Tuning of the product spectrum of vanillyl-alcohol oxidase by medium engineering.

The flavoenzyme vanillyl-alcohol oxidase (VAO) catalyzes the conversion of 4-alkylphenols through the initial formation of p-quinone methide intermediates. These electrophilic species are stereospecifically attacked by water to yield (R)-1-(4'-hydroxyphenyl)alcohols or rearranged in a competing reaction to 1-(4'-hydroxyphenyl)alkenes. Here, we show that the product spectrum of VAO can be controlled by medium engineering. When the enzymatic conversion of 4-propylphenol was performed in organic solvent, the concentration of the alcohol decreased and the concentration of the cis-alkene, but not the trans-alkene, increased. This change in selectivity occurred in both toluene and acetonitrile and was dependent on the water activity of the reaction medium. A similar shift in alcohol/cis-alkene product ratio was observed when the VAO-mediated conversion of 4-propylphenol was performed in the presence of monovalent anions that bind specifically near the enzyme active site.

Enzymatic synthesis of vanillin.

Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol is further oxidized to vanillin. Catalysis is limited by the formation of an abortive complex between enzyme-bound flavin and creosol. Moreover, in the second step of the process, the conversion of vanillyl alcohol is inhibited by the competitive binding of creosol. The VAO-catalyzed conversion of vanillylamine proceeds efficiently at alkaline pH values. Vanillylamine is initially converted to a vanillylimine intermediate product, which is hydrolyzed nonenzymatically to vanillin. This route to vanillin has biotechnological potential as the widely available principle of red pepper, capsaicin, can be hydrolyzed enzymatically to vanillylamine.

Peroxidase-mediated cross-linking of a tyrosine-containing peptide with ferulic acid.

The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase and hydrogen peroxide resulted in the formation of dehydrodimers. Kinetic studies of conversion rates of either the peptide or ferulic acid revealed conditions that allow formation of heteroadducts of GYG and ferulic acid. To a GYG-containing incubation mixture was added ferulic acid in small aliquots, therewith keeping the molar ratio of the substrates favorable for hetero-cross-linking. This resulted in a predominant product consisting of two ferulic acid molecules dehydrogenatively linked to a single peptide and, furthermore, two ferulic acids linked to peptide oligomers, ranging from dimers to pentamers. Also, mono- and dimers of the peptide were linked to one molecule of ferulic acid. A mechanism explaining the formation of all these products is proposed.

Enhanced sensitivity of Cypridina luciferin analogue (CLA) chemiluminescence for the detection of *O2(-) with non-ionic detergents.

Superoxide anion-triggered chemiluminescence of Cypridina luciferin analogue (CLA), 2-methyl-6-phenyl-3,7-dohydroimidazo[1,2-alpha]pyrazin-3-one, is enhanced by non-ionic detergents such as Tween 20, Triton X-100 and Tween 80. At the concentration of 0.6% (v/v) the largest increase (2.7-fold) of CLA light intensity was obtained with Tween 20, followed by Tween 80 and Triton X-100. Using this detergent-amplified CLA chemiluminescence, the detection limits of xanthine and xanthine oxidase were examined at pH 7.4 and reinvestigated at pH 5.5. At pH 5.5, concentrations of xanthine and xanthine oxidase as low as 5 nmol/L and 3.85 x 10(-7) U/mL, respectively, could be accurately determined, whereas, under the experimental conditions used, at pH 7.4 the lowest concentrations of xanthine and xanthine oxidase detectable were 10 nmol/L and 3.85 x 10(-6) U/mL. The lowest detectable values of xanthine and xanthine oxidase obtained at pH 5.5 are about 400 and 10 times lower than those previously reported. The detection limit of xanthine (5 nmol/L) by this chemiluminescent-based method is about 200 and 20 times more sensitive than the determination of xanthine by enzymatic means or by HPLC with detection limits of 1 micromol/L and 0.1 micromol/L, respectively. Our data suggest that this chemiluminescent probe can detect concentrations of superoxide anion below the nanomolar range.

Comparative studies of the chemiluminescent horseradish peroxidase-catalysed peroxidation of acridan (GZ-11) and luminol reactions: effect of pH and scavengers of reactive oxygen species on the light intensity of these systems.

