Direction of the reactivity of vanillyl-alcohol oxidase with 4-alkylphenols

The covalent flavoprotein vanillyl-alcohol oxidase (VAO) predominantly converts short-chain 4-alkylphenols, like 4-ethylphenol, to (R)-1-(4'-hydroxyphenyl)alcohols and medium-chain 4-alkylphenols, like 4-butylphenol, to 1-(4'-hydroxyphenyl)alkenes. Crystallographic studies have indicated that the active site residue Asp170 is involved in determining the efficiency of substrate hydroxylation. To test this hypothesis, we have addressed the reactivity of Asp170 variants with 4-alkylphenols. The substrate preference of Asp170Glu was similar to wild type VAO. However, Asp170Ser was most active with branched-chain 4-alkylphenols. The hydroxylation efficiency of the Asp170 variants was dependent on the bulkiness of the newly introduced side chain. The Glu170 mutation favored the production of alkenes, whereas the Ser170 mutation stimulated the formation of alcohols.     

Specificity of maltase to maltose in three different directions of reaction: Hydrolytic,vanillyl alcohol glucoside and vanillyl alcohol isomaltoside synthesis

Vanillyl alcohol glucoside is very attractive molecule due to its very powerful physiological activity. In this article, a detailed kinetic study of transglucosylation of vanillyl alcohol was performed. It was demonstrated that this reaction is very efficient (selectivity factor is 149) and occurred by a ping‐pong mechanism with inhibition by glucose acceptor. At low concentration of vanillyl alcohol one additional transglucosylation product was detected. Its structure was determined to be α‐isomaltoside of vanillyl alcohol, indicating that vanillyl alcohol glucoside is a product of the first transglucosylation reaction and a substrate for second, so the whole reaction mechanism was proposed. It was demonstrated that the rate of isomaltoside synthesis is two orders of magnitude smaller than glucoside synthesis, and that maltase has interestingly high K_m value to maltose when vanillyl alcohol glucoside is second transglucosylation substrate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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.     

Enantioselective benzylic microbial hydroxylation of indan and tetralin

A screening with 15 strains of bacteria and fungi targeted at the production of specific hydroxylated benzylic derivatives of indan 1 and tetralin 2 was carried out. Mortierella isabellina, Mortierella ramanniana and Beauveria bassiana were shown to mediate the respective conversions to 1-indanol (3) and 1-tetralol (4), the most satisfactory results being obtained with M. isabellina, which gave 78% conversion of 1 to (1R)-3 (64% yield, 86% ee) after a 2-day-incubation, and 52% conversion of 2 to (1R)-4 (38% yield, 92% ee) in a 4-day-incubation. Over-oxidation of alcohols 3 and 4 during the reactions resulted on the formation of 1-indanone and 2-tetralone, respectively.     

Enzymatic synthesis of a capsinoid by the acylation of vanillyl alcohol with fatty acid derivatives catalyzed by lipases

Capsinoids are a novel group of compounds produced by the Capsicum plant. We synthesized a capsinoid by the lipase-catalyzed esterification of vanillyl alcohol with fatty acid derivatives in an organic solvent. The use of seven out of 17 commercially available lipases, especially Novozym 435, was applicable to the synthesis of vanillyl nonanoate, a model compound of capsinoids. The yield of vanillyl nonanoate under the optimum conditions of 50 mM vanillyl alcohol and 50 mM methyl nonanoate in 500 microl of dioxane, using 20 mg of Novozym 435 and 50 mg of 4 A molecular sieves at 25 degrees C, was 86% in 20 h. Several capsinoid homologues having various acyl chain lengths (C6-C18) were synthesized at 64-86% yields from the corresponding fatty acid methyl ester. The natural capsinoids, capsiate and dihydrocapsiate, were obtained by a 400-fold-scale reaction at these optimum conditions in 60% and 59% isolated yields, respectively.     

