A critical study of lignin peroxidase activity assay by veratryl alcohol oxidation

Summary The effects of various parameters on Phanerochaete chrysosporium lignin peroxidase activity as obtained in ligninase assay based on the oxidation of veratryl alcohol were investigated. Marked differences in the ligninase activity were observed when the temperature and pH were varied within the ranges of 23 to 37°C and 2.5 to 4.0, respectively, reported to have been used by various research groups. Further, both veratryl alcohol, and hydrogen peroxide concentration had a significant effect on ligninase activity.     

The oxidation of veratryl alcohol, dimeric lignin models and lignin by lignin peroxidase: The redox cycle revisited

Abstract: The mechanism of oxidation of veratryl alcohol and β-0–4 dimeric lignin models is reviewed. Veratryl alcohol radicals are intermediates in both oxidation pathways. The possible role of the veratryl alcohol radical cation as a mediator is discussed. The lignin peroxidase (LIP) redox cycle is analyzed in terms of the Marcus theory of electron transfer. Reduction of both LiP-Compound I (LiP-I) and LiP-Compound II (LiP-II) by veratryl alcohol occurs in the endergonic region of the driving force. The reduction of LiP-II has a higher reorganization energy due to the change in spin state and the accompanying conformational change in the protein. It is suggested that a reversible nucleophilic addition of a carbohydrate residue located at the entrance of the active site channel plays a key role in the LiP redox cycle. Moreover. (polymeric) hydroxysubstituted benzyl radicals may reduce LiP-II via long-range electron transfer.     

The mediation of veratryl alcohol in oxidations promoted by lignin peroxidase: the lifetime of veratryl alcohol radical cation

The kinetics of decay of veratryl alcohol radical cation, generated by cerium(IV) ammonium nitrate induced oxidation of veratryl alcohol, have been followed spectrophotometrically in a stopped-flow apparatus. In acidic aqueous acetonitrile the radical cation was found to decay by a first-order process, due to deprotonation from the alpha-carbon leading to an alpha-hydroxybenzyl radical with the rate constant of 17.1+/-0.5 s(-1). This value is in full agreement with those obtained by pulse radiolysis studies but much lower than the value (1.2x10(3) s(-1)) indirectly determined by EPR experiments. The implications of these results with respect to the possible role of veratryl alcohol as a mediator in the oxidative biodegradation of lignin catalysed by lignin peroxidase are discussed.     

The roles of veratryl alcohol and nonionic surfactant in the oxidation of phenolic compounds by lignin peroxidase

Veratryl alcohol (VA) at higher concentration stimulated the lignin peroxidase (LiP)-catalyzed oxidation of phenolic compounds remarkably. This novel phenomenon was due to its competition with the phenols for the active site of the enzyme and to the high reactivity of the formed cation radical of VA (VA+*) which resulted in an additional oxidation of the phenols. The influence of the nonionic surfactant Tween 80 on the VA-enhanced LiP-catalyzed oxidation of phenols depended on its concentration. At lower concentration it had a small synergetic effect but at higher concentration it decreased the initial rate. Studies of the capillary electrophoretic behavior of LiP in the presence of Tween 80 showed that this effect was caused by the surfactant aggregation on LiP which, at higher surfactant concentrations, might impede the access of VA to its binding site on LiP and, consequently, the VA+* formation.     

Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: role of veratryl alcohol in lignin biodegradation

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H2O2-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H2O2 concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H2O2 to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H2O2 and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H2O2. AA competitively inhibited the oxidation of VA by LiP; the Ki for AA inhibition was 32 microM. Addition of VA to LiPIII* resulted in its conversion to the native enzyme. In contrast, AA did not convert LiPIII* to the native enzyme; instead, LiPIII* was bleached in the presence of AA. Thus, AA does not protect LiP from inactivation by H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aromatic ring oxidation of vanillyl and veratryl alcohols by Lentinus edodes: possible artifacts in the lignin peroxidase and veratryl alcohol oxidase assays

The degradation pathway of vanillyl and veratryl alcohol by Lentinus edodes extracellular enzymes was studied. In both cases several products of side chain oxidation and aromatic ring cleavage were isolated and characterized. We have observed that the products from veratryl alcohol degradation by Lentinus edodes are quite different from those isolated from incubations with other white-rot fungi which have veraraldehyde as the major product, in fact, this compound is not produced as final metabolite in L. edodes incubations. This behaviour could explain the apparent absence of lignin peroxidase and veratryl alcohol oxidase activities in L. edodes cultures, since such activities are usually measured by monitoring veratraldehyde formation during the veratryl alcohol oxidation; thus, it is suggested that additional assay methods should be developed, with preferably direct observation of aromatic ring oxidation products.     

