Oxidation Of Veratryl Alcohol Research Articles

Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases

Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn(2+), the manganese peroxidase (MnP) substrate (Mn(3+) being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these 'hybrid' properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn(2+) oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not beta-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.

Catalytic performance of lignin peroxidase in a novel reverse micelle

To enhance the catalytic activity of lignin peroxidase (LiP) in a reverse micelle, a synthesized two-tail nonionic surfactant N-gluconyl glutamic acid didecyl ester (GGDE) was used to formulate a novel reverse micelle. Based on the LiP catalyzed oxidation of veratryl alcohol (VA) in this novel GGDE/TritonX-100–cyclohexane–H 2O reverse micelle, the effects of the size of the reverse micelle, the buffer pH, and the concentration of H 2O 2 on the catalytic activity of LiP were investigated. Under the optimized conditions, the catalytic efficiency of LiP in the GGDE/TritonX-100 reverse micelle was 40 times higher than that in the AOT reverse micelle. The full expression of catalytic activity of LiP in this medium was mainly due to the lack of electrostatic interaction between LiP and the head group of GGDE and TritonX-100 and to the size fit between LiP and the inner water cavity of the reverse micelle.

Kinetic Studies on the Lignin Peroxidase Catalyzed Oxidation of Veratryl Alcohol by H2O2 in AOT/Isooctane/Toluene/Water Reverse Micelles

AbstractThe steady state kinetics of the lignin peroxidase (LiP) catalyzed oxidation of veratryl alcohol (VA) by H2O2 in a sodium bis(2‐ethylhexyl) sulfosuccinate (AOT)/isooctane/toluene/water reverse micellar medium was studied and a comparison with the corresponding aqueous medium was made to understand the effect of the reverse micellar medium on the catalytic mechanism and kinetic parameters. Results indicated that the model reaction in the AOT reverse micelle followed the ping‐pong mechanism with true kcat, Km,VA and K being 59.6 min−1, 13.9 mmol· L−1 and 94.8 µmol·L−1, respectively; inhibition of high level of H2O2 on LiP followed the reversible competitive pattern with Ki being 0.140 mmol·L−1. The reaction mechanism and inhibition pattern in the AOT reverse micellar medium were the same as those in bulk aqueous medium, but the kinetic parameters except K were greatly different in the two media. The kcat and Ki values in the reverse micelle were approximately 2 and 20 times smaller than the corresponding values in the aqueous solution, but the Michaelis constant of VA was approximately 100 times greater than that in the aqueous solution. The above mentioned differences in the kinetic parameters were caused by the microheterogeneity and the interface of the AOT reverse micelle, which resulted in the partitioning of VA and H2O2, and by the changes of the conformation of LiP and the reactivity of the substrates.

5,10,15,20-Tetrakis(4-sulfonato-phenyl)porphine-Mn(III) immobilized on imidazole-activated silica as a novel lignin-peroxidase-like biomimetic catalyst

In this study a new biomimetic lignin-peroxidase-like catalyst was investigated. Such a heterogeneous catalyst could be of potential interest for oxidative degradation of plant effluents, containing soluble lignin derivatives. 5,10,15,20-Tetrakis(4-sulfonato-phenyl)porphine-Mn(III) was coordinated to imidazole-bearing silica to give a stable adduct; by contrast, the metalloporphine did not significantly interact with plain silica gel or aminopropylsilica. The adduct showed a remarkable ability to catalyze veratryl alcohol oxidation at the expenses of H 2O 2. Kinetic and operational characterisation of the catalyst is also reported.

On In Situ Prepared Cu–Phenanthroline Complexes in Aqueous Alkaline Solutions and Their Use in the Catalytic Oxidation of Veratryl Alcohol

AbstractThe in situ prepared complex based on copper(II) sulfate and 1,10‐phenanthroline (phen) and its performance in the catalytic oxidation of veratryl alcohol (3,4‐dimethoxybenzyl alcohol) to veratraldehyde with O2 in alkaline aqueous solutions have been studied. In aqueous solution, the structure of Cu–phen complexes varies with reaction conditions; by changing either the copper‐to‐ligand molar ratio or the pH, the amount of coordinated 1,10‐phenanthroline and hydroxido ligands in the coordination sphere of copper can be altered in a controlled manner. The highest activities are achieved in the pH range 12.6–13.3, which correlates with the presence of the [Cu(phen)(OH)2] species according to complex distribution curves. According to the UV/Vis and EPR studies, the oxidation reaction is initiated by the reduction of [Cu(phen)(OH)2] to CuI species by veratryl alcohol, which can be seen as a disappearance of the Cu2+ EPR signal and as a shift of the UV/Vis absorbance maximum from about 690 nm to the 410 and 525 nm regions. When molecular oxygen is added, the CuI species is readily oxidised back to CuII species, and the catalytic formation of aldehyde is initiated. On the basis of these observations, a mechanism for the catalytic oxidation is proposed. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Biomimetic Oxidation of Veratryl Alcohol with H2O2 Catalyzed by Iron(III) Porphyrins and Horseradish Peroxidase in Ionic Liquid

