syringaldazine and 2-2--azino-di-(3-ethylbenzothiazoline)-6-sulfonic-acid

In this research we report the biological synthesis of electrically conducting polymer - Polypyrrole (Ppy). Cell-assisted enzymatic polymerization/oligomerization of Ppy was achieved using whole cell culture and cell-free crude enzyme extract from two white-rot fungal cultures. The selected fungal strains belong to Trametes spp., known laccase producers, commonly applied in bioremediation and bioelectrochemical fields. The biocatalytic reaction was initiated in situ through the copper-containing enzymes biosynthesized within the cell cultures under submerged aerobe cultivation conditions. The procedure was inspired by successful reports of laccase-catalyzed pyrrole polymerization. The usage of whole culture and/or crude enzyme extract has the advantage of overcoming enzyme purification and minimizing the liability of enzyme inactivation through improved stability of enzymes in their natural environment. Spectral and electrochemical techniques (UV-vis spectroscopy, infrared spectroscopy; cyclic voltammetry (CV)) and pH measurements provided insight into the evolution of pyrrole polymerization/oligomerization and the electrochemical features of the final product. Microscopy techniques (optical microscopy and scanning electron microscopy (SEM)) were primary tools for visualization of the formed Ppy particles. The relevance of our research is twofold: Ppy prepared in crude enzyme extract results in enzyme encapsulated within Ppy and/or Ppy-modified fungal cells can be formed when polymerization occurs in whole cell culture. The route of biocatalysis can be chosen according to the desired bioelectrochemical application. The reported study focuses on the improvement of charge transfer through the fungal cell membrane and/or cell wall by modification of the fungal cells with conducting polymer - polypyrrole.

Topics: Benzothiazoles; Biocatalysis; Cell Wall; Culture Media; Electric Conductivity; Electrochemical Techniques; Fermentation; Fungal Proteins; Guaiacol; Hydrazones; Hydrogen-Ion Concentration; Monophenol Monooxygenase; Polymers; Pyrroles; Sulfonic Acids; Trametes

Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme.

Topics: Aldehydes; Benzothiazoles; Enzyme Stability; Fungal Proteins; Hydrazones; Laccase; Molecular Weight; Polyethylene Glycols; Pyrogallol; Sulfonic Acids; Trametes

Accurate monitoring of the antioxidant status or of oxidative stress in patients is still a big challenge in clinical laboratories. This study investigates the possibility of applying a newly developed total antioxidant capacity assay method based on laccase or peroxidase oxidized syringaldazine [Tetramethoxy azobismethylene quinone (TMAMQ)] which is referred to here as SyrinOX, as a diagnostic tool for monitoring both oxidative stress and antioxidant status in patients. Attempts to adapt the Randox total antioxidant procedure [simultaneous incubation of the radical generating system (metmyoglobin and H(2) O(2) ) and antioxidant sample] for SyrinOX were abandoned after it was discovered that the H(2) O(2) reacted with enzymatically generated TMAMQ and ABTS radicals at a rate of 6.4 × 10(-2) /μM/s and 5.7 × 10(-3) /μM/s respectively. Thus this study for the first time demonstrates the negative effects of H(2) O(2) in the Randox system. This leads to erroneous results because the total antioxidant values obtained are the sum of radicals reduced by antioxidants plus those reacting with the radical generating system. Therefore they should be avoided not only for this particular method but also when using other similar methods. Consequently, SyrinOX is best applied using a three-step approach involving, production of TMAMQ, recovery and purification (free from enzyme and other impurities) and then using TMAMQ for measuring the total antioxidant capacity of samples. Using this approach, the reaction conditions for application of SyrinOX when measuring the total antioxidant capacity of plasma sample were determined to be 50% (v/v) ethanol/50 mM sodium succinate buffer pH 5.5, between 20 and 25 °C for at least 1 h.

Topics: Antioxidants; Benzothiazoles; Biological Assay; Biomarkers; Buffers; Calibration; Chromans; Ethanol; Humans; Hydrazones; Hydrogen Peroxide; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Metmyoglobin; Oxidation-Reduction; Oxidative Stress; Predictive Value of Tests; Reference Standards; Reproducibility of Results; Solvents; Spectrum Analysis; Succinic Acid; Sulfonic Acids; Temperature; Thiazoles

Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic archaeon Haloferax volcanii. LccA was secreted at high levels into the culture supernatant of a recombinant H. volcanii strain, with peak activity (170 +/- 10 mU.ml(-)(1)) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U.mg(-)(1) with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO(4). Optimal oxidation of ABTS and SGZ was at 45 degrees C and pH 6 and pH 8.4, respectively. The apparent K(m) values for SGZ, bilirubin, and ABTS were 35, 236, and 670 muM, with corresponding k(cat) values of 22, 29, and 10 s(-)(1), respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50 degrees C of 31.5 h.