On a revised mechanism of side product formation in the lignin peroxidase catalyzed oxidation of veratryl alcohol

The O2-dependent formation of side products during the oxidation of veratryl alcohol (VA) by lignin peroxidase has previously been proposed to start with the attack of H2O on the VA radical cation (VA*+). This initial reaction is unlikely since it would also lead to side product formation in the absence of O2, which is not the case. In the current mechanism VA* reacts first with O2, whereafter H2O attacks. Furthermore, this paper describes an alternative explanation for the inhibitory effect of Mn2+ on VA side product formation. It is proposed that Mn2+ reduces reactive intermediates back to VA.     

Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium. Role of veratryl alcohol in polymer oxidation.

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP-catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP-catalyzed polymerization of RNase in strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 microM H2O2, relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single-electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 x 10(4) M-1.s-1, compared to the second-order VA oxidation rate constant of 3.7 x 10(5) M-1.s-1. The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 x 10(4) M-1.s-1, compared to the first-order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPIi are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.     

Involvement of veratryl alcohol and active oxygen species in degradation of a quinone compound by lignin peroxidase

Degradation of 2-hydroxy-1,4-naphthoquinone (HNQ) by lignin peroxidase is discussed. Degradation rat was remarkably increased by an increase in veratryl alcohol concentration. Degradation is partly prevented by adding OH. scavenger (mannitol or DMSO) to the reaction mixture. Addition of O2-. scavenger (Mn2+) to the reaction mixture completely prevents the degradation. These results suggest that active oxygen species formed in the lignin peroxidase-H2O2-veratryl alcohol system play an important role in HNQ degradation.     

Thiol and Mn(2+)-mediated oxidation of veratryl alcohol by horseradish peroxidase.

Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).     

Reactions of lignin peroxidase compounds I and II with veratryl alcohol. Transient-state kinetic characterization

Stopped-flow techniques were utilized to investigate the kinetics of the reaction of lignin peroxidase compounds I and II (LiPI and LiPII) with veratryl alcohol (VA). All rate data were collected from single turnover experiments under pseudo first-order conditions. The reaction of LiPI with VA strictly obeys second-order kinetics over the pH range 2.72-5.25 as demonstrated by linear plots of the pseudo first-order rate constants versus concentrations of VA. The second-order rate constants are strongly dependent on pH and range from 2.62 x 10(6) M-1 s-1 (pH 2.72) to 1.45 x 10(4) M-1 s-1 (pH 5.25). The reaction of LiPII and VA exhibits saturation behavior when the observed pseudo first-order rate constants are plotted against VA concentrations. The saturation phenomenon is quantitatively explained by the formation of a 1:1 LiPII-substrate complex. Results of kinetic and rapid scan spectral analyses exclude the formation of a LiPII-VA cation radical complex. The first-order dissociation rate constant and the equilibrium dissociation constant for the LiPII reaction are also pH dependent. Binding of VA to LiPII is controlled by a heme-linked ionizable group of pKa approximately 4.2. The pH profiles of the second-order rate constants for the LiPI reaction and of the first-order dissociation constants for the LiPII reaction both demonstrate two pKa values at approximately 3.0 and approximately 4.2. Protonated oxidized enzyme intermediates are most active, suggesting that only electron transfer, not proton uptake from the reducing substrate, occurs at the enzyme active site. These results are consistent with the one-electron oxidation of VA to an aryl cation radical by LiPI and LiPII.     