In this study, the chemiluminescent horseradish peroxidase/H(2)O(2)-catalysed oxidation of acridan (GZ-11) substrate was compared with the well-characterized light-producing luminol reaction. p-Iodophenol and p-phenylphenol were used as enhancers, respectively, for the luminol and acridan reactions. These two light-producing systems showed significant differences in relation to the effect of pH, as well as the effect of scavengers of reactive oxygen species, on the light intensity. Light production measured could be as low as pH 2.6 in the acridan reaction, whereas light emission was not detected in the luminol system below pH 5.6. In contrast with the luminol system, it was found that superoxide dismutase does not inhibit the light intensity of the acridan system. This suggests that superoxide anion does not participate in the mechanism of the light-emitting steps of the acridan reaction. Addition of hydroxyl radical scavengers, mannitol and benzoate, to the acridan reaction medium had no appreciable effect on the chemiluminescent intensity, indicating that hydroxyl radicals do not interfere in light-emitting steps. In addition, the peroxidation of the acridan substrate was found to be very slow at pH 5.6 in the absence of the enhancer, p-phenylphenol, whereas in its presence a rapid degradation of the acridan substrate was observed. Therefore, it is suggested that the enhancer might be initially oxidized by the HRP/H(2)O(2) system, resulting in the formation of the enhancer radical, which could be the actual oxidizing agent of the acridan substrate. Together, the data presented in this paper indicate that the chemiluminescent horseradish peroxidase-catalysed peroxidation of acridan (GZ-11) is more specific than the luminol reaction for the reactive oxygen species involved in the light-emitting steps, i. e, H(2)O(2).

Characterization of different commercial soybean peroxidase preparations and use of the enzyme for N-demethylation of methyl N-methylanthranilate To produce the food flavor methylanthranilate.

The potential of different peroxidase preparations for the N-demethylation of methyl N-methylanthranilate to produce the food flavor methylanthranilate (MA) was investigated. All tested peroxidase preparations were able to catalyze the N-dealkylation. The tested soybean preparations vary widely with respect to their heme content. Furthermore, the operational stability of purified soybean peroxidase (SP) is at least 25-fold lower than that of horseradish peroxidase and only 5-fold higher than that of microperoxidase 8. Thus, the presence of a large protein chain around a porphyrin cofactor in a peroxidase is, by itself, insufficient to explain the observed differences in operational stability. Despite its relatively low operational stability, SP proved to be the most efficient biocatalyst for the production of MA with high yield and purity, especially observed at the high temperature and low pH values at which SP appeared to be optimally active.

Antioxidant activities of carotenoids: quantitative relationships between theoretical calculations and experimental literature data.

Quantitative structure activity relationships (QSARs) are described for the antioxidant activity of series of all-trans carotenoids. The antioxidant activity of the carotenoids is characterised by literature data for (i) their relative ability to scavenge the ABTS*+ radical cation, reflected by the so-called trolox equivalent antioxidant capacity (TEAC) value, (ii) their relative rate of oxidation by a range of free radicals, or (iii) their capacity to inhibit lipid peroxidation in multilamellar liposomes, leading to a decrease in formation of thiobarbituric acid reactive substances (TBARS). All these antioxidant values for radical scavenging action correlate quantitatively with computer-calculated ionisation potentials of the carotenoids. These correlations are observed both when the ionisation potential is calculated as the negative of the energy of the highest occupied molecular orbital (-E(HOMO)) of the molecule, or as the relative change in heat of formation (deltadeltaHF) upon the one-electron oxidation of the carotenoids. The calculations provide a theoretical assay able to characterise the intrinsic electron donating capacity of an antioxidant, in hydrophilic, hydrophobic or artificial membrane environment.

Conversion of pentahalogenated phenols by microperoxidase-8/H2O2 to benzoquinone-type products.