3,4-Dimethoxybenzyl alcohol (veratryl alcohol) is a model compound for lignin substructures and its oxidation with H 2 O 2 catalyzed by iron(III) porphyrins and horseradish peroxidase (HRP) in ionic liquids has been described. Veratraldehyde and 2-hydroxymethyl-5-methoxy-2,5-cyclohexadiene-1,4-dione were the two major products of the oxidation of veratryl alcohol at room temperature. The iron(III) porphyrin or HRP immobilized in ionic liquids showed increased catalytic activity and can be recycled and this is believed due to the stabilization of highvalent oxo-iron(IV) π-cation radical intermediate generated in the reaction.

Copper-2,2′-bipyridines: Catalytic performance and structures in aqueous alkaline solutions

2,2′-Bipyridine and ten modified 2,2′-bipyridines were complexed in situ with copper(II) sulphate in aqueous alkaline solution. In oxidation studies with these complexes, the substituents of the bipyridine ring were found to have a significant effect on the catalytic activity. Parallel screening revealed that Cu-complexes with the bipyridine ligands substituted in 4,4′- and 5,5′-positions were highly active in the oxidation of veratryl alcohol with molecular oxygen. More detailed studies with 4,4′-disubstituted bipyridines showed that the electron donating methyl and methoxy substituents enhance the oxidation activity of Cu–bipyridine. The most effective Cu-complexes in the oxidation of veratryl alcohol also catalysed the oxidation of 2,2′-biphenol and the oxidative depolymerisation of the carbohydrate model compound dextran. The performance of Cu-2,2′-bipyridines in oxygen delignification of softwood pulp were in accordance with the model compound studies; the catalysts lowered the lignin content of the pulp, but they also tended to damage the cellulose fibres. According to the oxidation results, the complex distribution calculations and the UV–vis studies the structures of the active species were [Cu(2,2′-bipyridine) 2OH] + and [Cu(4,4′-dimethoxy-2,2′-bipyridine) 2OH] +.

Mechanism of versatile peroxidase inactivation by Ca 2+ depletion

Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca 2+ depletion. In this work, the spectroscopic characterizations of Ca 2+-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca 2+ content. At pH 7.5, UV-Vis spectra indicate a heme-Fe 3+ transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca 2+-depleted form. This Fe 3+ hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca 2+-depleted enzyme has a high spin Fe 3+. EPR measurements on VP confirm the differences in the Fe 3+ spin states at pH 4.5 and at 7.5 for both, native and Ca 2+-depleted enzymes. In addition, EPR spectra recorded after the addition of H 2O 2 to Ca 2+-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca 2+-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca 2+-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted.

Electrochemistry and kinetics of fungal laccase mediators

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.

Bis(pyridine-2-carboxaldehyde oxime)copper(II) sulphate as catalyst precursor in the oxidation of veratryl alcohol by dioxygen

In situ generated [Cu II(pyridine-2-carboxaldehyde oxime) 2] complex catalyses selective oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) to aldehyde by molecular oxygen in aqueous solution. Under alkaline conditions, the catalyst has both oxime groups deprotonated and the solid state structure of the complex reveals a square pyramidal geometry established by two pyridine-2-carboxaldehyde oxime ligands and sulphate as a counter anion.

Insight into copper oxidation catalysts: Kinetics, catalytic active species and their deactivation

The catalytic properties of in situ formed copper(II) complexes of N, N, N′, N′-tetramethylethylenediamine (TMEDA), rac-1,2-diaminocyclohexane (DACH) and 9,10-diaminophenantrene (DAPHEN) were investigated under optimized reaction conditions in the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) with molecular oxygen in alkaline aqueous solutions. The kinetics of the reaction, catalytic species and their deactivation were studied, in order to clarify the reasons for low conversions in Cu(TMEDA) and Cu(DACH) catalyzed reactions. The most active catalyst was the scarcely soluble Cu(DAPHEN) with turn over number 5700 in the oxidation of veratryl alcohol. The catalytic precursors in Cu(TMEDA) catalyzed reactions is binuclear hydroxyl bridged complex (LCu-μ-(OH) 2-CuL) 2+. The main reason for the deactivation of these catalysts according to our results is the reactivity of the ligands under reaction conditions.

