Anisaldehyde and Veratraldehyde Acting as Redox Cycling Agents for H2O2 Production by Pleurotus eryngii

The existence of a redox cycle leading to the production of hydrogen peroxide (H₂O₂) 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 H₂O₂. 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. H₂O₂ levels were proportional to the initial concentrations of veratryl compounds (around 0.5%), and an equilibrium between aryl-alcohol oxidase and an unknown H₂O₂-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.     

Anisaldehyde production and aryl-alcohol oxidase and dehydrogenase activities in ligninolytic fungi of the genus Pleurotus.

A variety of simple aromatic compounds were identified in liquid cultures of the basidiomycetes Pleurotus cornucopiae, P. eryngii, P. floridanus, P. pulmonarius, P. ostreatus, and P. sajor-caju by using gas chromatography-mass spectrometry. Such compounds were detected in fungal cultures on lignin- and straw-containing media, but it was found that they were also produced in the absence of aromatic precursors. Anisylic and hydroxybenzylic compounds (such as alcohols, aldehydes, and acids) were identified, p-anisaldehyde being the most characteristic extracellular metabolite synthesized by these ligninolytic fungi. Small amounts of 3-chloro-p-anisaldehyde were also detected in several species. It is postulated that the balance between the more-or-less-oxidized aromatic compounds can be explained in terms of the activity of fungal enzymes, including aryl-alcohol oxidase and dehydrogenase. The former enzyme shows high affinity for p-anisyl alcohol, which is oxidized to p-anisaldehyde with production of H2O2. The aryl-alcohol dehydrogenase was detected only in the mycelium, where it reduces aromatic aldehydes in the presence of NADPH. Both enzymes could be involved in the redox cycling of these aromatic compounds, providing H2O2 to ligninolytic peroxidases.     

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.     

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.     

Purification and properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium

An intracellular aryl-alcohol dehydrogenase (previously referred to as aryl-aldehyde reductase) was purified from the white-rot fungus Phanerochaete chrysosporium. The enzyme reduced veratraldehyde to veratryl alcohol using NADPH as a cofactor. Other aromatic benzaldehydes were also reduced, but not aromatic ketones. Methoxy-substituted rings were better substrates than hydroxylated ones. The enzyme was also able to reduce a dimeric aldehyde (4-benzyloxy-3-methoxybenzaldehyde). The highest reduction rate was measured when 3,5-dimethoxybenzaldehyde was used as a substrate. On SDS/PAGE the purified enzyme showed one major band with a molecular mass of 47 kDa, whereas gel filtration suggested a molecular mass of 280 kDa. Polyclonal antibodies raised against the gel purified 47-kDa protein were able to immunoprecipitate the aryl-alcohol dehydrogenase indicating that its activity possibly resides entirely in this protein fragment. The pI of the enzyme was 5.2 and it was most active at pH 6.1. The aryl-alcohol dehydrogenase was partially inhibited by typical oxidoreductase inhibitors.     

Purification and characterization of a veratryl alcohol oxidase enzyme from the lignin degrading basidiomycete Pleurotus ostreatus

A veratryl alcohol oxidase (VAO) enzyme was discovered in cultures of Pleurotus ostreatus. The enzyme, which oxidizes veratryl alcohol to veratraldehyde reducing O2 to H2O2, was purified to homogeneity and its main structural and catalytic properties have been determined. The enzyme is a glycoprotein and contains FAD as a prosthetic group. The amino acid composition and carboxy- and amino-terminal sequences were determined. Primary aromatic alcohols with methoxy substituents in position four are good substrates for VAO; cinnamyl alcohol is the substrate which is oxidized faster whereas coniferyl alcohol is oxidized at a slower rate. The enzyme is moderately thermostable (t1/2(55 degrees C) about 1.5 h, apparent melting temperature about 60 degrees C). The enzyme stability in 50% water/organic solvents mixtures has also been studied.     