This study reports the microperoxidase-8 (MP8)/H2O2-catalyzed dehalogenation of pentafluorophenol and pentachlorophenol, compounds whose toxic effects and persistence in the environment are well documented. The primary products of this dehalogenation reaction appear to be the corresponding tetrahalo-p-benzoquinones. Under the conditions used, the fluorinated phenol and its intermediate products are more susceptible to degradation than the corresponding chlorinated analogue and its products. The main degradation products of tetrachloro-p-benzoquinone and tetrafluoro-p-benzoquinone were identified as trichlorohydroxy-p-benzoquinone and trifluorohydroxy-p-benzoquinone, respectively. This secondary conversion of tetrafluoro-p-benzoquinone and tetrachloro-p-benzoquinone was not mediated by MP8, but was driven by H2O2. Evidence is presented for a mechanism where H2O2 molecules and not hydroxide anions are the reactive nucleophilic species attacking the tetrahalo-p-benzoquinones. In addition to the formation of the trihalohydroxy-p-benzoquinones, the formation of adducts of the tetrahalo-p-benzoquinone products with ethanol, present in the incubation medium, was observed. The adduct from the reaction of tetrachloro-p-benzoquinone with ethanol was isolated and identified as trichloroethoxyquinone. Thus, the present paper describes a system in which the formation of tetrahalo-p-benzoquinone-type products by an oxidative heme-based catalyst could be unequivocally demonstrated.

Regio- and stereospecific conversion of 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase.

The regio- and stereospecific conversion of prochiral 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase was investigated. The enzyme was active, with 4-alkylphenols bearing aliphatic side chains of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol and 4-n-propylphenols. These short-chain 4-alkylphenols are stereoselectively hydroxylated to the corresponding (R)-1-(4'-hydroxyphenyl)alcohols (F. P. Drijfhout, M. W. Fraaije, H. Jongejan, W. J. H. van Berkel, and M. C. R. Franssen, Biotechnol. Bioeng. 59:171-177, 1998). (S)-1-(4'-Hydroxyphenyl)ethanol was found to be a far better substrate than (R)-1-(4'-hydroxyphenyl)ethanol, explaining why during the enzymatic conversion of 4-ethylphenol nearly no 4-hydroxyacetophenone is formed. Medium-chain 4-alkylphenols were exclusively converted by vanillyl-alcohol oxidase to the corresponding 1-(4'-hydroxyphenyl)alkenes. The relative cis-trans stereochemistry of these reactions was strongly dependent on the nature of the alkyl side chain. The enzymatic conversion of 4-sec-butylphenol resulted in two (4'-hydroxyphenyl)-sec-butene isomers with identical masses but different fragmentation patterns. We conclude that the water accessibility of the enzyme active site and the orientation of the hydrophobic alkyl side chain of the substrate are of major importance in determining the regiospecific and stereochemical outcome of vanillyl-alcohol oxidase-mediated conversions of 4-alkylphenols.

Regulation of the flavin redox potential by flavin-binding antibodies.

Single-chain Fv antibody fragments binding different flavin forms [10-(5'-carboxybutyl-)flavin (Fl[ox]) and 10-(5'-carboxybutyl)-1,5-dihydroflavin (Fl[red])] have been generated from an antibody phage-display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These 'flavobodies' are characterized by time-resolved and steady-state fluorescence spectroscopy, by competitive ELISA methods (mapping of the antigen-binding site), and by molecular modelling. The three-dimensional models of the antigen-binding sites are consistent with the experimental results. Binding of anti-Fl(red) 5 to flavin increases the redox potential, mainly due to an Arg residue interacting with the flavin N1. Thus anti-Fl(red) 5 shows an 'oxidase-like' redox-potential behaviour, confirming the idea that positively charged residues in the vicinity of N1 increase the redox potential. The results obtained with anti-Fl(ox), which do not resemble a natural flavoprotein, show that when the pyrimidine-like nucleus of the flavin is not involved in binding, the redox potential is not significantly affected. These results are in contrast to those obtained with chicken riboflavin-binding protein.

Motor and cognitive development in children with congenital hypothyroidism: a long-term evaluation of the effects of neonatal treatment.

