Being particularly remarkable in laccase-mediator remedies [58].electron absorption spectra confirmed the correct folding and cofactor incorporation.Native and derivatized softwood and hardwood ligninsConclusions Data from stopped-flow (single turnover) analyses and steady-state treatment options (the latter analyzed by SEC and 2D-NMR) of native and derivatized (nonphenolic) lignosulfonates unambiguously demonstrate that: (i) the minor phenolic moiety of lignin is preferentially degraded by ligninolytic VP; and (ii) a solvent exposed tryptophan residue (conserved in both VPs and LiPs) is essential for electron transfer involving the nonphenolic lignin and also the H2O2 activated enzyme. MethodsEnzyme productionTwo water-soluble sulfonated lignins had been employed within this study: softwood (Picea abies) and hardwood (Eucalyptus grandis) lignosulfonates kindly provided by G. E. Fredheim (Borregaard AS, Sapsborg, Norway). The lignosulfonate samples have been dialyzed in ten mM EDTA, 50 mM Tris (pH eight) with the aim of removing Mn2+ traces (which decrease H2O2-activated VP), after which in Milli-Q water. Lignosulfonates (50 mg) have been acetylated inside a 50-mL pear-shaped flask with 3 mL of a pyridine-acetic anhydride (1:1, vv) solution, stirring for 24 h at room temperature. Then, 10 mL of aqueous methanol (50 ) were added plus the mixture was evaporated to dryness under vacuum. The solvent treatment was repeated 3 times with toluene (3 10 mL), and once with methanol (10 mL). Finally, the acetylated lignosulfonates (605 mg) had been dried at 50 overnight. Acetylated lignosulfonates were applied as enzyme substrate, and for estimation of phenolic and alcoholic hydroxyl content material by NMR, as described below. For lignosulfonates O-methylation with methyl iodide [44, 68], 65 mg of sample were dissolved in 10 mL of dimethylsulfoxide (DMSO), methyl iodide (1 mL) and finely powdered NaOH (1 g) were added, and the mixture was vigorously vortexed for 10 min. Then, further NaOH (300 mg) and methyl iodide (1 mL) were added, the mixture was stirred for 1 h, and also the reaction quenched by adding ten mL of water and adjusting the pH under 7 with 1 M HCl. The methylated lignosulfonates (455 mg) had been dialyzed, concentrated beneath vacuum and freeze-dried.Enzyme (transientstate) kineticsNative VP from P. eryngii (mature protein-coding sequence of Hesperidin methylchalcone NF-��B isoenzyme VPL2, GenBank AF007222) and its W164S mutated variant [29] have been created in Escherichia coli and in vitro activated as reported elsewhere [65]. The mature protein-coding sequence of P. chrysosporium LiP-H8 (GenBank Y00262) was also developed in E. coli and in vitro activated [66, 67]. The recombinant enzymes had been purified by anionexchange chromatography (Resource Q column, GE Healthcare, Uppsala, Sweden) applying a 0.3 M NaCl gradient (2 mL min-1, 20 min) in 1 mM CaCl2-containing 10 mM tartrate, pH 5.5 (for VP and its W164S variant), or succinate, pH 6 (for LiP). The Rz (A410A280 4) values have been indicative in the purity with the enzymes, and theReduction of peroxidase CI and CII in 0.1 M tartrate (pH three) by softwood and hardwood lignosulfonates (native and derivatized samples) was followed within a stopped-flow speedy spectrophotometry equipment (Bio-Logic, Claix, France) with a three-syringe module (SFM300) synchronized to a diode array detector (J M, Essingen, Germany), and BioKine computer software. CI reduction was studied by mixing the enzyme (1 final concentration) with H2O2 (1 final concentration) for 0.six s, resulting in CI formati.
