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The screening of potential redox mediators for laccase was performed using homogeneous Trametes hirsuta laccase. Heterogeneous (electrochemical) and homogeneous (oxidation by laccase) reactions of the different types of the enhancers (mediators) of the enzyme were investigated. It was discovered that derivatives of phenyl-methyl-pyrazolones and benzoic acid, as well as N-hydroxynaphthalimide were efficient substrates for the laccase. The characterization of several representatives from each class was carried out using electrochemical and enzyme kinetics methods. The kinetic parameters for the oxidation of phenyl-methyl-pyrazolones and 3-(6-hylroxy)-aminobenzoic acid were comparable to those for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by the laccase, whereas the rate of enzymatic oxidation of N-hydroxynaphthalimide was sufficiently lower. Electrochemical experiments demonstrated that only oxidation of phenyl-methyl-pyrazolones and N-hydroxynaphthalimide yielded several high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, whereas derivatives of benzoic acid showed low-potential intermediate, which was not able to oxidized lignin model compound. Phenyl-methyl-pyrazolones was about 50% as effective in degrading veratryl alcohol compared to ABTS as judged from HPLC kinetic studies, whereas N-hydroxynaphthalimide showed the same efficiency as ABTS. Phenyl-methyl-pyrazolones and hydroxynaphthalimides may be of commercial interest for oxidoreductase-catalyzed biodegradation of different xenobiotics.

The reliable detection of a superoxide anion radical O2(*-) is complicated by its spontaneous dismutation reaction to H2O2 at acidic pHs. To simultaneously detect both O2(*-) and H2O2 produced in the course of its spontaneous dismutation, an electrochemical two-electrode system based on cytochrome c (cyt c) and recombinant horseradish peroxidase (rHRP) was applied. Therewith, a limited applicability of the cyt c system for the reliable monitoring of O2(*-) in acidic and neutral solutions was shown. It was demonstrated that both the reaction of O2(*-) dismutation to H2O2 and the reaction between the formed H2O2 and O2(*-) chemically decrease the amount of the initially present O2(*-), decreasing the sensitivity and reliability of the electrochemical detection at acidic pH. However, by appropriately varying solution pH, the concentration of O2(*-) initially injected in the system can be estimated from the analysis of calibration curves for H2O2 obtained with highly sensitive rHRP-modified electrode system at pH 6.0 and 7.0.

The tryptophanless mutant of horseradish peroxidase, W117F, has been constructed and expressed in Escherichia coli. The mutation affects enzyme folding and stability. The optimum composition of the refolding medium requires the presence of ammonium sulfate. The yield of mutant is ca. 8000 U per liter of the optimized refolding medium with the specific activity of 1100-1500 U/mg (compared to 25, 000 U per liter and 2000 U/mg for the recombinant wild-type enzyme). The mutant is more stable in acid media, in the reaction course and toward irradiation. The effect of hydrogen peroxide pretreatment on radiation-induced inactivation of the wild-type and mutant enzyme indirectly indicates participation of Trp-117 in electron transfer pathways through the enzyme molecule. This is in agreement with the steady-state kinetic data interpreted in terms of Trp-117 participation in electron transfer within the Michaelis complex., (Copyright 1999 Academic Press.)

The tobacco anionic peroxidase has been isolated from the leaves of transgenic Nicotiana sylvestris plants overproducing this enzyme. The plant expression system and the purification protocol developed allow the preparation of greater than 60 mg of homogeneous enzyme (M(r) 36 kDa, pI 3.5) from 1 kg of fresh leaves, which is an order of magnitude higher than for wild-type tobacco plants. The tobacco anionic peroxidase exhibits rather unusual catalytic properties in comparison with horseradish peroxidase (HRP C). Compound I is less active than Compound II in the tobacco enzyme. The enzyme is nearly inactive towards iodide, reflecting the peculiarities of its molecular structure. In particular, the presence of the negatively charged glutamate residue 141 at the entrance of the haeme-binding pocket seems to affect the stabilities of Compounds I, II and III, leading to a different enzyme substrate specificity than that of HRP C. Investigation of thermal stability towards a number of electron donors reveals the following 'order of stabilities': ferrocyanide > guaiacol > 2,2'-azino-bis(3-ethyl-6- benzothiazoline sulphonate) > iodide > o-dianisidine, which may indicate different binding sites and rate-limiting steps in the mechanism of the substrate oxidation.