Although neonatal thyroid screening programs have been of value in preventing cerebral damage, it is still controversial whether patients with congenital hypothyroidism achieve normal motor and cognitive skills. We studied the motor and cognitive skills of 72 children with early-treated congenital hypothyroidism and 35 control subjects at the ages of 7 1/2 and 9 1/2 years. The relative influence of cause, blood thyroxine concentration at the time of screening, and age at the start of thyroxine replacement therapy on motor and cognitive development was investigated. Despite having received treatment at a mean age of 23 days, children with low neonatal thyroxine concentrations (< 50 nmol/L) at screening, particularly children with thyroid agenesis, had significant motor problems and borderline intelligence scores as late as 9 1/2 years of age. Balance and gross motor functions seemed to decline between 7 1/2 and 9 1/2 years of age, whereas language and memory functions seemed to be maintained. Significant correlations between the start of therapy and both motor scores and performance IQ scores at the age of 7 1/2 years in children with severe hypothyroidism show the importance of early treatment for these patients.

Low-density lipoprotein receptors in rat adipocytes: regulation with fasting.

Adipose tissue metabolism is exquisitely sensitive to caloric intake. With increasing adiposity more triglyceride and cholesterol are stored within increasingly large adipocytes, whereas less triglyceride and cholesterol are stored as the size of the fat cell decreases. A portion of the uptake of cholesterol by adipocytes is mediated by low-density lipoprotein (LDL) receptors. The present studies addressed whether LDL receptors are differentially regulated in adipose tissue and the liver during fasting in the rat. Two days of fasting caused a reduction in body weight with an approximately 40% decrease in the epididymal fat depot and fat cell size. No changes in serum cholesterol were noted, but serum triglycerides fell approximately 55% with fasting. LDL receptors detected by immunoblotting decreased progressively with fasting to levels that were 95% below controls in adipocytes isolated from epididymal fat pads by 2-3 days. In contrast, hepatic LDL receptor expression was unaltered by fasting. After 2 days of fasting, the rate of synthesis of LDL receptors in isolated adipose cells was decreased approximately 35%, whereas levels of LDL receptor mRNA were diminished approximately 55%. It is concluded that the expression of LDL receptors in rat adipocytes is markedly downregulated during fasting through transcriptional and posttranscriptional mechanisms. Furthermore, LDL receptor expression is differentially regulated in adipose tissue and liver during fasting in the rat.

The pH in reversed micelles as imposed by the dihydrogen/proton redox couple and indicated by viologens and cytochrome c3 using hydrogenase as redox catalyst.

The pH values in reversed micelles were measured, making use of the hydrogenase enzyme as redox catalyst short-circuiting the viologen oxidized/semiquinone redox states. The hydrogenases from Desulfovibrio vulgaris (Hildenborough) and from Megasphaera elsdenii were applied. The observed pH values in reversed micelles were not dependent on the type of hydrogenase. Two cationic [cetyltrimethylammonium bromide and dodecylammonium propionate (DAP)] and two anionic sodiumdodecyl sulphate, sodium di(ethylhexyl)sulfosuccinate types of reversed micelles were used in combination with viologens having distinguishable valencies. It was observed that, in the cationic-reversed micelles, the dissociation constant for the semiquinone dimer had about the same value as compared to bulk water, while this value was significantly higher in the anionic-reversed micelles. Furthermore, the dissociation constant was independent of the concentration of viologen semiquinone in the reversed micelle, indicating that exchange kinetics are faster than the dimerisation process. With the exception of DAP, a linear relation exists, pH = a.pHrm + b, between the pH of the bulk water and the pH as measured in the reversed micelle (pHrm). In all these cases the value of a is smaller than unity, the value of b ranges between 1.6-2.7. For DAP the pHrm is always around 7. In DAP-reversed micelles, the counter-ion propionate probably serves as an internal buffer. Using cytochrome c3 as pH indicator in combination with N,N'-di(3-aminopropyl)-4,4'-bipyridinium)4+ to take care of electron transfer, in cetyltrimethylammonium-bromide-reversed micelles the pHrm is about the same as indicated by the viologen; in SDS-reversed micelles the pHrm is always lower than that indicated by N,N'-di(3-aminopropyl)4,4'-pyridinium4+. In contrast to cytochrome c3 from D. vulgaris, which in reversed micelles cannot become reduced directly by its D. vulgaris hydrogenase, the hydrogenase of M. elsdenii is able to reduce its ferredoxin directly.

Time-resolved fluorescence and circular dichroism of porphyrin cytochrome c and Zn-porphyrin cytochrome c incorporated in reversed micelles.

