Redox chemistry in laccase-catalyzed oxidation of N-hydroxy compounds

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.     

Redox Chemistry in Laccase-Catalyzed Oxidation of N-Hydroxy Compounds

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.     

Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound.

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3, 4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both kcat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect kcat and the oxidation rate of dimer I.     

Structure/redox potential relationship of simple organic compounds as potential precursors of dyes for laccase-mediated transformation

The aim of this study was to examine the ability of an extracellular fungal laccase (LAC) to form colored products from simple non-colored organic precursors. Thirty different phenolic and non-phenolic precursors (o-, m-, and p-methoxy-, hydroxy-, sulfonic-, and amino-substituted) were tested as single and coupled substrates in a LAC-catalyzed oxidation. The findings show that LAC catalyzes the formation of colored products (from yellow/brown to red and blue) by oxidation of single substrates that are benzene derivatives containing at least two substituents comprised of amino, hydroxy, and methoxy groups. All precursors were tested by cyclic voltammetry and the correlation between their structure and redox potential, and the possibility of their transformation into colored products by fungal LAC was found. Colored products were yielded from single substrates possessing a value of the oxidation peak (E(o)) lower than 1,150 mV vs. normal hydrogen electrode (NHE). Substrates with an oxidation peak higher than 1,150 mV vs. NHE were transformed by LAC into colored compounds only in the presence of an additional precursor characterized by a low value of E(o) and the presence of reactive substituents such as methoxy, hydroxy, and amino groups. Therefore, additional hydroxylation, methoxylation, and amination of phenolic and non-phenolic substrates may represent a strategy to increase the range of these compounds as potential dyes precursors.     

Cleavage and synthesis function of high and low redox potential laccases towards 4-morpholinoaniline and aminated as well as chlorinated phenols

Laccases are able to mediate both cleavage and synthesis processes. The basis for this dual reaction capability lies in the property of the enzyme laccase to oxidize phenolic, and to some extent non-phenolic substances, to reactive radicals which can undergo on the one hand separations of small substitutents or large molecule parts from the parent compound and on the other hand coupling reactions with other radicals or molecules which are not themselves oxidizable by laccase. The cleavage of the non-phenolic compound 4-morpholinoaniline as well as the deamination of 4-aminophenol and the dechlorination of 4-chlorophenol resulted in the formation of 1,4-hydroquinone which is immediately oxidized by laccase to 1,4-benzoquinone. The formation of the 1,4-hydroquinone/1,4-benzoquinone is the rate limiting step for the synthesis of the heteromolecular dimers and trimers composed of 1,4-benzoquinone and one or two molecules of morpholine. In addition to the synthesis of new compounds from the cleavage products, 4-morpholinoaniline polymerized probably via azo groups and C-N bonds to a homomolecular dimer and trimer. Similarities and differences in cleavage and synthesis reactions catalyzed by the low redox potential laccase of Myceliophthora thermophila (0.46 V) and the high redox potential laccase of Pycnoporus cinnabarinus (0.79 V) were determined. In addition, the dependency of the cleavage and synthesis efficiencies on the (a) structure and redox potential of the laccase, (b) structure and redox potential of the substrate, (c) pH value of the buffer used, (d) incubation temperature, (e) solvent concentration, and (f) laccase activity is discussed in general.     

Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both k_cat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect k_cat and the oxidation rate of dimer I.     

Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation.

Two laccase isoenzymes produced by Pleurotus eryngii were purified to electrophoretic homogeneity (42- and 43-fold) with an overall yield of 56.3%. Laccases I and II from this fungus are monomeric glycoproteins with 7 and 1% carbohydrate content, molecular masses (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 65 and 61 kDa, and pIs of 4.1 and 4.2, respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for laccase I was reached at 65 degrees C and pH 4, and that for laccase II was reached at 55 degrees C and pH 3.5. Both isoenzymes are stable at high pH, retaining 60 to 70% activity after 24 h from pH 8 to 12. Their amino acid compositions and N-terminal sequences were determined, the latter strongly differing from those of laccases of other basidiomycetes. Antibodies against laccase I reacted with laccase II, as well as with laccases from Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus floridanus. Different hydroxy- and methoxy-substituted phenols and aromatic amines were oxidized by the two laccase isoenzymes from P. eryngii, and the influence of the nature, number, and disposition of aromatic-ring substituents on kinetic constants is discussed. Although both isoenzymes presented similar substrate affinities, the maximum rates of reactions catalyzed by laccase I were higher than those of laccase II. In reactions with hydroquinones, semiquinones produced by laccase isoenzymes were in part converted into quinones via autoxidation. The superoxide anion radical produced in the latter reaction dismutated, producing hydrogen peroxide. In the presence of manganous ion, the superoxide union was reduced to hydrogen peroxide with the concomitant production of manganic ion. These results confirmed that laccase in the presence of hydroquinones can participate in the production of both reduced oxygen species and manganic ions.     

Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes.

Lignin peroxidase oxidizes non-phenolic substrates by one electron to give aryl-cation-radical intermediates, which react further to give a variety of products. The present study investigated the possibility that other peroxidative and oxidative enzymes known to catalyse one-electron oxidations may also oxidize non-phenolics to cation-radical intermediates and that this ability is related to the redox potential of the substrate. Lignin peroxidase from the fungus Phanerochaete chrysosporium, horseradish peroxidase (HRP) and laccase from the fungus Trametes versicolor were chosen for investigation with methoxybenzenes as a homologous series of substrates. The twelve methoxybenzene congeners have known half-wave potentials that differ by as much as approximately 1 V. Lignin peroxidase oxidized the ten with the lowest half-wave potentials, whereas HRP oxidized the four lowest and laccase oxidized only 1,2,4,5-tetramethoxybenzene, the lowest. E.s.r. spectroscopy showed that this congener is oxidized to its cation radical by all three enzymes. Oxidation in each case gave the same products: 2,5-dimethoxy-p-benzoquinone and 4,5-dimethoxy-o-benzoquinone, in a 4:1 ratio, plus 2 mol of methanol for each 1 mol of substrate. Using HRP-catalysed oxidation, we showed that the quinone oxygen atoms are derived from water. We conclude that the three enzymes affect their substrates similarly, and that whether an aromatic compound is a substrate depends in large part on its redox potential. Furthermore, oxidized lignin peroxidase is clearly a stronger oxidant than oxidized HRP or laccase. Determination of the enzyme kinetic parameters for the methoxybenzene oxidations demonstrated further differences among the enzymes.     

Oxidation of carbazole, N-ethylcarbazole, fluorene, and dibenzothiophene by the laccase of Coriolopsis gallica

Purified laccase from Coriolopsis gallica UAMH8260 oxidized carbazole, N-ethylcarbazole, fluorene, and dibenzothiophene in the presence of 1-hydroxybenzotriazole and 2,2-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) as free radical mediators. Susceptibility to laccase oxidation appears related to the ionization potential (IP) of the substrate: compounds with an IP above 8.52, dibenzofuran (IP = 8.77) and benzothiophene (IP = 8.73) were not attacked. Carbazole (IP = 7.68) was the most sensitive to oxidation with >99% transformed with 10 milliunits of laccase after 1 h, though most reactions were carried out for 18 h. 9-Fluorenone was identified as the product of fluorene (IP = 8.52) oxidation, and dibenzothiophene sulfone from dibenzothiophene (IP = 8.44). Although carbazole and N-ethylcarbazole were both completely removed within 18 h, no oxidation or condensation metabolites were detected. This investigation is the first to report the oxidation of dibenzothiophene, carbazole, and N-ethylcarbazole by laccase.     

Redox potentials, laccase oxidation, and antilarval activities of substituted phenols

Laccases are copper-containing oxidases that are involved in sclerotization of the cuticle of mosquitoes and other insects. Oxidation of exogenous compounds by insect laccases may have the potential to produce reactive species toxic to insects. We investigated two classes of substituted phenolic compounds, halogenated di- and trihydroxybenzenes and substituted di-tert-butylphenols, on redox potential, oxidation by laccase and effects on mosquito larval growth. An inverse correlation between the oxidation potentials and laccase activity of halogenated hydroxybenzenes was found. Substituted di-tert-butylphenols however were found to impact mosquito larval growth and survival. In particular, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl)phenol (15) caused greater than 98% mortality of Anophelesgambiae larvae in a concentration of 180nM, whereas 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-methylpropanal oxime (13) and 6,8-di-tert-butyl-2,2-dimethyl-3,4-dihydro-2H-chromene (33) caused 93% and 92% mortalities in concentrations of 3.4 and 3.7μM, respectively. Larvae treated with di-tert-butylphenolic compounds died just before pupation.     

Kinetics of electron transfer between cytochrome c and laccase.

