1-Hydroxybenzotriazole, violuric acid, and N-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an "outer-sphere" type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation.Laccases (EC 1.10.3.2) are multi-Cu oxidases that can catalyze the oxidation of a range of reducing substances with the concomitant reduction of O 2 (for recent reviews, see reference 24 and references therein). Because of their capability of catalyzing the oxidation of aromatic compounds, laccases are receiving increasing attention as potential industrial enzymes in various applications, such as pulp delignification, wood fiber modification, dye or stain bleaching, chemical or medicinal synthesis, and contaminated water or soil remediation (15, 37).Laccases contain one type 1 (T1) Cu center, one type 2 (T2) Cu center, and one type 3 (T3) Cu center. The T2 and T3 sites form a trinuclear Cu cluster onto which O 2 is reduced. The T1 Cu oxidizes the reducing substrate and transfers electrons to the T2 and T3 Cu. Laccase is able to oxidize certain phenols with E 0 values higher than its own (0.5 to 0.8 V versus the normal hydrogen electrode [NHE]) (36). However, many inorganic and organic compounds with comparable E 0 values (such as 1,2,3,5-tetramethoxybenzene [18]) are not laccase substrates due to unfavorable kinetics. Under certain conditions, however, these compounds can be indirectly oxidized by laccase through the mediation of small, redox-active laccase substrates. 2,2Ј-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was the first compound found capable of efficiently mediating the laccase oxidation of high-E 0 , nonsubstrate lignin model compounds (such as veratryl alcohol and nonphenolic lignin model dimers) (8). Based on product structure analysis, it has been proposed that laccase-oxidized ABTS can abstract an H atom from the lignin model compounds, leading to indirect laccase catalysis upon the oxidation of the compounds (25). To date, other types of mediators, particularly phenoxazines and N-OH compounds, also have been recognized for their mediation function in laccase catalysis (1, 6,17,29).Mediated laccase catalysis has been applied to a wide range of applications, such as pulp delignification (9, 10, 12, 22, 32), textile dye bleaching (31), polycyclic aromatic h...

Comparison of conventional and newly developed laccase-based electrocatalytic systems for oxygen reduction.

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A mathematical model of amperometric biosensors has been developed. The model is based on non-stationary diffusion equations containing a non-linear term related to Michaelis-Menten kinetics of the enzymatic reaction. Using digital simulation, the influence of the thickness of enzyme membrane on the biosensor response was investigated. The digital simulation of the biosensor operation showed the non-monotonous change of the maximal biosensor current versus the membrane thickness at the various maximal enzymatic rates. Digital simulation was carried out using the finite difference technique. Results of the numerical simulation was compared with known analytical solutions. This paper presents a framework for selection of the membrane thickness, ensuring the sufficiently stable sensitivity of a biosensor in a required range of the maximal enzymatic rate.

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