Interactions between fluorescent horse heart cytochrome c derivatives (e. g. porphyrin cytochrome c and Zn-porphyrin cytochrome c) with surfactant interfaces in reversed micellar solutions have been studied, using different spectroscopic techniques. Anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] and cationic (cetyltrime-thylammonium bromide, CTAB) surfactant solutions have been used in order to investigate the effects of charge interactions between proteins and interfaces. Circular dichroism reveals that much of the protein secondary structure is lost in AOT-reversed micelles, especially when the molar water/surfactant ratio, wo, is high (wo = 40), whereas in CTAB-reversed micelles secondary structure seems to be preserved. Time-resolved fluorescence measurements of the porphyrin in the cytochrome c molecule yields information about the changes in structure and the dynamics of the protein upon interaction with surfactant assemblies both in aqueous and in hydrocarbon solutions. With AOT as surfactant a strong interaction between protein and interface can be observed. The effects found in aqueous AOT solution are of the same kind as in hydrocarbon solution. In the CTAB systems the interactions between protein and surfactant are much less pronounced. The measured effects on the fluorescence properties of the proteins are different in aqueous and hydrocarbon solutions. In general, the observations can be explained by an electrostatic attraction between the overall positively charged protein molecules and the anionic AOT interface. Electrostatic attraction can also occur between the cytochrome c derivatives and CTAB because there is a negatively charged zone on the surface of the proteins. From the fluorescence anisotropy decays it can be concluded that in the CTAB-reversed micellar system these interactions are not important, whereas in an aqueous CTAB solution the proteins interact with surfactant molecules.

Spectroscopic properties of horse liver alcohol dehydrogenase in reversed micellar solutions.

Catalytic and spectroscopic properties of alcohol dehydrogenase from horse liver, incorporated in reversed micellar media, have been studied. Two different reversed micellar systems have been used, one containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT], the other containing a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. With 1-hexanol as substrate the turnover number of the enzyme in AOT-reversed micelles is strongly dependent on the water content of the system. At low wo ([H2O]/[surfactant]) (wo less than 20) no enzymatic activity can be detected whereas at high wo (wo = 40) the turnover is only slightly lower than in aqueous solution. In CTAB-reversed micelles the dependence of the turnover number on wo is much less. The enzymatic activity is in this case significantly lower than in aqueous solution and increases only slightly with an increasing water content of the reversed micelles. Possible interactions of the protein with the surfactant interfaces in the reversed micellar media were studied via circular dichroism and fluorescence measurements. From the circular dichroism of the protein backbone it is observed that the protein secondary structure is not significantly affected upon incorporation in the reversed micelles since the far-ultraviolet spectrum is not altered. Results from time-resolved fluorescence anisotropy experiments indicate that, especially in AOT-reversed micelles, interactions between the protein and the surfactant interface are largely electrostatic in nature, as evident from the dependence on the pH of the buffer used. In CTAB-reversed micellar solutions such interactions appear to be much less pronounced than in AOT.

Rules for optimization of biocatalysis in organic solvents.

General rules for the optimization of different biocatalytic systems in various types of media containing organic solvents are derived by combining data from the literature, and the logarithm of the partition coefficient, log P, as a quantitative measure of solvent polarity. (1) Biocatalysis in organic solvents is low in polar solvents having a log P < 2, is moderate in solvents having a log P between 2 and 4, and is high in a polar solvents having a log P > 4. It was found that this correlation between polarity and activity parallels the ability of organic solvents to distort the essential water layer that stabilizes the biocatalysts. (2) Further optimization of biocatalysis in organic solvents is achieved when the polarity of the microenvironment of the biocatalyst (log P(i)) and the continuous organic phase (log P(cph)) is tuned to the polarities of both the substrate (log P(s)) and the product (log P(p)) according to the following rules: |log P(i) - log P(s)| and |log P(cph) - log P(p)| should be minimal and |log P(cph) - log P(s)| and |log P(i) - log P(p)| should be maximal, with the exception that in the case of substrate inhibition log P(i), should be optimized with respect to log P(s) In addition to these simple optimization rules, the future developments of biocatalysis in organic solvents are discussed.