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     

Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2-producing ligninolytic enzyme

Aryl-alcohol oxidase provides H(2)O(2) for lignin biodegradation, a key process for carbon recycling in land ecosystems that is also of great biotechnological interest. However, little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme, which oxidizes a variety of polyunsaturated alcohols. Different alcohol substrates were docked on the aryl-alcohol oxidase molecular structure, and six amino acid residues surrounding the putative substrate-binding site were chosen for site-directed mutagenesis modification. Several Pleurotus eryngii aryl-alcohol oxidase variants were purified to homogeneity after heterologous expression in Emericella nidulans, and characterized in terms of their steady-state kinetic properties. Two histidine residues (His502 and His546) are strictly required for aryl-alcohol oxidase catalysis, as shown by the lack of activity of different variants. This fact, together with their location near the isoalloxazine ring of FAD, suggested a contribution to catalysis by alcohol activation, enabling its oxidation by flavin-adenine dinucleotide (FAD). The presence of two aromatic residues (at positions 92 and 501) is also required, as shown by the conserved activity of the Y92F and F501Y enzyme variants and the strongly impaired activity of Y92A and F501A. By contrast, a third aromatic residue (Tyr78) does not seem to be involved in catalysis. The kinetic and spectral properties of the Phe501 variants suggested that this residue could affect the FAD environment, modulating the catalytic rate of the enzyme. Finally, L315 affects the enzyme k(cat), although it is not located in the near vicinity of the cofactor. The present study provides the first evidence for the role of aryl-alcohol oxidase active site residues.     

A search for ligninolytic peroxidases in the fungus pleurotus eryngii involving alpha-keto-gamma-thiomethylbutyric acid and lignin model dimers

Because there is some controversy concerning the ligninolytic enzymes produced by Pleurotus species, ethylene release from alpha-keto-gamma-thiomethylbutyric acid (KTBA), as described previously for Phanerochaete chrysosporium lignin peroxidase (LiP), was used to assess the oxidative power of Pleurotus eryngii cultures and extracellular proteins. Lignin model dimers were used to confirm the ligninolytic capabilities of enzymes isolated from liquid and solid-state fermentation (SSF) cultures. Three proteins that oxidized KTBA in the presence of veratryl alcohol and H2O2 were identified (two proteins were found in liquid cultures, and one protein was found in SSF cultures). These proteins are versatile peroxidases that act on Mn2+, as well as on simple phenols and veratryl alcohol. The two peroxidases obtained from the liquid culture were able to degrade a nonphenolic beta-O-4 dimer, yielding veratraldehyde, as well as a phenolic dimer which is not efficiently oxidized by P. chrysosporium peroxidases. The former reaction is characteristic of LiP. The third KTBA-oxidizing peroxidase oxidized only the phenolic dimer (in the presence of Mn2+). Finally, a fourth Mn2+-oxidizing peroxidase was identified in the SSF cultures on the basis of its ability to oxidize KTBA in the presence of Mn2+. This enzyme is related to the Mn-dependent peroxidase of P. chrysosporium because it did not exhibit activity with veratryl alcohol and Mn-independent activity with dimers. These results show that P. eryngii produces three types of peroxidases that have the ability to oxidize lignin but lacks a typical LiP. Similar enzymes (in terms of N-terminal sequence and catalytic properties) are produced by other Pleurotus species. Some structural aspects of P. eryngii peroxidases related to the catalytic properties are discussed.     

De-novo biosynthesis of chlorinated aromatics by the white-rot fungus Bjerkandera sp. BOS55. Formation of 3-chloro-anisaldehyde from glucose.

The white-rot fungus Bjerkandera sp. BOS55 produced de-novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi, other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde and a yet unknown compound containing two chlorine atoms. Additionally GC/MS analysis revealed the production of small amounts of anisyl alcohol and 3-chloro-anisyl alcohol. After 14 days, the extracellular fluid of Bjerkandera BOS55 contained 100 microM veratraldehyde and 50 microM 3-chloro-anisaldehyde. This is the first report of de-novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. Anisaldehyde and 3-chloro-anisaldehyde were also produced by Bjerkandera adusta but not by Phanerochaete chrysosporium.     

Respiratory activity and naphthoquinone synthesis in the fungus Fusarium decemcellulare exposed to oxidative stress

The effect of oxidants (hydrogen peroxide and juglone) on the growth, respiration, and naphthoquinone synthesis in the fungus Fusarium decemcellulare was studied. The addition of the oxidants to the exponential-phase fungus inhibited cell respiration (either partially or completely, depending on the oxidant concentration), culture growth, and naphthoquinone synthesis. The treatment of fungal cells with nonlethal concentrations of H2O2 (below 0.25 mM) and juglone (below 0.1 mM) induced the resistance of cell respiration to cyanide. The residual respiration in the presence of cyanide could be inhibited by benzohydroxamic acid, indicating the occurrence of alternative oxidase. Increased concentrations of oxidants (0.25 mM juglone and 0.5 mM H2O2) rapidly and irreversibly inhibited cell respiration. These observations suggest that the mitochondrial respiratory chain of fungal cells exposed to oxidative stress is subject to the action of active oxygen species. The treatment of fungal cells with nonlethal concentrations of H2O2 and juglone activated cellular glutathione reductase and glucose-6-phosphate dehydrogenase, which are protective enzymes against oxidative stress.     

