Enzymatic activity was determined at room temperature. The assay solution contained 5 mM ABTS as substrate in 50 mM tartaric acid buffer pH 4, and transparent polystyrene 96-well microplates (Nunc) were used. The reaction was followed with a microplate reader at 420 nm, and enzymatic activity was calculated with a molar extinction coefficient for oxidized ABTS of 36'000 M−1 cm−1.
The pH optimum and range of bacterial and eukaryotic LMCOs was determined in triplicate using the McIlvaine citrate buffer row series in the pH range 2.2–8.0. In the range of pH 8.5–9.5 100 mM Tris-hydroxyaminomethane-HCl was used as buffer, while in the range of pH 10.0–12.0 200 mM sodium phosphate buffer was used. The substrates ABTS and 2,6-DMP each were added to a final concentration of 5 mM, and the assays were performed in parallel in 96-well microtiter plates at room temperature. Relative activities for 2,6-DMP could not be reliably determined above pH 8.0 due to autooxidation of the substrate.
The substrate range of bacterial and commercial LMCOs and laccase was determined as single measurement in 96-well plates. Therefore, potential laccase substrates were dissolved in the appropriate solvent at a concentration of 10 mM and diluted to a final concentration of 1 mM in the assay. Routinely, substrates were dissolved in water containing 5% (v/v) DMSO, except for trans-cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, syringic acid, methylsyringate, resveratrol, quercetin and phenolphtalein, which were dissolved in ethanol, 4-(dimethylamino) benzoic acid, 3-hydroxyanthranilic acid, 4-amino salicylic acid, N-hydroxyphthalimide, epicatechin, 3,5-demethoxy-benzonitrile, which were dissolved in ethanol and methanol 1∶5 (v/v), syringaldazine and phenothiazine, which were dissolved in 100% DMSO and triphenylamine, which was dissolved in THF.
The 200 µL reactions were performed in 96-well plates in 0.1 M potassium phosphate buffer, pH 6.0 at 37°C with shaking at 100 rpm. The reaction was initiated by adding 10 µL CFE or solutions of commercial laccase or LMCO. Control reactions for recombinant bacterial LMCOs contained 10 µL CFE of the empty vector strain. Commercial laccase and LMCO preparations were prepared as stock solutions in ddH2O with 100 U/mL of R. vernificera laccase, T. versicolor LMCO (f-TvL), M. thermophila LMCO (f-MtL) and Cucurbita ascorbate oxidase (p-Cur). Control reactions contained the appropriate inactivated enzyme, which was previously incubated at 95°C for 10 min. A blank reaction lacking mediator in the co-solvents was also monitored. A UV-Vis scan between 230–700 nm was recorded prior to enzyme addition and after 24 hours reaction time.
The pH optimum and range of bacterial and eukaryotic LMCOs was determined in triplicate using the McIlvaine citrate buffer row series in the pH range 2.2–8.0. In the range of pH 8.5–9.5 100 mM Tris-hydroxyaminomethane-HCl was used as buffer, while in the range of pH 10.0–12.0 200 mM sodium phosphate buffer was used. The substrates ABTS and 2,6-DMP each were added to a final concentration of 5 mM, and the assays were performed in parallel in 96-well microtiter plates at room temperature. Relative activities for 2,6-DMP could not be reliably determined above pH 8.0 due to autooxidation of the substrate.
The substrate range of bacterial and commercial LMCOs and laccase was determined as single measurement in 96-well plates. Therefore, potential laccase substrates were dissolved in the appropriate solvent at a concentration of 10 mM and diluted to a final concentration of 1 mM in the assay. Routinely, substrates were dissolved in water containing 5% (v/v) DMSO, except for trans-cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, syringic acid, methylsyringate, resveratrol, quercetin and phenolphtalein, which were dissolved in ethanol, 4-(dimethylamino) benzoic acid, 3-hydroxyanthranilic acid, 4-amino salicylic acid, N-hydroxyphthalimide, epicatechin, 3,5-demethoxy-benzonitrile, which were dissolved in ethanol and methanol 1∶5 (v/v), syringaldazine and phenothiazine, which were dissolved in 100% DMSO and triphenylamine, which was dissolved in THF.
The 200 µL reactions were performed in 96-well plates in 0.1 M potassium phosphate buffer, pH 6.0 at 37°C with shaking at 100 rpm. The reaction was initiated by adding 10 µL CFE or solutions of commercial laccase or LMCO. Control reactions for recombinant bacterial LMCOs contained 10 µL CFE of the empty vector strain. Commercial laccase and LMCO preparations were prepared as stock solutions in ddH2O with 100 U/mL of R. vernificera laccase, T. versicolor LMCO (f-TvL), M. thermophila LMCO (f-MtL) and Cucurbita ascorbate oxidase (p-Cur). Control reactions contained the appropriate inactivated enzyme, which was previously incubated at 95°C for 10 min. A blank reaction lacking mediator in the co-solvents was also monitored. A UV-Vis scan between 230–700 nm was recorded prior to enzyme addition and after 24 hours reaction time.
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