Topics: Amino Acid Sequence; Benzothiazoles; Carbohydrates; Dimerization; Glycoproteins; Half-Life; Haloferax volcanii; Hydrazones; Hydrogen-Ion Concentration; Kinetics; Laccase; Molecular Sequence Data; Oxidation-Reduction; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Sulfonic Acids

Electrodes modified with sol-gel encapsulated laccase (isolated from Cerrenaunicolor) exhibiting mediated or mediatorless bioelectrocatalytic dioxygen reduction activity were inspected using confocal laser scanning microscopy, atomic force microscopy and scanning electrochemical microscopy. Potential-driven leaching of the redox mediator 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) from carbon ceramic electrodes covered by hydrophilic silicate-encapsulated laccase was detected during electrocatalytic action. Strongly non-homogeneous lateral distribution of the activity towards dioxygen reduction was found by redox competition mode of scanning electrochemical microscopy using a similar electrode with syringaldazine as redox mediator. Hydrogen peroxide formation at these electrodes is detected at potentials lower than 0.05V. It is ascribed to the electrochemical oxygen reduction at the carbon material while laccase-catalyzed oxygen reduction occurs below 0.35V without hydrogen peroxide formation. The scanning electrochemical microscopy images of electrodes consisting of single-walled carbon nanotubes non-covalently modified with pyrenesulfonate and laccase encapsulated in a sol-gel processed silicate film confirm direct electron transfer electrocatalysis in redox competition mode experiments and show that the enzyme is evenly distributed in the composite film. In conclusion scanning electrochemical microscopy proved to be useful for mapping of enzyme activity on different materials.

Topics: Benzothiazoles; Biocatalysis; Ceramics; Coriolaceae; Electrochemistry; Electrodes; Enzymes, Immobilized; Hydrazones; Laccase; Microscopy; Nanotubes, Carbon; Oxidation-Reduction; Oxygen; Silicates; Sulfonic Acids

Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase (CotA) from Bacillus sp. HR03 and then expressed it under microaerobic conditions and decreased temperature in order to obtain high amounts of soluble protein. The laccase was purified and its biochemical properties were investigated using three common laccase substrates, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). K(M) and k(cat) were calculated 535 microM and 127 s(-1) for ABTS, 53 microM and 3 s(-1) for 2, 6-DMP and 5 microM and 20 s(-1) for SGZ when the whole reactions were carried out at room temperature. Laccase activity was also studied when the enzyme was preincubated at 70 and 80 degrees C. With SGZ as the substrate, the activity was increased three-fold after 50 min preincubation at 70 degrees C and 2.4-fold after 10 min preincubation at 80 degrees C. Preincubation of the enzyme in 70 degrees C for 30 min raised the activity four-fold with ABTS as the substrate. Also, L-dopa was used as a substrate. The enzyme was able to oxidize L-dopa with the K(M) and k(cat) of 1,493 microM and 194 s(-1), respectively.

Topics: Aerobiosis; Bacillus; Bacterial Proteins; Benzothiazoles; Cloning, Molecular; Enzyme Activation; Gene Expression; Hydrazones; Kinetics; Laccase; Levodopa; Molecular Sequence Data; Oxidation-Reduction; Pyrogallol; Recombinant Proteins; Sequence Analysis, DNA; Sulfonic Acids; Temperature

To select Trichoderma strains for enhanced laccase production in Pleurotus ostreatus or Agaricus bisporus cultures.. Laccase production by P. ostreatus and A. bisporus was evaluated in liquid (axenic) and solid (dual cultures) malt extract medium. Oxidation of ABTS, DMP and syringaldazine was evaluated in order to assess the potential of Trichoderma strains to enhance laccase production by basidiomycetes. Selected Pleurotus-Trichoderma interactions yielded higher increases in laccase volumetric activity and an additional laccase isoform was produced. By contrast, Agaricus-Trichoderma interactions lead to smaller increases on laccase volumetric activity, probably as result of repression (or degradation) towards one of the laccases isoforms.. The strains of P. ostreatus and A. bisporus assessed in this work showed good potential as laccase producers. The Trichoderma-mediated biological stimulation of laccase production by P. ostreatus and A. bisporus is relevant in order to develop highly productive processes.. Extracellular laccases from basidiomycetes are produced only in small amounts. It is therefore important to increase process productivity for potential industrial applications. The results from this study enable the selection Trichoderma strains capable of increasing laccase production by P. ostreatus or A. bisporus in dual cultures.

