetic study were analyzed using Graph Pad Prism 7 and Lineweaver-Burk plot to obtain kinetic constants ( k m and Vmax ) which were used to calculate substrate specificity. Amongst all the substrates tested, ABTS had the highest specificity-constant (181.51 M −1 s −1 ), and therefore, the most preferred substrate was followed by α-naphthol, o -dianisidine, guaiacol, pyrogallol, and catechol. Resorcinol, orcinol, and veratryl alcohol did not display any considerable chemical shift in the presence of Trametes polyzona WRF03 laccase. Also, oxidation of phenolic substrates appeared to be dependent on the nature of the substituent groups and their relative position on the aromatic nucleus. Since most of these substrates are structural analogs of lignin and many recalcitrant environmental pollutants, the enzyme may find application in delignification, treatment of wastewater containing dyes, and polycyclic aromatic hydrocarbons (PAHs).

The effect of thermal treatment on the activity of laccase from Trametes polyzona WRF03 was studied at pH and temperature ranges of 3.0 to 6.5 and of 40 to 70 oC respectively. Kinetic data revealed that the heat inactivation of Trametes polyzona WRF03 laccase (TpL) was pH dependent and followed first-order kinetics. There was a positive correlation between activation energy (Ea) for thermal inactivation of TpL and the reaction pH. Highest activation energy, Ea, value of 175.49 kJ/mol was obtained at pH 6.0. On the contrary, the z-value decreased with a lowest value of 12.37 oC at pH 6.0. The high Ea value and low z-value were indicative of the thermo-stable nature of TpL which suggests that pH 6.0 had a compensatory stabilizing effect on TpL against its thermal denaturation. There was a gradual decrease in the enthalpy of denaturation (∆Ho) and Gibb’s free-energy with every 10 % rise in temperature within the investigated pH range, suggesting that TpL was more stable at 40 oC. Positive values of entropy of inactivation (ΔSº) at each temperature indicated that there was no aggregation during the inactivation processes. Thus, these results provided useful information about the behaviour of TpL under certain pH and temperature combination with respect to biotechnological application. Thus, the kinetic and thermodynamic data could be used to design a model to predict the thermal inactivation of TpL during industrial application.