Degradation of veratryl alcohol by Penicillium simplicissimum

Summary Several bacteria, yeast and fungi selectively isolated from paper-mill waste-water grew on veratryl alcohol, a key intermediate of lignin metabolism. Penicillium simplicissimum oxidized veratryl alcohol via a NAD(P)⁺-dependent veratryl alcohol dehydrogenase to veratraldehyde, which was further oxidized to veratric acid in a NAD(P)⁺-dependent reaction. Veratric-acid-grown cells contained NAD(P)H-dependent O-demethylase activity for veratrate, vanillate and isovanillate. Protocatechuate was cleaved by a protocatechuate 3,4-dioxygenase. Offprint requests to: E. de Jong     

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.     

Electroenzymatic oxidation of veratryl alcohol by lignin peroxidase

This paper reports the formation of veratraldehyde by electroenzymatic oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) hybridizing both electrochemical and enzymatic reactions and using lignin peroxidase. The novel electroenzymatic method was found to be effective for replacement of hydrogen peroxide by an electrochemical reactor, which is essential for enzyme activity of lignin peroxidase. The effects of operating parameters such as enzyme dosage, pH, and electric potential were investigated. Further, the kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to oxidation when hydrogen peroxide was supplied externally.     

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.     

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.     

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.     

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.     

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.     

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.     

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

Phenolic compounds as enhancers in enzymatic and electrochemical oxidation of veratryl alcohol and lignins

Sixteen phenolic compounds, 14 of which naturally occurring, were compared to the synthetic 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and violuric acid (VA) in terms of their ability to act as mediators/enhancers in: (1) laccase oxidation of veratryl alcohol as a lignin model compound, and (2) electrochemical oxidation of kraft and flax lignins. HPLC analysis revealed that the syringyl-type phenols methyl syringate and acetosyringone were the most efficient natural enhancers in the laccase oxidation of veratryl alcohol. Both compounds, though far from the performance of ABTS were able to generate veratraldehyde in amount similar to that obtained with VA. By contrast, the best performing phenolic enhancers for the electrochemical oxidation of lignins were sinapinaldehyde, vanillin, acetovanillone, and syringic acid. Catalytic efficiencies close to those achieved with ABTS and VA were calculated for these phenolic compounds.     

Purification and characterization of dye degrading of veratryl alcohol oxidase from Pseudomonas aeruginosa strain BCH

In the present study we have purified the intracellular veratryl alcohol oxidase (VAO) enzyme from Pseudomonas aeruginosa strain BCH to evaluate its dye decolorizing potential. The enzyme was purified by ion exchange chromatography using DEAE cellulose followed by gel filtration chromatography using Biogel P-100. The molecular weight of the purified enzyme was estimated by polyacrylamide gel electrophoresis (PAGE) analysis. The VAO was purified up to 12 and 16.3-fold by ion exchange and gel filtration chromatography respectively. VAO was estimated to be about 85 kDa by SDS–PAGE. The optimum pH and temperature for purified VAO was 3 and 55°C respectively. The purified enzyme exerted its optimal activity with veratryl alcohol and also oxidized various other substrates, whereas diminished activity was noted in case of tryptophan and xylidine. The metal ions Mn⁺⁺ and Hg⁺⁺ were found to suppress the oxidase activity. The purified enzyme decolorized different dyes with variable decolorization rates and efficiencies. Decolorization mechanism of Remazol Black by purified enzyme was studies in detail using various analytical techniques like HPLC, GC–MS and FTIR. This study is useful for understanding the precise role of Pseudomonas aeruginosa strain BCH in the decolorization of textile dyes containing industrial wastewater.     

Lignin and veratryl alcohol are not inducers of the ligninolytic system of Phanerochaete chrysosporium.

Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. In this work, we investigated the roles of veratryl alcohol and lignin in the ligninolytic system of P. chrysosporium BKM-F-1767 cultures grown under nitrogen-limited conditions. Cultures supplemented with 0.4 to 2 mM veratryl alcohol showed increased lignin peroxidase activity. Addition of veratryl alcohol had no effect on Mn-dependent peroxidase activity and inhibited glyoxal oxidase activity. Azure-casein analysis of acidic proteases in the extracellular fluid showed that protease activity decreased during the early stages of secondary metabolism while lignin peroxidase activity was at its peak, suggesting that proteolysis was not involved in the regulation of lignin peroxidase activity during early secondary metabolism. In cultures supplemented with lignin or veratryl alcohol, no induction of mRNA coding for lignin peroxidase H2 or H8 was observed. Veratryl alcohol protected lignin peroxidase isozymes H2 and H8 from inactivation by H2O2. We conclude that veratryl alcohol acts as a stabilizer of lignin peroxidase activity and not as an inducer of lignin peroxidase synthesis.     

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.     

Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium

The lignin-degrading basidiomycete Phanerochaete chrysosporium synthesizes veratryl alcohol (3,4-dimethoxybenzyl alcohol) via phenylalanine, 3,4-dimethoxycinnamyl alcohol and veratrylglycerol. Study of the conversion of 3,4-dimethoxycinnamyl alcohol to veratrylglycerol and veratryl alcohol showed is to be (a) catalyzed by a secondary metabolic system, (b) markedly suppressed by culture agitation, and (c) strongly inhibited by l-glutamate. The amount of veratryl alcohol synthesized de novo was positively correlated with the O₂ concentration after primary growth. Other work has shown that the cinnamyl alcohol terminal residue in a lignin substructure model compound is degraded via arylglycerol and benzyl alcohol structures in ligninolytic cultures of P. chrysosporium, and that the ligninolytic system exhibits traits (a)-(c) above. Ligninolytic activity is also strongly and positively correlated with O₂ concentration. The results here suggest, therefore, that the actual biosynthetic secondary metabolic product is 3,4-dimethoxycinnamyl alcohol, but that this is degraded by the ligninolytic system to veratryl alcohol via veratrylglycerol. Veratryl alcohol is only slowly metabolized by the fungus, and accumulates.Non-standard abbreviation tlc thin layer chromatography     

Ligninolytic enzyme activity: A comparison of the veratryl alcohol and Azure B assay systems

Summary Ligninolytic enzyme activities in cell-free culture filtrates of Coriolus versicolor, Fusarium solani and Phanaerochaete chrysosporium were assessed using a veratryl alcohol oxidation assay system and results compared with those obtained using a relatively new assay system based on the oxidation of Azure B.