Anisaldehyde and Veratraldehyde Acting as Redox Cycling Agents for H(2)O(2) Production by Pleurotus eryngii

The existence of a redox cycle leading to the production of hydrogen peroxide (H(2)O(2)) in the white rot fungus Pleurotus eryngii has been confirmed by incubations of 10-day-old mycelium with veratryl (3,4-dimethoxybenzyl) and anisyl (4-methoxybenzyl) compounds (alcohols, aldehydes, and acids). Veratraldehyde and anisaldehyde were reduced by aryl-alcohol dehydrogenase to their corresponding alcohols, which were oxidized by aryl-alcohol oxidase, producing H(2)O(2). Veratric and anisic acids were incorporated into the cycle after their reduction, which was catalyzed by aryl-aldehyde dehydrogenase. With the use of different initial concentrations of either veratryl alcohol, veratraldehyde, or veratric acid (0.5 to 4.0 mM), around 94% of veratraldehyde and 3% of veratryl alcohol (compared with initial concentrations) and trace amounts of veratric acid were found when equilibrium between reductive and oxidative activities had been reached, regardless of the initial compound used. At concentrations higher than 1 mM, veratric acid was not transformed, and at 1.0 mM, it produced a negative effect on the activities of aryl-alcohol oxidase and both dehydrogenases. H(2)O(2) levels were proportional to the initial concentrations of veratryl compounds (around 0.5%), and an equilibrium between aryl-alcohol oxidase and an unknown H(2)O(2)-reducing system kept these levels steady. On the other hand, the concomitant production of the three above-mentioned enzymes during the active growth phase of the fungus was demonstrated. Finally, the possibility that anisaldehyde is the metabolite produced by P. eryngii for the maintenance of this redox cycle is discussed.     

Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungusPleurotus eryngii

Summary Production of extracellular hydrogen peroxide by fungal oxidases is been investigated as a requirement for lignin degradation. Aryl-alcohol oxidase activity is described in extracellular liquid and mycelium ofPleurotus eryngii and studied under non-limiting nitrogen conditions. This aryl-alcohol oxidase catalyses conversion of primary aromatic alcohols to the corresponding aldehydes and H₂O₂, showing no activity with aliphatic and secondary aromatic alcohols. The enzyme is stable at pH 4.0–9.0, has maximal activity at 45°–50°C and pH 6.0–6.5, is inhibited by Ag⁺, Pb²⁺ and NaN₃, and has aK _m of 1.2 mM using veratryl alcohol as substrate. A single protein band with aryl-alcohol oxidase activity was found in zymograms of extracellular and intracellular crude enzyme preparations fromP. eryngii.     

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H₂O₂-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw.     

Hydrogen-peroxide-producing system ofPleurotus eryngii involving the extracellular enzyme aryl-alcohol oxidase

The effect of benzyl alcohol, benzaldehyde and benzoic acid on the production of extracellular hydrogen peroxide (H₂O₂) by the ligninolytic fungusPleurotus eryngii was investigated. It was found that an equilibrium between oxidative and reductive reactions of these compounds is established, leading to the continuous production of H₂O₂. A multienzymatic cyclic system is proposed in which H₂O₂ is produced extracellularly by the action of aryl-alcohol oxidase on benzyl alcohol, the most abundant compound after redox reactions, and to a lower extent on benzaldehyde. The oxidation products of these reactions, benzaldehyde and benzoic acid, are reduced by intracellular dehydrogenases.     

Enzymatic versatility and thermostability of a new aryl-alcohol oxidase from Thermothelomyces thermophilus M77