Novel scheme for biosynthesis of aryl metabolites from L-phenylalanine in the fungus Bjerkandera adusta

Aryl metabolite biosynthesis was studied in the white rot fungus Bjerkandera adusta cultivated in a liquid medium supplemented with L-phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors ((14)C- and (13)C-labelled L-phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in B. adusta cultures. Metabolite formation required de novo protein biosynthesis. Our results show that L-phenylalanine was deaminated to trans-cinnamic acid by a phenylalanine ammonia lyase and trans-cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from L-phenylalanine to aryl metabolites. Additionally, trans-cinnamic acid can also be transformed via beta-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a beta-oxidation degradation intermediate. To our knowledge, this is the first time that a beta-oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from L-phenylalanine is proposed.     

Inhibitory and enhancing effects of NO on H(2)O(2) toxicity: dependence on the concentrations of NO and H(2)O(2)

Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H(2)O(2)) toxicity and protect cells against H(2)O(2) toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H(2)O(2) toxicity in cultured liver endothelial cells over a wide range of NO and H(2)O(2) concentrations. NO was generated by spermine NONOate (SpNO, 0.001-1 mM), H(2)O(2) was generated continuously by glucose/glucose oxidase (GOD, 20-300 U/l), or added as a bolus (200 microM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H(2)O(2)-induced cell death. SpNO concentrations >0.1 mM were injurious with low H(2)O(2) concentrations, but protective at high H(2)O(2) 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 H(2)O(2) steady-state levels in line with inhibition of H(2)O(2) degradation. Thus, the overall effect of NO on H(2)O(2) toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H(2)O(2) levels and enhancement being predominant with high NO and low H(2)O(2) levels.     

关于巯基和Mn~(2+)介导豆壳过氧化物酶氧化藜芦醇的研究

藜芦醇作为非酚型木素模型物具有较高的氧化还原电位,豆壳过氧化物酶(soybeanhullperoxidase,SHP,EC.1.11.1.7)通过依赖于过氧化氢的正常过氧化物酶催化循环不能氧化藜芦醇,但在还原型谷胱甘肽、Mn2+和有机酸络合剂存在下却可以通过不依赖于过氧化氢的氧化酶反应途径完成对藜芦醇的氧化,产物为藜芦醛,反应最适pH为4.2。动力学研究表明该反应遵循顺规序列反应机制;对藜芦醇的表观KM值为4.3mmol/L,对谷胱甘肽的表观KM值为4.8mmol/L。巯基还原剂二硫苏糖醇、L-半胱氨酸和β-巯基乙醇亦可替代还原型谷胱甘肽促进藜芦醇氧化     

Metabolism of cyanide by Phanerochaete chrysosporium

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.     

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.     

Comparison of the Efficacies of Chloromethane, Methionine, and S-Adenosylmethionine as Methyl Precursors in the Biosynthesis of Veratryl Alcohol and Related Compounds in Phanerochaete chrysosporium

The effect on veratryl alcohol production of supplementing cultures of the lignin-degrading fungus Phanerochaete chrysosporium with different methyl-(sup2)H(inf3)-labelled methyl precursors has been investigated. Both chloromethane (CH(inf3)Cl) and l-methionine caused earlier initiation of veratryl alcohol biosynthesis, but S-adenosyl-l-methionine (SAM) retarded the formation of the compound. A high level of C(sup2)H(inf3) incorporation into both the 3- and 4-O-methyl groups of veratryl alcohol occurred when either l-[methyl-(sup2)H(inf3)]methionine or C(sup2)H(inf3)Cl was present, but no significant labelling was detected when S-adenosyl-l-[methyl-(sup2)H(inf3)]methionine was added. Incorporation of C(sup2)H(inf3) from C(sup2)H(inf3)Cl was strongly antagonized by the presence of unlabelled l-methionine; conversely, incorporation of C(sup2)H(inf3) from l-[methyl-(sup2)H(inf3)]methionine was reduced by CH(inf3)Cl. These results suggest that l-methionine is converted either directly or via an intermediate to CH(inf3)Cl, which is utilized as a methyl donor in veratryl alcohol biosynthesis. SAM is not an intermediate in the conversion of l-methionine to CH(inf3)Cl. In an attempt to identify the substrates for O methylation in the metabolic transformation of benzoic acid to veratryl alcohol, the relative activities of the SAM- and CH(inf3)Cl-dependent methylating systems on several possible intermediates were compared in whole mycelia by using isotopic techniques. 4-Hydroxybenzoic acid was a much better substrate for the CH(inf3)Cl-dependent methylation system than for the SAM-dependent system. The CH(inf3)Cl-dependent system also had significantly increased activities toward both isovanillic acid and vanillyl alcohol compared with the SAM-dependent system. On the basis of these results, it is proposed that the conversion of benzoic acid to veratryl alcohol involves para hydroxylation, methylation of 4-hydroxybenzoic acid, meta hydroxylation of 4-methoxybenzoic acid to form isovanillic acid, and methylation of isovanillic acid to yield veratric acid.     