Nano-assembly of manganese peroxidase and lignin peroxidase from P. chrysosporium for biocatalysis in aqueous and non-aqueous media

Development, characterization, and activity studies of nano-assemblies of lignin peroxidase (LiP), and manganese peroxidase (MnP) from Phanerochaete c hrysosporium on flat surfaces as well as colloidal particles have been investigated. These assemblies of LiP and MnP were fabricated with polyelectrolytes—poly(ethylenimine) (PEI), poly(dimethyldiallylammonium chloride) (PDDA), and poly(allylamine) (PAH)—using a layer-by-layer self-assembly technique (LbL). Characterization of these assemblies on flat surfaces was monitored using quartz crystal microbalance (QCM), while assemblies on microparticles such as melamine formaldehyde (MF) were carried out with zeta potential analyzer (ZPA). A unique dynamic adsorption–desorption of the enzyme layers is observed during the assembly. All the nano-assemblies of LiP and MnP can effectively oxidize veratryl alcohol (VA) to its aldehyde for an extended period of time. The effect of different polyions and the number of polyion layers on the activities of LiP and MnP nano-assembly was also examined. It is observed that drying of enzyme layer during the assembly and the use of non-aqueous media, such as acetone can significantly reduce the activity of the enzymes. Enzyme activity reaches a minimum when the concentration of acetone is increased to 30%; however, the activity can be restored to its original value by increasing the concentration of aqueous media. Preliminary studies using assemblies of LiP and MnP on MF microparticles further demonstrate the feasibility of developing potential systems for degradation of environmental pollutants.

Characteristics of novel lignin peroxidases produced by white-rot fungus Phanerochaete sordida YK-624

We characterized a lignin peroxidase (YK-LiP2) isolated from shaking culture inoculated with the white-rot fungus Phanerochaete sordida YK-624. The YK-LiP2 enzyme was identified and purified to homogeneity by anion-exchange chromatography and gel permeation chromatography. The molecular weight of YK-LiP2 was approximately 45 kDa, and its absorption spectrum was almost the same as that of the LiP (Pc-LiP) from P. chrysosporium. Steady-state kinetics of veratryl alcohol (VA) oxidation by YK-LiP2 revealed an ordered bi-bi ping-pong mechanism, although the Pc-LiP oxidation of ferrocytochrome c obeys peroxidase ping-pong kinetics rather than ordered bi-bi ping-pong kinetics. Degradation of dimeric lignin model compounds by YK-LiP2 was more effective than that by Pc-LiP. Moreover, YK-LiP2 and YK-LiP1, which was previously isolated from static culture inoculated with P. sordida YK-624, oxidized VA under a higher concentration of hydrogen peroxide (>2.5 mM) although Pc-LiP could not oxidize VA in the presence of 2.5 mM hydrogen peroxide.

Direct oxidation of polymeric substrates by multifunctional manganese peroxidase isoenzyme from Pleurotus ostreatus without redox mediators.

VPs (versatile peroxidases) sharing the functions of LiP (lignin peroxidase) and MnP (manganese peroxidase) have been described in basidiomycetous fungi Pleurotus and Bjerkandera. Despite the importance of this enzyme in polymer degradation, its reactivity with polymeric substrates remains poorly understood. In the present study, we first report that, unlike LiP, VP from Pleurotus ostreatus directly oxidized two polymeric substrates, bovine pancreatic RNase and Poly R-478, through a long-range electron pathway without redox mediators. P. ostreatus produces several MnP isoenzymes, including the multifunctional enzyme MnP2 (VP) and a typical MnP isoenzyme MnP3. MnP2 (VP) depolymerized a polymeric azo dye, Poly R-478, to complete its catalytic cycle. Reduction of the oxidized intermediates of MnP2 (VP) to its resting state was also observed for RNase. RNase inhibited the oxidation of VA (veratryl alcohol) in a competitive manner. Blocking of the exposed tryptophan by N-bromosuccinimide inhibited the oxidation of RNase and VA by MnP2 (VP), but its Mn2+-oxidizing activity was retained, suggesting that Trp-170 exposed on an enzyme surface is a substrate-binding site both for VA and the polymeric substrates. The direct oxidation of RNase and Poly R by MnP2 (VP) is in sharp contrast with redox mediator-dependent oxidation of these polymers by LiP from Phanerochaete chrysosporium. Molecular modelling of MnP2 (VP) revealed that the differences in the dependence on redox mediators in polymer oxidation by MnP2 (VP) and LiP were explained by the anionic microenvironment surrounding the exposed tryptophan.