Kinetic studies on veratryl alcohol transformation by horseradish peroxidase

A number of peroxidases, such as lignin peroxidase and manganese peroxidase have proved to be useful for industrial applications. Some studies on the effects of temperature and pH stability have been carried out. It is known that veratryl alcohol increases their stability in the range 28-50 degrees C and is oxidized, leading to veratryl aldehyde formation. Similar results with horseradish peroxidase (HRP) in the presence of cofactors were found, but the oxidation of veratryl alcohol in the absence of cofactors was extremely labile at acid pH and inactivated in a few minutes. Considering the growing industrial application of HRP, knowledge of its stability and denaturation kinetics is required. In this study, horseradish peroxidase pool (HRP-VI) and its isoenzymes HRP-VIII (acid) and HRP-IX (basic) have been shown to catalyze the oxidation of veratryl alcohol to veratryl aldehyde in the presence of hydrogen peroxide at pH 5.8 in the 35-45 degrees C range and in the absence of any cofactors. Heat and pH denaturation experiments in the presence and absence of veratryl alcohol incubation were conducted with HRP-VI and HRP-IX isoenzymes. HRP-IX was the most active isoenzyme acting on veratryl alcohol but HRP-VI was the most stable for the temperature range tested. At 35 degrees C the HRP pool presented decay constant (Kd) values of 5.5 x 10(-2) h(-1) and 1.4 10(-2) h(-1) in the absence and presence of veratryl alcohol, respectively, with an effective ratio of 3.9. These results present a new feature of peroxidases that opens one more interesting application of HRP to industrial processes.     

On the mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 by EDTA.

The mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 (LiPH2) by EDTA was investigated. It was found that EDTA was decarboxylated and that cytochrome c, nitro blue tetrazolium, ferric iron, and molecular oxygen were reduced in a reaction mixture containing LiPH2, H2O2, veratryl alcohol, and EDTA. The reductive activity observed with LiPH2 followed first order kinetics with respect to the concentration of EDTA. Stoichiometry studies showed that in the presence of sufficient EDTA, 1.7 mol of ferric iron were reduced per mole of H2O2 added to the reaction mixture. Superoxide- and EDTA-derived radicals were detected by ESR spin trapping upon incubation of LiPH2 with H2O2, veratryl alcohol, and EDTA. The Km values of veratryl alcohol and H2O2 remained the same for both the oxidative and reductive activities of LiPH2. Reductive activity was also observed with LiPH2 and EDTA using other free radical mediators in the place of veratryl alcohol, such as 1,4-dimethoxybenzene, 1,2,3- and 1,2,4-trimethoxybenzenes, and 1,2,4,5-tetramethoxybenzene. EDTA reduced the cation radical of 1,2,4,5-tetramethoxybenzene formed by LiPH2 in the presence of H2O2. Hence, it is proposed that the apparent inhibition of the veratryl alcohol oxidase activity of LiPH2 by EDTA is due to the reduction of the veratryl alcohol cation radical intermediate back to veratryl alcohol by EDTA. The reduction of cytochrome c, nitro blue tetrazolium, ferric ion, and molecular oxygen appears to be mediated by the EDTA radical formed by reduction of the veratryl alcohol cation radical.     

Synthesis of veratraldehyde from veratryl alcohol by lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electrochemical reactor

We report the synthesis of veratraldehyde from veratryl alcohol by Phanerochaete chrysosporium lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electroenzymatic reactor. The effects of operating parameters such as enzyme level, pH, and electrical potential on the efficiency of veratryl alcohol oxidation were investigated. Furthermore, we compared direct addition of hydrogen peroxide with electrogeneration of the material during enzymatic oxidation of veratryl alcohol. The electroenzymatic method using in situ-generated hydrogen peroxide was found to be effective for oxidation of veratryl alcohol by lignin peroxidase. The new method may be easily applied to biodegradation systems.     

Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium

Several aromatic compounds increased initial lignin degradation rates in cultures of Phanerochaete chrysosporium. This activation was connected to increased H₂O₂ production and glucose oxidation rates. Veratryl alcohol, a natural secondary metabolite of P. chrysosporium, also activated the lignin-degrading system. In the presence of added veratryl alcohol the ligninolytic system appeared 6–8 h earlier than in reference cultures. This effect was only seen when lignin was added after the primary growth was completed because lignin itself also caused earlier appearance of the degradative system. In cultures which received no added lignin or veratryl alcohol the ligninolytic activity only appeared once the alcohol started to accumulate. The degradation patterns of veratryl alcohol and lignin were similar. The activity levels of lignin degradation and glucose oxidation could be regulated by veratryl alcohol concentration. It is suggested that either veratryl alcohol itself or a metabolite derived from it is actually responsible for the low levels of ligninolytic activity in glucose grown cultures.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.     