Topics: Agaricus; Benzothiazoles; Biomass; Culture Techniques; Hydrazones; Laccase; Mycelium; Pleurotus; Pyrogallol; Sulfonic Acids

A new laccase gene (cotA) was cloned from Bacillus licheniformis and expressed in Escherichia coli. The recombinant protein CotA was purified and showed spectroscopic properties, typical for blue multi-copper oxidases. The enzyme has a molecular weight of approximately 65 kDa and demonstrates activity towards canonical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants KM and kcat for ABTS were of 6.5+/-0.2 microM and 83 s(-1), for SGZ of 4.3+/-0.2 microM and 100 s(-1), and for 2,6-DMP of 56.7+/-1.0 microM and 28 s(-1). Highest oxidizing activity towards ABTS was obtained at 85 degrees C. However, after 1 h incubation of CotA at 70 degrees C and 80 degrees C, a residual activity of 43% and 8%, respectively, was measured. Furthermore, oxidation of several phenolic acids and one non-phenolic acid by CotA was investigated. CotA failed to oxidize coumaric acid, cinnamic acid, and vanillic acid, while syringic acid was oxidized to 2,6-dimethoxy-1,4-benzoquinone. Additionally, dimerization of sinapic acid, caffeic acid, and ferulic acid by CotA was observed, and highest activity of CotA was found towards sinapic acid.

Topics: Bacillus; Bacterial Proteins; Benzothiazoles; Cloning, Molecular; Dimerization; Escherichia coli; Genes, Bacterial; Hydrazones; Hydroxybenzoates; Laccase; Substrate Specificity; Sulfonic Acids

The gene, Aquifex aeolicus AAC07157.1, encoding a multicopper oxidase (McoA) and localized in the genome as part of a putative copper-resistance determinant, has been cloned, over-expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. The purified enzyme shows spectroscopic and biochemical characteristics typical of the well-characterized multicopper oxidase family of enzymes. McoA presents higher specificity (k(cat)/K(m)) for cuprous and ferrous ions than for aromatic substrates and is therefore designated as a metallo-oxidase. Addition of copper is required for maximal catalytic efficiency. A comparative model structure of McoA has been constructed and a striking structural feature is the presence of a methionine-rich region (residues 321-363), reminiscent of those found in copper homeostasis proteins. The kinetic properties of a mutant enzyme, McoADeltaP321-V363, deleted in the methionine-rich region, provide evidence for the key role of this region in the modulation of the catalytic mechanism. McoA has an optimal temperature of 75 degrees C and presents remarkable heat stability at 80 and 90 degrees C, with activity lasting for up to 9 and 5 h, respectively. McoA probably contributes to copper and iron homeostasis in A. aeolicus.

Topics: Amino Acid Sequence; Bacteria; Benzothiazoles; Cloning, Molecular; Copper; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Escherichia coli; Hydrazones; Iron; Laccase; Models, Molecular; Molecular Sequence Data; Oxidoreductases; Sequence Alignment; Sulfonic Acids

Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l(-1)) were produced in response to Cu2+. Highest laccase levels (19,000 U l(-1)) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M (w) of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability. Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60 degrees C, and at 5 degrees C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any activity during 6 months storage at 4 degrees C. Kinetic constants (K (m), k (cat)) were determined for 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine.

Topics: Benzothiazoles; Chromatography, Ion Exchange; Copper; Culture Media; Enzyme Stability; Hydrazones; Hydrogen-Ion Concentration; Isoelectric Point; Isoenzymes; Kinetics; Laccase; Molecular Weight; Polyporales; Pyrogallol; Resins, Synthetic; Spectrum Analysis; Sulfonic Acids; Temperature; Ultrafiltration

The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps, its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants. Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately 85 kDa at pH 5. A band also appeared at a molecular mass of approximately 200 kDa when the gels were treated with dopamine, L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7. The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase. However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU). Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200 kDa). Laccase activity was detected in a holidic sucrose diet that was fed on for 16 h by two females, but only a trace of catechol oxidase activity was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase exhibited an optimum pH of 4.75-5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the watery saliva.

Topics: Animals; Benzothiazoles; Catechol Oxidase; Catechols; Dihydroxyphenylalanine; Dopamine; Electrophoresis, Polyacrylamide Gel; Hemiptera; Histological Techniques; Hydrazones; Hydrogen-Ion Concentration; Hydroquinones; Laccase; Oxidation-Reduction; Salivary Glands; Salivary Proteins and Peptides; Sulfonic Acids