Mediator role of veratryl alcohol in the lignin peroxidase-catalyzed oxidative decolorization of Remazol brilliant blue R

Lignin peroxidase produced by Trametes versicolor decolorizes Remazol brilliant blue R (RBBR) in the presence as well as in the absence of veratryl alcohol (VA). VA enhances and stabilizes the RBBR-decolorization rates by lignin peroxidase. RBBR has better substrate reactivity than VA for LiP. RBBR is also decolorized directly by LiP and competitively inhibits VA oxidation by LiP. In the presence of higher concentrations of RBBR (i) RBBR decolorization rates improve, (ii) veratryl aldehyde appears after a lag and (iii) VA oxidation rates decrease. The lag is due to consumption of VA cation radical (VA +) generated upon LiP-catalyzed VA oxidation, during RBBR oxidation. That may result in the formation of compound III in the absence of VA + and contributes to the inhibitory influence of RBBR on LiP activity.

Mediator role of veratryl alcohol in the lignin peroxidase-catalyzed oxidative decolorization of Remazol Brilliant Blue R

Lignin peroxidase (LiP) produced by Trametes versicolor decolorizes Remazol Brilliant Blue R (RBBR) in the presence as well as in the absence of veratryl alcohol (VA). VA enhances and stabilizes the RBBR-decolorization rates by lignin peroxidase. RBBR has better substrate reactivity than VA for LiP. RBBR is also decolorized directly by LiP and competitively inhibits VA oxidation by LiP. In the presence of higher concentrations of RBBR (i) RBBR decolorization rates improve, (ii) veratryl aldehyde appears after a lag and (iii) VA oxidation rates decrease. The lag is due to consumption of VA cation radical (VA +) generated upon LiP-catalyzed VA oxidation, during RBBR oxidation. That may result in the formation of compound III in the absence of VA + and contributes to the inhibitory influence of RBBR on LiP activity.

Purification and characterization of manganese peroxidase from wood-degrading fungus Trichophyton rubrum LSK-27

A manganese peroxidase (MnP) from a wood-degrading fungus Trichophyton rubrum LSK-27 was purified to homogeneity by anion-exchange chromatography followed by gel filtration. Molecular mass of the purified enzyme was determined to be about 42 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Spectrophotometric analysis of the enzyme revealed one Soret maximum at 407 nm, and two visible peaks at 502 and 644 nm, which are consistent with photometric spectra of other MnPs. Mass spectrometric analysis of the digested protein revealed that it had a very high homology to a unique peroxidase (a hybrid of MnP and lignin peroxidase) from Bjerkandera sp. B33/3. Bjerkandera MnP was able to oxidize veratryl alcohol, whereas T. rubrum LSK-27 MnP could not. T. rubrum LSK-27 MnP had the highest p I of 8.2 among MnPs reported so far. The enzyme was stable at rather high temperatures, and when compared with other MnPs, this MnP was more stable in the presence of high concentrations of H 2O 2.

An efficient method to investigate metal–ligand combinations for oxygen bleaching

Catalytic activities of 189 metal–ligand combinations in aqueous alkaline solution were studied in the oxidation of veratryl alcohol with molecular oxygen. The parallel screening revealed high oxidation activity of copper ions with dimethylglyoxime, 2,2′-bipyridine and 1,10-phenanthroline ligands. Further study with these in situ prepared catalysts indicated that ionic complexes with two ligands are probably the most active. The maximum activities of the three catalysts were achieved at different pH and metal–ligand molar ratio. In 3 h reaction with catalyst–substrate molar ratio 1:1000, the highest turnover numbers obtained were 420 for the Cu-dimethylglyoxime catalyst, 830 for Cu-bipyridine and 760 for Cu-phenanthroline. These most effective Cu-complexes catalysed not only the oxidation of lignin model compounds but also the depolymerisation of the carbohydrate model compound dextran. Results achieved in oxygen delignification of softwood pulp were in accordance with the model compound studies; the catalysts effectively lowered the kappa number, but they also tended to damage the cellulose fibres.

Lifetime of Veratryl Alcohol Radical Cation Electrogenerated in Acetonitrile

Abstract The electrochemical oxidation of veratryl alcohol at a carbon fiber microdisk electrode with relatively high sweep rates in acetonitrile shows a chemically reversible response. Analysis of the cathodic and anodic current ratio allows one to estimate the lifetime of the short-lived radical cation in the solution.

Biotransformation of lignin polymers derived from beech wood pulping by Sporobolomyces roseus isolated from leafy material.

The ability of the yeast, Sporobolomyces roseus, isolated from leafy material, to modify lignin derived from beechwood pulping was examined by FTIR and 13C NMR spectroscopy, which revealed oxidative cleavage of the Calpha-Cbeta linkages between lignin units. Using veratryl alcohol as a model substrate confirmed that Sp. roseus could oxidize veratryl alcohol into veratric acid. This yeast might be suitable for the pretreatment of lignocellulosic materials and/or for biotransformation of technical lignins.