Veratryl alcohol oxidases from the lignin-degrading basidiomycete Pleurotus sajor-caju.

The basidiomycete Pleurotus sajor-caju mineralizes ring-14C-labelled lignin (dehydrogenative polymer) when grown in mycological broth. Under these conditions, two veratryl alcohol oxidase (VAO) enzymes were found in the culture medium. They oxidized a number of aromatic alcohols to aldehydes and reduced O2 to H2O2. The enzymes were purified by ion-exchange and gel-permeation chromatography. The final step of purification on Mono Q resolved the activity into two peaks (VAO I and VAO II). Both enzymes had the same Mr, approx. 71,000, but their isoelectric points differed slightly, 3.8 for VAO I and 4.0 for VAO II. Their amino acid compositions were similar except for aspartic acid/asparagine and glycine. Both enzymes are glycoproteins and contain flavin prosthetic groups. Their pH optima were around 5, and kinetic constants and specificities were similar. 4-Methoxybenzyl alcohol was oxidized the most rapidly, followed by veratryl alcohol. Not all aromatic alcohols were oxidized, neither were non-aromatic alcohols. Cinnamyl alcohol was oxidized at the gamma position. The VAO enzymes thus represent a significantly different route for veratryl alcohol oxidation from that catalysed by the previously found lignin peroxidases from Phanerochaete chrysosporium. The role of the oxidases in biodegradation might be to produce H2O2 during oxidation of lignin fragments.     

Cloning, expression and characterization of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767

ABSTRACT: BACKGROUND: The white-rot fungus Phanerochaete chrysosporium is among the small group of fungi that can degrade lignin to carbon dioxide while leaving the crystalline cellulose untouched. The efficient lignin oxidation system of this fungus requires cyclic redox reactions involving the reduction of aryl-aldehydes to the corresponding alcohols by aryl-alcohol dehydrogenase. However, the biochemical properties of this enzyme have not been extensively studied. These are of most interest for the design of metabolic engineering/synthetic biology strategies in the field of biotechnological applications of this enzyme. RESULTS: We report here the cloning of an aryl-alcohol dehydrogenase cDNA from the white-rot fungus Phanerochaete chrysosporium, its expression in Escherichia coli and the biochemical characterization of the encoded GST and His6 tagged protein. The purified recombinant enzyme showed optimal activity at 37 degreesC and at pH 6.4 for the reduction of aryl- and linear aldehydes with NADPH as coenzyme. NADH could also be the electron donor, while having a higher Km (220 muM) compared to that of NADPH (39 muM). The purified recombinant enzyme was found to be active in the reduction of more than 20 different aryl- and linear aldehydes showing highest specificity for mono- and dimethoxylated Benzaldehyde at positions 3, 4, 3,4 and 3,5. The enzyme was also capable of oxidizing aryl-alcohols with NADP + at 30degreesC and an optimum pH of 10.3 but with 15 to 100-fold lower catalytic efficiency than for the reduction reaction. CONCLUSIONS: In this work, we have characterized the biochemical properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. We show that this enzyme functions in the reductive sense under physiological conditions and that it displays relatively large substrate.     

Preparation of (1S)-verbenone, aromatic and alicyclic carboxylic acids by oxidation of aldehydes, primary and secondary alcohols with Nocardia corallina

(lS)-Verbenone, (S)-perillyl acid, cinnamic acid, meta-nitrocinnamic acid, veratric acid and 2-naphthoic acid were prepared, at 1 mM scale, from the corresponding alcohols or aldehydes with whole cells of Nocardia corallina B-276, in yields from 19 to 71% (w/w). Similar microbiological oxidations gave poor yields with the heterocyclic alcohols: 3-pyridylmethanol, 4-flavanol and 4-chromanol.