Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Peroxidase Can Perform the Hydroxylation Step in the "oxidative Cascade" during Oxidation of Tea Catechins
    Verloop, Annewieke J.W. ; Vincken, Jean Paul ; Gruppen, Harry - \ 2016
    Journal of Agricultural and Food Chemistry 64 (2016)42. - ISSN 0021-8561 - p. 8002 - 8009.
    black tea - hydroxylation - peroxidase - theatridimensins - tyrosinase - UHPLCâMS/MS

    The formation of black tea thearubigins involves at least two of the following oxidation steps: (i) oligomerization, (ii) rearrangement, and (iii) hydroxylation. The first two are mainly catalyzed by polyphenol oxidase (PPO), whereas the enzyme responsible for hydroxylation has not yet been identified. Two main oxidative activities, peroxidase (POD) and PPO, occur in tea leaves. POD was hypothesized to be responsible for hydroxylation. Model systems with horseradish POD and mushroom tyrosinase were used investigating hydroxylation of theaflavins (TFs). POD was found capable of hydroxylation. TFs with up to five extra hydroxyl groups were annotated by their MS2 data. Hydroxylation by POD was also shown for theanaphtoquinones, theatridimensins, and dehydrodicatechins. The H2O2 concentration influenced the extent of hydroxylation, decreasing it at concentrations above 0.01 mM. TFs with up to five extra hydroxyl groups and traces of other hydroxylated oligomeric catechins could be annotated in black tea without any sample pretreatment, using a selective screening method with reversed-phase ultrahigh-performance liquid chromatography mass spectrometry.

    Oligomerization and hydroxylation of green tea catechins by oxidative enzymes
    Verloop, J.W. - \ 2016
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789462577770 - 146
    green tea - oxidation - theaflavine - peroxidase - oxygenases - flavanols - phenolic compounds - catechol oxidase - mass spectrometry - maldi-tof - process control - groene thee - oxidatie - theaflavine - peroxidase - oxygenasen - flavanolen - fenolverbindingen - catechol oxidase - massaspectrometrie - maldi-tof - procesbewaking

    Black teas are known for their characteristic brown colour, bitter taste and astringent mouth feel. These sensory characteristics are mainly influenced by the phenolic oxidation products present in black tea. The oxidation of phenolics from green tea leaves during black tea manufacturing is an uncontrolled process. With the objective to make tea oxidation a more controlled process, the aim of this thesis was to understand the enzymatic oxidation reactions occurring during tea oxidation, and to enable more rapid analysis of complex mixtures of phenolics. By incubating green tea catechins with an exogenous tyrosinase, a black tea-like phenolic profile was obtained, enriched in theaflavins, which are important for quality of tea. Further oxidation of theaflavins yielded theatridimensins, in which an epicatechin is coupled to the benzotropolone ring of theaflavin. By using MS/MS on selected ions these theatridimensins were shown to occur in black tea. This MS method could also be used to distinguish isomeric procyanidins and dehydrocatechins based on MS2 fragments, as well as the different interflavanic configurations occurring in dehydrodicatechins. The dehydrocatechins were shown to occur in black tea as well. Besides these oligomerization reactions mediated by tyrosinase, oxidation of tea phenolics also comprised hydroxylation. The enzymatic activity from tea leaves responsible for this hydroxylation reaction, was found to be peroxidase. All findings were condensed into a new version of the ‘oxidative cascade hypothesis’, describing the oxidation reactions towards formation of a black tea.

    Mesoscale structure and techno-functional properties of enzymatically cross-linked a-lactalbumin nanoparticles
    Dhayal, S.K. - \ 2015
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Peter Wierenga. - Wageningen : Wageningen University - ISBN 9789462572812 - 152
    enzymatische cross-linking - eiwitten - nanotechnologie - deeltjes - functionele eigenschappen - polymerisatie - peroxidase - enzymatic cross-linking - proteins - nanotechnology - particles - functional properties - polymerization - peroxidase

    Abstract

    The aim of this thesis is to understand the connection between molecular, meso and macroscales of enzymatically cross-linked proteins. It was hypothesised that the techno-functional properties at macroscale, such as bulk rheology and foam stability, are affected by the structure of nanoparticles at mesoscale. The approach was to make α-lactalbumin (α-LA) nanoparticles by using two different enzymes, horseradish peroxidase (HRP) or microbial transglutaminase (mTG), to produce an open and compact mesoscale structure, respectively. In addition to the control over the mesoscale structure, the size of the nanoparticles can be independently controlled by varying the dosage of hydrogen peroxide in the case of HRP and by thermal inactivation in the case of mTG. The other important parameters determining the size are protein concentration and ionic strength. The size (radius of gyration) range that could be achieved by varying the above mentioned control parameters is 20 – 200 nm. The polydispersed nanoparticles were separated by asymmetrical flow field flow fractionation (AF4) and characterised inline with multi angle light scattering (MALS). Polymerization of apo α-LA with HRP and mTG proceeds in a step growth way i.e. first monomers react to form oligomers and the oligomers are cross-linked to form polymers (nanoparticles). Extensive cross-linking of α-LA with HRP gives rise to not only di-tyrosine cross-links, but also tri–octa tyrosine cross-links, which was hitherto unknown. The two different mesoscale structures result in gels of different storage moduli. The storage modulus of gels made by concentrating the α-LA/mTG nanoparticles was around ten times higher than that made with open nanoparticles. The half-life time (t0.5) of the foam made with α-LA nanoparticles was two to six times higher than that of the monomeric α-LA. The higher foam-stability of the α-LA nanoparticles as compared to the monomeric α-LA is due to their higher thickness of the interfacial layer and thin films. In conclusion, it is shown that the techno-functional properties of α-LA are directly correlated to the size and meso-scale structures of the nanoparticles and enzymatic cross-linking is an effective way to control them.