BackgroundFungal aryl-alcohol oxidases (AAOx) are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids, with concomitant production of hydrogen peroxide (H₂O₂) as by-product. The H₂O₂ can be provided to lignin degradation pathway, a biotechnological property explored in biofuel production. In the thermophilic fungus Thermothelomyces thermophilus (formerly Myceliophthora thermophila), just one AAOx was identified in the exo-proteome.MethodsThe glycosylated and non-refolded crystal structure of an AAOx from T. thermophilus at 2.6 Å resolution was elucidated by X-ray crystallography combined with small-angle X-ray scattering (SAXS) studies. Moreover, biochemical analyses were carried out to shed light on enzyme substrate specificity and thermostability.ResultsThis flavoenzyme harbors a flavin adenine dinucleotide as a cofactor and is able to oxidize aromatic substrates and 5-HMF. Our results also show that the enzyme has similar oxidation rates for bulky or simple aromatic substrates such as cinnamyl and veratryl alcohols. Moreover, the crystal structure of MtAAOx reveals an open active site, which might explain observed specificity of the enzyme.ConclusionsMtAAOx shows previously undescribed structural differences such as a fully accessible catalytic tunnel, heavy glycosylation and Ca²⁺ binding site providing evidences for thermostability and activity of the enzymes from AA3_2 subfamily.General significanceStructural and biochemical analyses of MtAAOx could be important for comprehension of aryl-alcohol oxidases structure-function relationships and provide additional molecular tools to be used in future biotechnological applications.     

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     

Degradation of veratraldehyde by Alcaligenes paradoxus

Abstract The degradation of veratraldehyde by Alcaligenes paradoxus was studied. Three products, veratric acid, vanillic acid and a minor amount of veratryl alcohol, were identified. The effect of various metabolic inhibitors on the uptake of veratraldehyde, veratric and vanillic acid showed the uptake process to be energy-dependent. The NAD⁺-dependent enzyme responsible for the conversion of veratraldehyde to veratric acid has been separated from veratryl alcohol-oxidizing enzyme.     

Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid

Transformation of veratric (3,4-dimethoxybenzoic) acid by the white rot fungus Phlebia radiata was studied to elucidate the role of ligninolytic, reductive, and demeth(ox)ylating enzymes. Under both air and a 100% O₂ atmosphere, with nitrogen limitation and glucose as a carbon source, reducing activity resulted in the accumulation of veratryl alcohol in the medium. When the fungus was cultivated under air, veratric acid caused a rapid increase in laccase (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) production, which indicated that veratric acid was first demethylated, thus providing phenolic compounds for laccase. After a rapid decline in laccase activity, elevated lignin peroxidase (ligninase) activity and manganese-dependent peroxidase production were detected simultaneously with extracellular release of methanol. This indicated apparent demethoxylation. When the fungus was cultivated under a continuous 100% O₂ flow and in the presence of veratric acid, laccase production was markedly repressed, whereas production of lignin peroxidase and degradation of veratryl compounds were clearly enhanced. In all cultures, the increases in lignin peroxidase titers were directly related to veratryl alcohol accumulation. Evolution of ¹⁴CO₂ from 3-O¹⁴CH₃-and 4-O¹⁴CH₃-labeled veratric acids showed that the position of the methoxyl substituent in the aromatic ring only slightly affected demeth(ox)ylation activity. In both cases, more than 60% of the total ¹⁴C was converted to ¹⁴CO₂ under air in 4 weeks, and oxygen flux increased the degradation rate of the ¹⁴C-labeled veratric acids just as it did with unlabeled cultures.     

Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii.

The production in a 5-1 fermenter of the extracellular enzymes laccase and aryl-alcohol oxidase by the fungus Pleurotus eryngii was studied. The latter enzyme has been purified 50-fold by Sephacryl S-200 and Mono Q chromatography. Purified aryl-alcohol oxidase is a unique flavoprotein with 15% carbohydrate content, a molecular mass of 72.6 kDa (SDS/PAGE) and a pI of 3.9. The enzyme presents wide specificity, showing activity on benzyl, cinnamyl, naphthyl and aliphatic unsaturated alcohols. Neither activity nor inhibition of veratryl alcohol oxidation was found with saturated alcohols, but competitive inhibition was produced by aromatic compounds which were not aryl-alcohol oxidase substrates, such as phenol or 3-phenyl-1-propanol. From these results, it was apparent that a double bond conjugated with a primary alcohol is necessary for substrate recognition by aryl-alcohol oxidase, and that activity is increased by the presence of additional conjugated double bonds and electron donor groups. Both affinity and maximal velocity during enzymic oxidation of methoxybenzyl alcohols were affected in a similar way by ring substituents, increasing from benzyl alcohol (Km = 0.84 mM, Vmax = 52 U/mg) to 4-methoxybenzyl alcohol (Km = 0.04 mM, Vmax = 208 U/mg). Aryl-alcohol oxidase presents also a low oxidase activity with aromatic aldehydes, but the highest activity was found in the presence of electron-withdrawing groups.     