Production of catalase-free alcohol oxidase by Hansenula polymorpha

Summary Many of the potential technical applications of alcohol oxidase (MOX; EC 1.1.3.13) are limited by the presence of high activities of catalase in the enzyme preparations. In order to circumvent laborious and costly purification or inactivation procedures, the induction of MOX in a catalase-negative mutant of Hansenula polymorpha has been studied. Emphasis was laid on the induction of activities of MOX and the dissimilatory enzymes in continuous cultures grown on various mixtures of formate/glucose and formaldehyde/glucose. In continuous cultures of the catalase-negative mutant grown on these mixtures, MOX can be induced efficiently. To obtain a stable and productive process, the ratio of the substrates is of critical importance. The optimal ratios of the mixtures for the catalase-negative strain for formate/glucose and formaldehyde/glucose were 3:1 and 1–2:1, respectively. Under identical cultivation conditions the wild-type strain showed similar induction patterns for MOX and the dissimilatory enzymes formaldehyde dehydrogenase (FaDH) and formate dehydrogenase (FoDH). The MOX levels in the catalase-negative strain were approx. 50% of those in the wild-type strain.     

Colorimetric alcohol assays with alcohol oxidase

A method is described for manufacturing crude alcohol oxidase (EC 1.1.3.13) preparations which are suitable for application in colorimetric alcohol assays. The procedure involves a one-step removal of catalase activity from a partially purified preparation of alcohol oxidase from the yeast Hansenula polymorpha via dialysis against 3-amino-1,2,4-triazole and hydrogen peroxide. Thus, the irreversible inactivation of more than 90% of the catalase present was achieved, which is prerequisite for the use of alcohol oxidase preparations in colorimetric alcohol assays via peroxidase-mediated oxidation of dyes. This type of assay was shown to be rapid, accurate and sensitive. The influence of the relative concentrations of the various assay constituents such as alcohol oxidase, catalase and peroxidase is discussed. It is concluded that this colorimetric alcohol assay is particularly suitable for the determination of ethanol in fermentation broths, both in qualitative and in quantitative tests.     

Oxidation of formaldehyde by alcohol oxidase of Candida boidinii

A formaldehyde oxidase activity was found in cell-free extracts of methanol-grown yeast Candida boidinii. Loss of alcohol oxidase activity in a mutant, 4₈, led to loss of the formaldehyde oxidase activity, indicating that the same enzyme is probably responsible for both activities. This could be demonstrated with the purified alcohol oxidase which oxidizes, besides lower primary alcohols, formaldehyde to formate. The K _m value for formaldehyde is 5.7 mM. It seems that alcohol oxidase is not implicated in formaldehyde oxidation in vivo.     

By-passing of unwanted vanillyl alcohol formation using selective adsorbents to improve vanillin production with Phanerochaete chrysosporium

World Journal of Microbiology and Biotechnology -     

Lipase-catalyzed synthesis of capsiate analog using vanillyl alcohol and conjugated linoleic acid under vacuum system

This study focused on the application of vacuum system to synthesize capsiate analogs. The capsiate analogs containing conjugated linoleic acid (CLA) was successfully synthesized in solvent free system via lipase-catalyzed esterification. This esterification was carried out using vanillyl alcohol and CLA as substrates, and Lipozyme RM IM from Rhizomucor miehei as a biocatalyst. The best reaction condition was a molar ratio of 1:2 (vanillyl alcohol to CLA), a reaction temperature of 50 °C, and a lipase loading of 10% (w/w, based on total substrates). Application of vacuum increased the yield of capsiate analog as well as the reaction rate. When the vacuum levels were between 66.7 kPa and 1.3 kPa, an equilibrium yield of 100 mol% was achieved. The maximum yield was approached after only 3 h of reaction at the vacuum levels of higher than 13.3 kPa. The content of 9c,11t-CLA in capsiate analog synthesized was higher than that of 10t,12c-CLA.     

Coniferyl alcohol oxidase — a catechol oxidase?

The physico-chemical properties of coniferyl alcohol oxidase (CAO), a copper containing glycoprotein spatiotemporally associated with lignification in conifers, is reported here. By electron paramagnetic resonance spectroscopy, only type 3 copper was indicated in CAO. CAO oxidizes several laccase substrates; however, it is not a blue-copper protein and monoclonal antibodies against both native and deglycosylated CAO did not recognize any of several laccases. The N-terminal sequence of CAO, H₂N-X E L A Y S P P Y X P S, was non-homologous with known enzymes. Transparent copper, tetrameric structure, aminoacid composition, phenylhydrazine and tropolone inhibition, and SDS enhancement of CAO activity indicate that CAO is an o-diphenol oxidase.     

