Kinetic evidence for the interactive inhibition of laccase from Trametes versicolor by pH and chloride.

The interactive inhibitory effects of pH and chloride on the catalysis of laccase from Trametes versicolor were investigated by studying the alteration of inhibition characteristics of sodium chloride at different pHs for the oxidation of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid). At pH 3.0, the addition of sodium chloride (50 mM) brought about a 40-fold increase in Km(app) and a 4-fold decrease in Vmax(app). As the pH increased to 7.0, the inhibitory effects of sodium chloride became significantly weakened. The mixed-inhibition mechanism was successfully used to quantitatively estimate the competitive and uncompetitive inhibition strengths by chloride at two different pHs (pH 3.0 and 6.0). At pH 3.0, the competitive inhibition constant, Ki, was 0.35 mM, whereas the uncompetitive inhibition constant, Ki', was 18.1 mM, indicating that the major cause of the laccase inhibition by chloride is due to the competitive inhibition step. At a higher pH of 6.0, where the inhibition of the laccase by hydroxide ions takes effect, the inhibition of the laccase by chloride diminished to a great extent, showing increased values of both the competitive inhibition constant (Ki= 23.7 mM) and uncompetitive inhibition constant (Ki' = 324 mM). These kinetic results evidenced that the hydroxide anion and chloride share a common mechanism to inhibit the laccase activity.

Partial uncompetitive inhibition of horseradish peroxidase by a water-miscible ionic liquid [BMIM][MeSO4].

The ionic liquid, 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MeSO(4)]), was used to investigate the catalytic mechanism of horseradish peroxidase (HRP). The ionic liquid decreased both K(m) and k(cat) values for the HRP-catalyzed oxidation of guaiacol (2-methoxyphenol) by H(2)O(2). These studies imply that [BMIM][MeSO(4)] inhibits the enzyme in an uncompetitive manner. The incorporation of substrate stabilization effects measured by a thermodynamic method into the partial uncompetitive inhibition scheme successfully describes HRP-catalysis in the presence of [BMIM][MeSO(4)], which participates as the inhibitor. The inhibition constant of the ionic liquid was 0.051 M. The turn-over number of the native HRP was almost 14-times higher than that of the HRP-ionic liquid complex indicating that [BMIM][MeSO(4)] does not form a dead-end complex with HRP.

Strong substrate-stabilizing effect of a water-miscible ionic liquid [BMIM][BF(4)] in the catalysis of horseradish peroxidase.

The effects of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF(4)]), on both thermodynamics and kinetic mechanism of the horseradish peroxidase (HRP)-catalyzed oxidation of guaiacol (2-methoxyphenol) by H(2)O(2) were investigated. The ionic liquid stabilized the ground state of guaiacol by causing an 8-fold increase of K(m) from 3 to 23 mM upon the addition of 25% (v/v) [BMIM][BF(4)]. In addition, the effect of [BMIM][BF(4)] in decreasing the k(cat) value of HRP catalysis was described by a non-competitive inhibition mechanism. The value of the inhibition constant of [BMIM][BF(4)] was 2.9 M indicating that the ionic liquid plays the role of a weak non-competitive inhibitor for HRP catalysis.

Effect of ionic liquid on the kinetics of peroxidase catalysis.

The effect of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), on the horseradish peroxidase (HRP)-catalyzed oxidation of 2-methoxyphenol (guaiacol) with hydrogen peroxide (H2O2) was investigated. HRP maintains its high activity in the aqueous mixtures containing various concentrations of the ionic liquid and even in 90% (v/v) ionic liquid. In order to minimize the effect of solution viscosity on the kinetic constants of HRP catalysis, the enzymatic reactions in the subsequent kinetic study were performed in water-ionic liquid mixtures containing 25% (v/v) ionic liquid at maximum. As the concentration of [BMIM][BF4] increased for the oxidation of guaiacol by HRP, the K(m) value increased with a slight decrease in the k(cat) value: The K(m) value increased from 2.8 mM in 100% (v/v) water to 22.5 mM in 25% (v/v) ionic liquid, indicating that ionic liquid significantly weakens the binding affinity of guaiacol to HRP.

Immobilization of horseradish peroxidase on multi-wall carbon nanotubes and its electrochemical properties.

Horseradish peroxidase (HRP) was immobilized on carboxylated multi-wall carbon nanotubes in the presence of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The immobilized HRP maintained its oxidative activity for guaiacol over a broad range of pH values (4-9). An electrode of graphite rod, 6 mm diam. was fabricated using the immobilized HRP. Cyclic voltammetry of the enzyme electrode confirmed electron transfer between the immobilized HRP and the electrode in the presence of H2O2 but without an added mediator or a reducing substrate.