Stability of crude ligninases: Effect of different hydrogen peroxide concentrations in the presence of aromatic alcohols

Summary In absence of veratryl alcohol (VA),Phanerochaete chrysosporium ligninases were extensively inactivated by H₂O₂ concentrations as low as 5.0 μM (1 hr exposure time, pH 4.5, 38°C). In the presence of 2.5 mM VA (but not 2.5 mM benzyl alcohol), protection occurred below 500 μM H₂O₂.     

In situ Inactivation of the Oxidase Activity of Xylem Peroxidases by H2O2 in the H2O2-Producing Xylem of Zinnia elegans

Zinnia elegans stems with 3,3′, 5, 5′-tetramethylbenzidine (TMB) in the presence and in the absence of catalase reveals the presence of xylem oxidase activities in the H₂O₂-producing lignifying xylem cells. This staining of lignifying xylem cells with TMB is the result of two independent mechanisms: one is the catalase-sensitive (H₂O₂-dependent) peroxidase-mediated oxidation of TMB, and the other the catalase-insensitive (H₂O₂-independent) oxidation of TMB, probably due to the oxidase activity of xylem peroxidases. The response of this TMB-oxidase activity of xylem peroxidases to different exogenous H₂O₂ concentrations was studied, and the results showed that H₂O₂ at high concentrations (100–1,000 mM) clearly acted as an inactivator of this xylem TMB-oxidase activity, although some inhibitory effect could still be appreciated at 10 mM H₂O₂. This xylem TMB-oxidase activity resided in a strongly basic cell wall-bound peroxidase (pl about 10.5). Given such a scenario, it may be concluded that this TMB-oxidase activity of peroxidase is located in tissues capable of sustaining H₂O₂ production, and that the in situ oxidase activity shown by this enzyme is inactivated by high H₂O₂ concentrations. Received 20 April 1999/ Accepted in revised form 16 August 1999     

Inhibitory and enhancing effects of NO on H2O2 toxicity: Dependence on the concentrations of NO and H2O2

Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H₂O₂) toxicity and protect cells against H₂O₂ toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H₂O₂ toxicity in cultured liver endothelial cells over a wide range of NO and H₂O₂ concentrations. NO was generated by spermine NONOate (SpNO, 0.001–1 mM), H₂O₂ was generated continuously by glucose/glucose oxidase (GOD, 20–300 U/l), or added as a bolus (200 μM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H₂O₂-induced cell death. SpNO concentrations >0.1 mM were injurious with low H₂O₂ concentrations, but protective at high H₂O₂ concentrations. Protection appeared to be mainly due to inhibition of lipid peroxidation, for which SpNO concentrations as low as 0.01 mM were sufficient. SpNO in high concentration (1 mM) consistently raised H₂O₂ steady-state levels in line with inhibition of H₂O₂ degradation. Thus, the overall effect of NO on H₂O₂ toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H₂O₂ levels and enhancement being predominant with high NO and low H₂O₂ levels.     

Physiological Role of Chlorinated Aryl Alcohols Biosynthesized De Novo by the White Rot Fungus Bjerkandera sp. Strain BOS55

The white rot fungus Bjerkandera sp. strain BOS55 produces veratryl, anisyl, 3-chloroanisyl, and 3,5-dichloroanisyl alcohol and the corresponding aldehydes de novo from glucose. All metabolites are produced simultaneously with the extracellular ligninolytic enzymes and have an important physiological function in the fungal ligninolytic system. Both mono- and dichlorinated anisyl alcohols are distinctly better substrates for the extracellular aryl alcohol oxidases than veratryl alcohol. The aldehydes formed are readily recycled by reduction by washed fungal mycelium, thus creating an extracellular H₂O₂ production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols either in the absence or in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly according to the growth conditions, indicating that production of veratryl alcohol and the production of the (chlorinated) anisyl metabolites are independently regulated. We conclude that the chlorinated anisyl metabolites biosynthesized by the white rot fungus Bjerkandera sp. strain BOS55 can be purposefully produced for ecologically significant processes such as lignin degradation.     