    A duplex approach for immunochemical staining and typing of protein in western blots
    Kuczius, T. ; Brandstädter, L. ; Karch, H. ; Langeveld, J.P.M. - \ 2011
    Analytical Biochemistry 409 (2011)2. - ISSN 0003-2697 - p. 260 - 266.
    cellular prion proteins - bovine spongiform encephalopathy - electrophoretic transfer - nitrocellulose - scrapie - peroxidase - brain - prpsc - visualization - sensitivity
    The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a specific banding pattern. For protein characterization, several antibodies that recognize different epitopes within the protein sequence are used. However, repeated or parallel gel runs are needed. Here we describe a sequential determination of prion proteins in healthy and pathological states that both consist of di-, mono-, and nonglycosylated isoforms using a single blot with two antibodies from two species that recognize one antigen with two epitopes. The band signals are visualized by using different chemiluminescent substrate reactions. This application can be used in the fields of diagnostics and public health to detect full-length and fragmented proteins and can also be used for characterization of overlaying proteins.
    Peroxidase-mediated cross-linking of bovine a-lactalbumin
    Heijnis, W.H. - \ 2010
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Willem van Berkel; Peter Wierenga. - S.l. : s.n. - ISBN 9789085858324 - 120
    alfa-lactalbumine - peroxidase - schuimen - schuim - enzymatische cross-linking - alpha-lactalbumin - peroxidase - foaming - foams - enzymatic cross-linking
    The research presented in this thesis aimed at controlling the horseradish peroxidase-catalyzed cross-linking of bovine α lactalbumin and the implications of this cross-linking for the foam stabilizing properties. Attention is also given to microreactors and their potential to control the enzymatic cross-linking of proteins.
    The proportion of cross-linked α lactalbumin dimers, oligomers and polymers could be directed by variations in ionic strength, pH, H2O2, and temperature.
    Covalent α lactalbumin dimers were proteolytic digested. FTMS analysis of the peptide mixture resulted in the unambiguous identification of a Tyr18 Tyr50 dityrosine cross-link. Structural modeling of the α lactalbumin dimer indicated that favorite electrostatics direct the selectivity of the cross-linking reaction and, hence, the formation of an intermolecular cross-link. The formation of the Tyr18-Tyr50 cross-link suggests that further cross-linking of α lactalbumin dimers enables the formation of linear polymers.
    A microreactor system was set up to obtain control over the reaction conditions to cross-link proteins. The enzymatic cross-linking of α lactalbumin was analyzed as a function of enzyme and substrate(s) feed. The increase in absorption at 318 nm due to dityrosine formation was found to be directly correlated to the decrease in monomeric α lactalbumin and was shown to be a good tool to monitor the cross-linking reaction.
    The α lactalbumin oligomers produced were investigated for their foam stabilizing properties. Cross-linked α lactalbumin oligomers did not stabilize foams, whereas α lactalbumin polymers acted as an anti-foam, destabilizing other protein films.