Radical intermediates in veratryl alcohol oxidation by ligninase. NMR evidence

Proton nuclear magnetic resonance (NMR) spectra of veratryl alcohol (3,4-dimethoxybenzyl alcohol) were obtained during its oxidation by ligninase. It was observed that a substantial increase in the linewidths of the resonances occurred only in the presence of both the enzyme and hydrogen peroxide. Quenching the reaction by the addition of alkali immediately restored the normal linewidths of the resonances. Furthermore, inversion-recovery experiments showed a decrease in the longitudinal relaxation time of the substrate when the enzyme was actively turning over. Changes in both these NMR parameters are consistent with the generation of radical intermediates during the ligninase-catalysed oxidation of veratryl alcohol.     

Catalytic activity of lignin peroxidase and partition of veratryl alcohol in AOT/isooctane/toluene/water reverse micelles

The activity of lignin peroxidase (LiP) and the partition of its optimum substrate veratryl alcohol (VA) in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/toluene/water reverse micelles were studied in this paper to understand the microheterogeneous effect of the medium on the catalytic properties of LiP hosted in the reverse micelle. Results showed that LiP from Phanerochaete chrysosporium could express its activity in the reverse micelles, but its activity depended, to a great extent, on the composition of the reverse micelles. Optimum activity occurred at a molar ratio of water to AOT (ω₀) of 11, a pH value of 3.6, and a volume ratio of isooctane to toluene of 7–9. Under optimum conditions, the half-life of LiP was circa 12 h. The dependence of LiP activity on the volume fraction of water in the medium (θ), at a constant ω₀ value of 11, indicated that VA was mainly solubilized in the pseudophase of the reverse micelle. Based on the pseudobiphasic model and the corresponding kinetic method, a linear line can be obtained in a plot of apparent Michaelis constant of VA vs θ, and the partition coefficient of VA between the pseudophase and the organic solvent phase was determined to be 35.8, which was higher than that (22.3) between bulk water and the corresponding mixed organic solvent. H₂O₂ inhibited LiP at concentrations higher than 80 μM; this concentration value seems to be different from that in aqueous solution (about 3 mM). The differences mentioned above should be ascribed to the microheterogeneity and the interface of the AOT reverse micelle.     

Veratryl alcohol-mediated oxidation of isoeugenyl acetate by lignin peroxidase.

The mechanism of the veratryl alcohol (VA)-mediated oxidation of isoeugenyl acetate (IEA) by lignin peroxidase, and the subsequent spontaneous Calpha-Cbeta cleavage of IEA to vanillyl acetate were studied. IEA oxidation only occurred in the presence of VA. It probably did not bind to lignin peroxidase as evidenced by an unaffected Km for VA in the presence of IEA, and by the fact that a 10-fold molar excess of the unreactive IEA counterpart, eugenyl acetate, did not affect the IEA oxidation rate. IEA was very efficient in recycling VA. Up to 34 mol of IEA were oxidized per mol VA. Formation of the predominant VA oxidation product, veratraldehyde, was postponed until IEA was almost completely oxidized. Together these findings suggest that IEA was oxidized by VA.+ rather than directly by lignin peroxidase. Thus, VA functioned as a redox mediator during IEA oxidation which is remarkable considering the high calculated ionization potential of 8.81 eV. Regardless of the presence of O2, approximately 2 mol of IEA were consumed per mol H2O2, which indicated that IEA was enzymatically oxidized by one electron to the putative radical cation (IEA.+). After formation of IEA.+, a series of O2-dependent chemical reactions were responsible for Calpha-Cbeta cleavage to the major oxidation product vanillyl acetate, as evidenced by the observation that an N2 atmosphere did not inhibit IEA oxidation, but almost completely inhibited vanillyl acetate formation. GC-MS analyses revealed that under an air atmosphere 1-(4'-acetoxy-3'-methoxyphenyl)-2-propanone, 1-(4'-acetoxy-3'-methoxyphenyl)-1-hydroxy-2-propanone, and 1-(4'-acetoxy-3'-methoxyphenyl)-2-hydroxy-1-propanone were also formed. Formation of the latter two was diminished under an N2 atmosphere.     

Combinatorial evaluation of laccase-mediator system in the oxidation of veratryl alcohol

Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both commercially prepared and crude extracts, for their ability to oxidize veratryl alcohol in the presence of various solvents and mediators. Screening revealed complete conversion of veratryl alcohol to veratraldehyde catalyzed by a crude preparation of the laccase from Trametes versicolor ATCC 11235 and the mediator TEMPO in 20 % (v/v) tert-butanol.