Enhancing the Production of Hydroxyl Radicals by Pleurotus eryngii via Quinone Redox Cycling for Pollutant Removal

The induction of hydroxyl radical (OH) production via quinone redox cycling in white-rot fungi was investigated to improve pollutant degradation. In particular, we examined the influence of 4-methoxybenzaldehyde (anisaldehyde), Mn²⁺, and oxalate on Pleurotus eryngii OH generation. Our standard quinone redox cycling conditions combined mycelium from laccase-producing cultures with 2,6-dimethoxy-1,4-benzoquinone (DBQ) and Fe³⁺-EDTA. The main reactions involved in OH production under these conditions have been shown to be (i) DBQ reduction to hydroquinone (DBQH₂) by cell-bound dehydrogenase activities; (ii) DBQH₂ oxidation to semiquinone (DBQ⁻) by laccase; (iii) DBQ⁻ autoxidation, catalyzed by Fe³⁺-EDTA, producing superoxide (O₂⁻) and Fe²⁺-EDTA; (iv) O₂⁻ dismutation, generating H₂O₂; and (v) the Fenton reaction. Compared to standard quinone redox cycling conditions, OH production was increased 1.2- and 3.0-fold by the presence of anisaldehyde and Mn²⁺, respectively, and 3.1-fold by substituting Fe³⁺-EDTA with Fe³⁺-oxalate. A 6.3-fold increase was obtained by combining Mn²⁺ and Fe³⁺-oxalate. These increases were due to enhanced production of H₂O₂ via anisaldehyde redox cycling and O₂⁻ reduction by Mn²⁺. They were also caused by the acceleration of the DBQ redox cycle as a consequence of DBQH₂ oxidation by both Fe³⁺-oxalate and the Mn³⁺ generated during O₂⁻ reduction. Finally, induction of OH production through quinone redox cycling enabled P. eryngii to oxidize phenol and the dye reactive black 5, obtaining a high correlation between the rates of OH production and pollutant oxidation.The degradation of lignin and pollutants by white-rot fungi is an oxidative and rather nonspecific process based on the production of substrate free radicals (36). These radicals are produced by ligninolytic enzymes, including laccase and three kinds of peroxidases: lignin peroxidase, manganese peroxidase, and versatile peroxidase (VP) (23). The H₂O₂ required for peroxidase activities is provided by several oxidases, such as glyoxal oxidase and aryl-alcohol oxidase (AAO) (9, 18). This free-radical-based degradative mechanism leads to the production of a broad variety of oxidized compounds. Common lignin depolymerization products are aromatic aldehydes and acids, and quinones (34). In addition to their high extracellular oxidation potential, white-rot fungi show strong ability to reduce these lignin depolymerization products, using different intracellular and membrane-bound systems (4, 25, 39). Since reduced electron acceptors of oxidized compounds are donor substrates for the above-mentioned oxidative enzymes, the simultaneous actions of both systems lead to the establishment of redox cycles (35). Although the function of these redox cycles is not fully understood, they have been hypothesized to be related to further metabolism of lignin depolymerization products that require reduction to be converted in substrates of the ligninolytic enzymes (34). A second function attributed to these redox cycles is the production of reactive oxygen species, i.e., superoxide anion radicals (O₂⁻), H₂O₂, and hydroxyl radicals (OH), where lignin depolymerization products and fungal metabolites act as electron carriers between intracellular reducing equivalents and extracellular oxygen. This function has been studied in Pleurotus eryngii, whose ligninolytic system is composed of laccase (26), VP (24), and AAO (9). Incubation of this fungus with different aromatic aldehydes has been shown to provide extracellular H₂O₂ on a constant basis, due to the establishment of a redox cycle catalyzed by an intracellular aryl-alcohol dehydrogenase (AAD) and the extracellular AAO (7, 