Catalytic reaction profile for alcohol oxidation by galactose oxidase

Galactose oxidase is a remarkable enzyme containing a metalloradical redox cofactor capable of oxidizing a variety of primary alcohols during enzyme turnover. Recent studies using 1-O-methyl alpha-D-galactopyranoside have revealed an unusually large kinetic isotope effect (KIE) for oxidation of the alpha-deuterated alcohol (kH/kD = 22), demonstrating that cleavage of the 6,6'-di[2H]hydroxymethylene C-H bond is fully rate-limiting for oxidation of the canonical substrate. This step is believed to involve hydrogen atom transfer to the tyrosyl phenoxyl in a radical redox mechanism for catalysis [Whittaker, M. M., Ballou, D. P., and Whittaker, J. W. (1998) Biochemistry 37, 8426-8436]. In the work presented here, the enzyme's unusually broad substrate specificity has allowed us to extend these investigations to a homologous series of benzyl alcohol derivatives, in which remote (meta or para) substituents are used to systematically perturb the properties of the hydroxyl group undergoing oxidation. Quantitative structure-activity relationship (QSAR) correlations over the steady state rate data reveal a shift in the character of the transition state for substrate oxidation over this series, reflected in a change in the magnitude of the observed KIE for these reactions. The observed KIE values have been shown to obey a log-linear correlation over the substituent parameter, Hammett sigma. For the relatively difficult to oxidize nitro derivative, the KIE is large (kH/kD = 12.3), implying rate-limiting C-H bond cleavage for the oxidation reaction. This contribution becomes less important for more easily oxidized substrates (e.g., methoxy derivatives) where a much smaller KIE is observed (kH/kD = 3.6). Conversely, the solvent deuterium KIE is vanishingly small for 4-nitrobenzyl alcohol, but becomes significant for the 4-methoxy derivative (kH2O/kD2O = 1.2). These experiments have allowed us to develop a reaction profile for substrate oxidation by galactose oxidase, consisting of three components (hydroxylic proton transfer, electron transfer, and hydrogen atom transfer) comprising a single-step proton-coupled electron transfer mechanism. Each component exhibits a distinct substituent and isotope sensitivity, allowing them to be identified kinetically. The proton transfer component is unique in being sensitive to the isotopic character of the solvent (H2O or D2O), while hydrogen atom transfer (C-H bond cleavage) is independent of solvent composition but is sensitive to substrate labeling. In contrast, electron transfer processes will in general be less sensitive to isotopic substitution. Our results support a mechanism in which initial proton abstraction from a coordinated substrate activates the alcohol toward inner sphere electron transfer to the Cu(II) metal center in an unfavorable redox equilibrium, forming an alkoxy radical which undergoes hydrogen atom abstraction by the tyrosine-cysteine phenoxyl free radical ligand to form the product aldehyde.     

Oxidation of formaldehyde by alcohol oxidase of Candida boidinii.

A fromaldehyde oxidase activity was found in cellfree extracts of methanol-grown yeast Candida boidinii. Loss of alcohol oxidase activity in a mutant, 48, led to loss of the formaldehyde oxidase activity, indicating that the same enzyme is probably responsible for both activities. This could be demonstrated with the purified alcohol oxidase which oxidizes, besides lower primary alcohols, formaldehyde to formate. The Km value for formaldehyde is 5.7 mM. It seems that alcohol oxidase is not implicated in formaldehyde oxidation in vivo.     