The white-rot fungus Daedalea quercina produced the ligninolytic enzymes laccase and Mn-dependent peroxidase. Laccase was purified using anionexchange and size-exclusion chromatographies. SDS-PAGE showed the purified laccase to be a monomeric protein of 69 kDa (71 kDa using gel filtration) with an isoelectric point near 3.0. The optimum pH for activity was below 2.0 for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (K(m)=38 microM), 4.0 for 2,6-dimethoxyphenol (K(m)=48 microM), 4.5 for guaiacol (K(m)=93 microM) and 7.0 for syringaldazine (K(m)=131 microM). The temperature optimum was between 60 and 70 degrees C depending on the pH and buffer used. The enzyme was stable up to 45 degrees C, and stability was higher at alkaline pH. Enzyme activity was increased by the addition of Cu(2+) and inhibited by Mn(2+), sodium azide, dithiothreitol, and cysteine. Laccase from Daedalea quercina was able to decolorize the synthetic dyes Chicago sky blue, poly B-411, remazol brilliant blue R, trypan blue and reactive blue 2.

Topics: Anthraquinones; Azo Compounds; Benzothiazoles; Chromatography, Gel; Chromatography, Ion Exchange; Color; Coloring Agents; Enzyme Activators; Enzyme Inhibitors; Enzyme Stability; Guaiacol; Hydrazones; Hydrogen-Ion Concentration; Isoelectric Point; Laccase; Lignin; Metals; Molecular Weight; Peroxidases; Polyporales; Pyrogallol; Substrate Specificity; Sulfonic Acids; Temperature; Triazines; Trypan Blue

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.

Topics: Benzothiazoles; Directed Molecular Evolution; Glycosylation; Hydrazones; Laccase; Models, Molecular; Oxidoreductases; Recombination, Genetic; Saccharomyces cerevisiae; Sordariales; Substrate Specificity; Sulfonic Acids

Laccase-like activity was detected in melanin-producing strains of Sinorhizobium meliloti mainly in cells at the stationary growth phase when copper was added to the medium. The laccase showed both syringaldazine and ABTS (2,2'-azino-bis-ethylbenzthiazoline-6-sulfonic acid) oxidase activities and was activated by the addition of 1.7 mM sodium dodecyl sulfate. Activity was totally inhibited by the addition of 1.0 mM EDTA, suggesting that the enzyme is a metal-dependent one. The enzyme was found to be cytosolic having an optimum pH of 5.0, an estimated molecular mass of 95 kDa and a K(m) of 4 microM for syringaldazine. Both laccase and tyrosinase activities were detected in melanin-producing S. meliloti strains. Plant growth-promoting (PGP) effect in rice by a laccase-producing S. meliloti strain when co-inoculated with Azospirillum brasilense Cd was observed. PGP effect by co-inoculation significantly increased plant yield compared to A. brasilense by itself. To the best of our knowledge this is the first report on laccase production in rhizobia and cooperation between Azospirillum and Sinorhizobium in rice.

Topics: Azospirillum brasilense; Bacterial Proteins; Benzothiazoles; Copper; Edetic Acid; Enzyme Activation; Hydrazones; Hydrogen-Ion Concentration; Laccase; Melanins; Molecular Weight; Monophenol Monooxygenase; Oryza; Oxidoreductases; Sinorhizobium meliloti; Sodium Dodecyl Sulfate; Species Specificity; Substrate Specificity; Sulfonic Acids; Symbiosis

The white-rot fungus Phellinus ribis produced a single form of laccase, which was purified to apparent electrophoretic homogeneity from cultures induced with 2,5-xylidine. This protein was a dimer, consisting of two subunits of 76 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that the enzyme contained about 28% carbohydrate content. The laccase appeared to be different from other known laccases by the UV-visible absorption spectrum analysis. One enzyme molecule contained one copper, one manganese, and two zinc atoms. The laccase showed optimal activity at pH 4.0-6.0, 5.0, and 6.0 with 2,6-dimethoxyphenol, ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], and syringaldazine, respectively. The enzyme preferably oxidized dimethoxyphenol and aromatic amine compounds. The stability of the laccase was low at acidic pH, whereas it showed high stability at neutral pH and mild temperature. The N-terminal amino acid sequence revealed a very low homology with other microbial laccases. With some substrates, the addition of manganese and H2O2 resulted in a remarkable increase in the oxidation rate. Without an appropriate phenolic substrate, the enzyme could not oxidize Mn(II) in the presence of H2O2 or pyrophosphate.

Topics: Amino Acid Sequence; Basidiomycota; Benzothiazoles; Carbohydrates; Catalysis; Dimerization; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fungal Proteins; Hydrazones; Hydrogen Peroxide; Hydrogen-Ion Concentration; Laccase; Lectins; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Molecular Sequence Data; Oxidoreductases; Oxygen; Pyrogallol; Sequence Homology, Amino Acid; Spectrophotometry; Sulfonic Acids; Temperature; Time Factors; Ultraviolet Rays