10). The process was termed aromatic aldehyde redox cycling, and 4-methoxybenzaldehyde (anisaldehyde) serves as the main Pleurotus metabolite acting as a cycle electron carrier (13). A second cyclic system, involving a cell-bound quinone reductase activity (QR) and laccase, was found to produce O₂⁻ and H₂O₂ during incubation of P. eryngii with different quinones (11). The process was described as the cell-bound divalent reduction of quinones (Q) by QR, followed by extracellular laccase oxidation of hydroquinones (QH₂) into semiquinones (Q⁻), which autoxidized to some extent, producing O₂⁻ (Q⁻ + O₂ ⇆ Q + O₂⁻). H₂O₂ was formed by O₂⁻ dismutation (O₂⁻ + HO₂ + H⁺ → O₂ + H₂O₂). In an accompanying paper, we describe the extension of this O₂⁻ and H₂O₂ generation mechanism to OH radical production by the addition of Fe³⁺-EDTA to incubation mixtures of several white-rot fungi with different quinones (6). Among them, those derived from 4-hydroxyphenyl, guaiacyl, and syringyl lignin units were used: 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), and 2,6-dimethoxy-1,4-benzoquinone (DBQ), respectively. Semiquinone autoxidation under these conditions was catalyzed by Fe³⁺-EDTA instead of being a direct electron transfer to O₂. The intermediate Fe²⁺-EDTA reduced not only O₂, but also H₂O₂, leading to OH radical production by the Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺).Although OH radicals are the strongest oxidants produced by white-rot fungi (2, 14), studies of their involvement in pollutant degradation are quite scarce. In this context, the objectives of this study were to (i) determine possible factors enhancing the production of OH radicals by P. eryngii via quinone redox cycling and (ii) test the validity of this inducible OH production mechanism as a strategy for pollutant degradation. Our selection of possible OH production promoters was guided by two observations (6). First, the redox cycle of benzoquinones working with washed P. eryngii mycelium is rate limited by hydroquinone oxidation, since the amounts of the ligninolytic enzymes that remained bound to the fungus under these conditions were not large. Second, H₂O₂ is the limiting reagent for OH production by the Fenton reaction.With these considerations in mind, anisaldehyde and Mn²⁺ were selected to increase H₂O₂ production. As mentioned above, anisaldehyde induces H₂O₂ production in P. eryngii via aromatic aldehyde redox cycling (7). Mn²⁺ has been shown to enhance H₂O₂ production during the oxidation of QH₂ by P. eryngii laccase by reducing the O₂⁻ produced in the semiquinone autoxidation reaction (Mn²⁺ + O₂⁻ → Mn³⁺ + H₂O₂ + 2 H⁺) (26). Mn²⁺ was also selected to increase the hydroquinone oxidation rate, since this reaction has been shown to be propagated by the Mn³⁺ generated in the latter reaction (QH₂ + Mn³⁺ → Q⁻ + Mn²⁺ + 2 H⁺). The replacement of Fe³⁺-EDTA by Fe³⁺-oxalate was also planned in order to increase the QH₂ oxidation rate above that resulting from the action of laccase. Oxalate is a common extracellular metabolite of wood-rotting fungi to which the function of chelating iron and manganese has been attributed (16, 45). The use of Fe³⁺-oxalate and nonchelated Fe³⁺, both QH₂ oxidants, has been proven to enable quinone redox cycling in fungi that do not produce ligninolytic enzymes, such as the brown-rot fungus Gloeophyllum trabeum (17, 40, 41). Finally, phenol and the azo dye reactive black 5 (RB5) were selected as model pollutants.     

Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes

Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H₂O₂ (2 mM) for optimum activity. The apparent K_m values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H₂O₂ were 37, 298, 1,001, 2,367 and 1,313 μM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.     