The expression of catechol oxidase activity during the hydroxylation of p-coumaric acid by spinach-beet phenolase

1. The conditions under which oxygen consumption in excess of that required for the hydroxylation of p-coumaric acid to caffeic acid, catalysed by spinach-beet phenolase, can be suppressed, have been examined. 2. With dimethyltetrahydropteridine as electron donor, oxygen uptake was exactly equivalent to the caffeic acid produced, provided that p-coumaric acid was in excess, but with excess of reductant, oxygen uptake caused by the further oxidation of caffeic acid was also observed. 3. With equal concentrations of ascorbate and p-coumaric acid, equivalent oxygen uptake and caffeic acid production was found only in the first stages of the reaction, whereas with NADH substituted for ascorbate, oxygen uptake was in excess throughout. 4. When ascorbate was used, the period of the reaction over which this equivalence was found was decreased at high reaction rates and not observed at all with aged enzyme preparations; equivalence was restored by adding bovine serum albumin to these aged preparations. 5. Equivalence between oxygen consumption and caffeic acid production was observed with NADH, if small quantities of dimethyltetrahydropteridine were also added. 6. It is concluded that hydroxylation proceeds without the concomitant production of caffeic acid only if the enzyme is stabilized for hydroxylation by p-coumaric acid and the reductant, and is protected from attack by o-quinones.     

Biosensing approach for alcohol determination using immobilized alcohol oxidase

Alcohol oxidase (AOD) was immobilized in polypyrrole (PPy) and a random copolymer containing 3-methylthienyl methacrylate and p-vinylbenzyloxy poly(ethyleneoxide) matrices. Immobilization of enzyme was performed via entrapment in conducting polymers during electrochemical polymerization of pyrrole through the thiophene moiety of the copolymer. Three different alcohols, namely methanol, ethanol and n-propanol, were used as substrates. Maximum reaction rates, Michaelis–Menten constants, optimum temperature and pH values, operational stabilities and shelf life of the enzyme electrodes were investigated.     

Enantioselective recognition mechanism of secondary alcohol by surfactant-coated lipases in nonaqueous media.

The enantioselective recognition mechanism of secondary alcohol by lipases originated from Candida rugosa and Pseudomonas cepacia was elucidated on the basis of the kinetic study of the esterification of alcohol with lauric acid in isooctane. To obtain inherent kinetic parameters, we utilized a surfactant-coated lipase whose conformation is considered to be an "open" form in a homogeneous organic solvent. Based on the experimental results, the enantioselectivity of lipases was found to be derived from the difference in the V(max) values between the two enantiomers. The same result was observed when lipases of different origin and substrates with different molecular structures were applied. © 1999 John Wiley & Sons, Inc.     

Anticonvulsive and free radical scavenging activities of vanillyl alcohol in ferric chloride-induced epileptic seizures in Sprague-Dawley rats

Vanillyl alcohol (VA) is a component of Gastrodia elata Bl. (GE), which is a traditional Chinese herb widely used to treat convulsive disorders or dizziness. This study examined the role of VA in the anticonvulsive properties of GE in a Sprague-Dawley rat model of epilepsy. The anticonvulsive and free radical scavenging activities of VA were examined after intracortical injection of ferric chloride (100 mM, 8 microl) to induce epileptic seizures. These seizures were verified by behavioral observations and electroencephalographic (EEG) and electromyographic (EMG) recordings. Ferric chloride injection resulted in increased lipid peroxide levels in the ipsilateral and contralateral cerebral cortex, and increased luminol-chemiluminescence (CL) and lucigenin-CL counts in the peripheral blood. Intraperitoneal injection (i.p.) of VA (200 mg/kg or 100 mg/kg) or phenytoin 10 mg/kg prior to ferric chloride administration significantly inhibited wet dog shakes (WDS) and lipid peroxide levels in the bilateral cerebral cortex. VA 200 mg/kg also significantly reduced luminol-CL and lucigenin-CL counts in the peripheral blood, but no significant effect was observed following administration of VA 100 mg/kg or phenytoin. These data indicate that VA has both anticonvulsive and suppressive effects on seizures and lipid peroxidation induced by ferric chloride in rats. Data from the present study also demonstrate that VA has free radical scavenging activities, which may be responsible for its anticonvulsive propertics. This finding is consistent with the results from previous studies that generation of superoxide radical evoked by injection of iron salt into rat brain plays a critical role in ferric chloride-induced seizures. In addition, the results of the present study suggest that the anticonvulsive effect of GE may be attributable, at least in part, to its VA component.