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting Rhus vernicifera laccase as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 125 M-1 s-1 at 25 degrees C in 0.2 M phosphate buffer at pH 6.0 with the activation parameters delta H++ = 16.2 kJ mol-1 and delta S++ = 28.9 J mol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that electron transfer from cytochrome c to laccase is favored by the local electrostatic interaction (ZAZB = -0.9 at pH 6 and -1.3 at pH 4.8) between the basic proteins with positive net charges. From the increase of the rate of electron transfer with decreasing pH, one of the driving forces of the reaction was suggested to be the difference in the redox potentials between the type 1 copper in laccase and the central iron in cytochrome c. Further, on addition of one hexametaphosphate anion per cytochrome c molecule, the rate of the electron transfer was increased, probably because the association of both proteins became more favorable.     

Laccase-catalysed iodide oxidation in presence of methyl syringate

The kinetics of potassium triiodide (KI(3)) formation during fungal laccase action was investigated in presence of methyl syringate (MS). The recombinant forms of Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL), and Rhizoctonia solani (rRsL) laccases were used. The triiodide formation rate reached 6.1, 5.5, 6.0, and 2.1 microM/min at saturated rPpL, rCcL, rRsL, and rMtL concentration, respectively, in acetate buffer solution pH 5.5 and in presence of 10 microM of MS and 1 mM of potassium iodide. The triiodide formation rate increased if pH decreased from 6.5 to 4.5. The scheme of laccase-catalysed iodide oxidation includes stadium of MS interaction with oxidized laccase with concomitant production of MS(ox). The reaction of MS(ox) with iodide produced triiodide. The turnover number of MS was 93 and 44 at pH 5.5 for rPpL and rMtL, respectively. The scheme also contained a stadium of reversible reduction of laccase active centre with the mediator explaining the different saturation rate of triiodide production. The fitting kinetic data revealed that the reversibility of the reaction increased for laccases containing lower redox potential of copper type I.     

Characterization of a low redox potential laccase from the basidiomycete C30.

A new exocellular laccase was purified from the basidiomycete C30. LAC2 is an acidic protein (pI = 3.2) preferentially produced upon a combined induction by copper and p-hydroxybenzoate. The spectroscopic signature (UV/visible and EPR) of this isoform is typical of multicopper oxidases, but its enzymatic and physico-chemical properties proved to be markedly different from those of LAC1, the constitutive laccase previously purified from the same organism. In particular, the LAC2 kcat values observed for the oxidation of the substrates syringaldazine (kcat = 65 600 min-1), ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (kcat = 41 000 min-1) and guaiacol (kcat = 75 680 min-1) are 10-40 times those obtained with LAC1 and the redox potential of its T1 copper is 0.17 V lower than that of LAC1 (E degrees = 0.73 V). This is the first report on a single organism producing simultaneously both a high and a low redox potential laccase. The cDNA, clac2, was cloned and sequenced. It encodes a protein of 528 amino acids that shares 69% identity (79% similarity) with LAC1 and 81% identity (95% similarity) with Lcc3-2 from Polyporus ciliatus (AF176321-1), its nearest neighbor in database. Possible reasons for why this basidiomycete produces, in vivo, enzyme forms with such different behaviors are discussed.     

Characterization of a laccase from a wood‐feeding termite,Coptotermes formosanus

Coptotermes formosanus Shiraki is a wood‐feeding termite which secretes a series of lignolytic and cellulolytic enzymes for woody biomass degradation. However, the lignin modification mechanism in the termite is largely elusive, and the characteristics of most lignolytic enzymes in termites remain unknown. In this study, a laccase gene lac1 from C. formosanus was heterogeneously expressed in insect Sf9 cells. The purified Lac1 showed strong activities toward hydroquinone (305 mU/mg) and 2,6‐dimethoxyphenol (2.9 mU/mg) with low K_m values, but not veratryl alcohol or 2,2’‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid). Lac1 could function well from pH 4.5 to 7.5, and its activity was significantly inhibited by H₂O₂ at above 4.85 mmol/L (P < 0.01). In addition, the lac1 gene was found to be mainly expressed in the salivary glands and foregut of C. formosanus, and seldom in the midgut or hindgut. These findings suggested that Lac1 is a phenol‐oxidizing laccase like RflacA and RflacB from termite Reticulitermes flavipes, except that Lac1 was found to be more efficient in phenol oxidation, and it did not require H₂O₂ for its function. It is suspected that this kind of termite laccase might only be able to directly oxidize low redox‐potential substrates, and the high redox‐potential groups in lignin might be oxidized by other enzymes in the termite or by using the Fenton reaction.     