2-Chloro-1,4-Dimethoxybenzene as a Novel Catalytic Cofactor for Oxidation of Anisyl Alcohol by Lignin Peroxidase

2-Chloro-1,4-dimethoxybenzene (2Cl-14DMB) is a natural compound produced de novo by several white rot fungi. This chloroaromatic metabolite was identified as a cofactor superior to veratryl alcohol (VA) in the oxidation of anisyl alcohol (AA) by lignin peroxidase (LiP). Our results reveal that good LiP substrates, such as VA and tryptophan, are comparatively poor cofactors in the oxidation of AA. Furthermore, we show that a good cofactor does not necessarily serve a role in protecting LiP against H₂O₂ inactivation. 2Cl-14DMB was not a direct mediator of AA oxidation, since increasing AA concentrations did not inhibit the oxidation of 2Cl-14DMB at all. However, the high molar ratio of anisaldehyde formed to 2Cl-14DMB consumed, up to 13:1, indicates that a mechanism which recycles the cofactor is present.     

H2O2 Involvement in Polyamine-Induced Cell Death in Tobacco Leaf Discs

The response of tobacco (Nicotiana tabacum L.) wild-type SR1 leaf discs in terms of reactive oxygen species (ROS) formation and cell death occurrence was evaluated after exposure to the polyamines (PAs) putrescine (Put), spermidine (Spd), and spermine (Spm). Although NADPH oxidase-like enzyme activity was inhibited by all PAs at 3 or 21 h of treatment, H₂O₂ content increased significantly in a time- and concentration-dependent manner, suggesting that H₂O₂ accumulation was linked to the activity of other ROS-generating enzymes. Polyamine oxidase (PAO) activity, which increased markedly upon application of Spd or Spm, is a prime candidate for the increased H₂O₂ accumulation. Except for 0.1 mM Put, which maintained guaiacol peroxidase (GPOX) and catalase (CAT) activities at the same level as the control, the other PA treatments decreased CAT, ascorbate peroxidase, and GPOX activities at 21 h, contributing to the H₂O₂ increase. Esterase activity and Evans blue staining, two cell death parameters, were negatively affected at 3 h of treatment with 1 mM Spd and with both concentrations of Spm, whereas at 21 h there was an increase in cell death with both concentrations of the three PAs, except for 0.1 mM Put, which did not alter those parameters. The expression of the senescence-associated cysteine protease gene CP1 was measured to monitor senescence, a physiological cell death process. Application of all PAs increased the expression of the gene, except for 0.1 mM Put, which decreased its expression at 21 h. This result was in agreement with the prevention of cell death exerted by Put and evidenced by Evans blue staining, esterase activity, and electrolyte release.     

Studies on homoveratric acid transformation by the ligninolytic fungus Pleurotus eryngii

Homoveratric acid (HVA) degradation was observed in cultures of Pleurotus eryngii lacking lignin peroxidase (LiP) activity. Extracellular enzymes seemed responsible for this transformation, and the lack of activity after ultrafiltration of the culture liquid suggests that the presence of some low-molecular-size compounds is required. This hypothesis is supported by rapid HVA transformation after addition of the synthetic laccase substrate 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to the ultrafiltered liquid. HVA transformation by the extracellular enzymes from P. eryngii takes place via C-C breakdown and formation of veratryl alcohol, which is further transformed into veratraldehyde. The same major compounds were found during HVA transformation by LiP from Phanerochaete chrysosporium, but this reaction was not stimulated by ABTS. Although the involvement of other enzymes cannot be ruled out, purified laccase from Pleurotus eryngii caused the same HVA transformation pattern in presence of ABTS. Moreover, veratryl alcohol oxidation by P. eryngii laccase was demonstrated in the presence of ABTS. These results suggest that enzymatic systems lacking LiP could be responsible for natural degradation of lignin.     

Stability of crude ligninases: Effect of different hydrogen peroxide concentrations in the presence of aromatic alcohols

Summary In absence of veratryl alcohol (VA), Phanerochaete chrysosporium ligninases were extensively inactivated by H₂O₂ concentrations as low as 5.0 M (1 hr exposure time, pH 4.5, 38°C). In the presence of 2.5 mM VA (but not 2.5 mM benzyl alcohol), protection occurred below 500 M H₂O₂.