Decolorization and detoxification of textile dyes with a laccase from Trametes hirsuta

Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC(50)) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (DeltaE*) below 1.1 were measured for most dyes.     

Phenylpyrazolones, Novel Oxidoreductase Redox Mediators for Degradation of Xenobiotics

An approach was developed for screening organic compounds as putative redox mediators of oxidoreductases, including laccases and peroxidases, applicable for xenobiotic degradation. The study was carried out with a homogeneous laccase preparation from the basidiomycete Trametes hirsuta and horseradish peroxidase. Compounds belonging to 1-phenyl-3-methylpyrazolones were selected. Spectroscopic and electrochemical investigation of two of the compounds, sodium 1-phenyl-2,3-dimethyl-4-aminopyrazolone 5n(4)-methanesulfonate (PPNa) and 1-(3-sulfophenyl)-3-methylpyrazolone (SPP), was performed. Electrochemical oxidation of both PPNa and SPP gave rise to high-potential intermediates capable of oxidizing veratryl alcohol, a lignin-modeling compound. Kinetic parameters of these compounds were determined in enzymatic reactions in the presence of laccase. It was shown that enzymatic oxidation of SPP by laccase produced high-potential intermediates capable of oxidizing veratryl alcohol to veratric acid. Veratryl alcohol was not oxidized during enzymatic oxidation of SPP by peroxidase. This points to a difference between the mechanisms of enzymatic oxidation of PPNa and SPP by laccase and peroxidase.     

Degradation of phenanthrene by Trametes versicolor and its laccase

Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46% and 65% of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30 degrees C. Although the removal percentage of phenanthrene was highest (76.7%) at 10 mg/l of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of phenanthrene by laccase about 40% and 30%, respectively.     

Index

A synergistic effect of polyoxometalate and laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) has been observed in the aerobic oxidation of a hydrazonaphthalene type colorant. The considerable increase in the decolorization rate of Solophenyl Blue GL dye with the new catalytic system composed by laccase and heptamolybdopentovanadophosphate heteropolyanion, when compared with the treatment with enzyme or heteropolyanion alone, was tentatively explained by the redox mediation action of the heteropolyanion.     

Titrations with ferrocyanide of japanese-lacquer-tree (Rhus vernicifera) laccase and of the type 2 copper-depleted enzyme. Interrelation of the copper sites.

1. Redox titrations are reported of the metal centres in Japanese-lacquer-tree (Rhus vernicifera) laccase with ferrocyanide. 2. The redox potential of Type 1 Cu was found to increase with ferrocyanide concentration up to a limiting value similar to that for the Type 1 Cu in Type 2 Cu-depleted enzyme (which is independent of ferrocyanide concentration). 3. The redox potential of the two-electron acceptor (Type 3 Cu) is also independent of ferrocyanide concentration in Type 2 Cu-depleted enzyme and lower than values reported for the native enzyme. 4. The two-electron acceptor is present in the oxidized state in the Type 2 Cu-depleted enzyme, though the latter lacks the 330 nm absorption band. 5. The redox potential of Type 2 Cu also depends on ferrocyanide concentration, at least in the presence of azide. 6. The redox potentials are affected by freezing the solutions and/or addition of azide, the latter binding to Type 2 Cu with affinity dependent on the redox state of the two-electron acceptor.     

Homologous cloning,expression, and characterisation of a laccase from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> and enzymatic decolourisation of an indigo dye

The lack of a commercially available robust and inexpensive laccase is a major barrier to the widespread application of this enzyme in various industrial sectors. By using an efficient system developed in Streptomyces lividans, we have produced by homologous expression 350 mg L(-1) of a bacterial laccase with a high purity and without any extensive purification. This is the highest production yield reported in the literature for a bacterial laccase. The secreted enzyme achieved oxidation under a wide pH range depending on the substrate: 4.0 for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and 9.0 for 2,6-dimethoxyphenol. Furthermore, this bacterial laccase was found to be quite resistant under various conditions. It withstands pH from 3.0 to 9.0, shows a great thermostability at 70 degrees C and was highly resistant toward conventional inhibitors. For instance, while the laccase of Trametes versicolor was completely inhibited by 1 mM NaN(3), the laccase of Streptomyces coelicolor was fully active under the same conditions. To assess application potential of this laccase, we have investigated its ability to decolourise Indigo carmine. This enzyme was able to rapidly decolourise the dye in the presence of syringaldehyde as a redox mediator.