    Effect of medium-pH and MES on adventitious root formation from stem disks of apple
    Klerk, G.J.M. de; Hanecakova, J. ; Jasik, J. - \ 2008
    Plant Cell, Tissue and Organ Culture: an international journal on in vitro culture of higher plants 95 (2008)3. - ISSN 0167-6857 - p. 285 - 292.
    tracheary element differentiation - culture-medium ph - extracellular ph - acid - cell - microcuttings - peroxidase - induction - buffers - metabolism
    We have examined the effect of medium-pH on rooting using 1-mm slices cut from stems of apple microshoots. Before autoclaving, the pH of the rooting medium was set at various pH values between 4.5 and 8.0. During autoclaving, the pH drifted in particular in the alkaline region. Additional changes occurred during culture and the range set at 4.5-8.0 had shifted to 5.2-6.0 after autoclaving and 3 weeks of culture. When 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) had been added as buffering agent, the pH was stable when set at 5.0-6.5. Highest rooting was achieved at pH similar to 5.3 with and without MES (pH measured after autoclaving). This maximum did not correlate with highest auxin uptake. MES inhibited adventitious root formation during the initial phase of root formation when the meristemoids are being formed (ca. 30% reduction at 10 mM) but was promotive during outgrowth of the meristemoids to roots (30% increase at 10 mM). Inhibition and promotion by MES were not related to its buffering action as they were observed at all pHs.
    Covalent interactions between proteins and oxidation products of caffeoylquinic acid (chlorogenic acid)
    Prigent, S.V.E. ; Voragen, A.G.J. ; Visser, A.J.W.G. ; Koningsveld, G.A. van; Gruppen, H. - \ 2007
    Journal of the Science of Food and Agriculture 87 (2007)13. - ISSN 0022-5142 - p. 2502 - 2510.
    bovine serum-albumin - physicochemical characterization - proteolytic digestion - polyphenol oxidase - model solutions - caffeic acid - derivatives - tyrosine - systems - peroxidase
    BACKGROUND: The interactions between phenolic compounds and proteins can modify protein properties important in the food industry. To understand the effects of these interactions, the covalent interactions between caffeoylquinic acid (chlorogenic acid, CQA) oxidised by polyphenol oxidase (PPO) at acidic pH 6 (pH 6) and -lactalbumin, lysozyme and bovine serum albumin (BSA) were compared with non-enzymatically induced covalent interactions at alkaline pH (pH 9). The effects of these modifications on protein properties were examined. RESULTS: Both ways of modification seemed to result in protein modification mainly via dimeric rather than monomeric CQA quinones. These modifications led to a decrease in the number of free primary amino groups of the proteins. Modification with CQA alone induced a low degree of protein dimerisation, which also occurred through the action of PPO alone. Modification drastically reduced the solubility of lysozyme over a broad pH range, whereas that of -lactalbumin was strongly reduced only at pH values close to its pI. The solubility of BSA was much less affected than that of the other proteins and only at acidic pH. CONCLUSION: These results indicate some similarities between modifications at pH 6 and 9 and that both modifications clearly change the functional properties of globular proteins.
    Effects of germination on the activities of amylases and phenolic enzymes in sorghum varieties grouped according to food end-use properties
    Dicko, M.H. ; Gruppen, H. ; Zouzouho, O.C. ; Traore, A.S. ; Berkel, W.J.H. van; Voragen, A.G.J. - \ 2006
    Journal of the Science of Food and Agriculture 86 (2006)6. - ISSN 0022-5142 - p. 953 - 963.
    phenylalanine ammonia-lyase - polyphenol oxidase - beta-amylase - cyanide contents - alpha-amylase - burkina-faso - peroxidase - cultivars - malt - viscosity
    Fifty sorghum varieties were screened to determine the effects of germination on levels of starch, -amylase, -amylase, phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO). Germination decreased starch content, with amylose being more degraded than amylopectin. In germinated grain, -amylase activity increased several-fold in all varieties, whereas -amylase activity did not increase uniformly and even decreased in some varieties. Activity of the key enzyme in phenolic biosynthesis, PAL, was detected in only half of the varieties before germination but in all of them after germination. PPO was not activated in germinated sorghum grains, whereas POX activity increased up to tenfold in some varieties. Zymography revealed that germination induced de novo synthesis of several POX isoenzymes, among which an anionic POX isoenzyme (pI 3.1) was ubiquitously present. Amylase and phenolic enzyme activities could be correlated with grain and plant agronomic characteristics. The use of sorghum varieties for local dishes such as tô, dolo, couscous and thin porridge could be correlated with amylase and phenolic enzyme activities and the contents of their substrates. The biochemical constituents determined are useful markers for selection of varieties for food utilisation with special emphasis on infant porridges.
    Structural features of a hyperthermostable endo-beta-1,3-glucanase in solution and adsorbed on 'invisible' particles
    Koutsopoulos, S. ; Oost, J. van der; Norde, W. - \ 2005
    Biophysical Journal 88 (2005)1. - ISSN 0006-3495 - p. 467 - 474.
    archaeon pyrococcus-furiosus - time-resolved fluorescence - circular-dichroism - proteins - adsorption - peroxidase - interfaces - stability - mechanism
    Conformational characteristics and the adsorption behavior of endo-ß-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus were studied by circular dichroism, steady-state and time-resolved fluorescence spectroscopy, and calorimetry in solution and in the adsorbed state. The adsorption isotherms were determined on two types of surfaces: hydrophobic Teflon and hydrophilic silica particles were specially designed so that they do not interact with light and therefore do not interfere with spectroscopic measurements. We present the most straightforward method to study structural features of adsorbed macromolecules in situ using common spectroscopic techniques. The enzyme was irreversibly adsorbed and immobilized in the adsorbed state even at high temperatures. Adsorption offered further stabilization to the heat-stable enzyme and in the case of adsorption on Teflon its denaturation temperature was measured at 133°C, i.e., the highest experimentally determined for a protein. The maintenance of the active conformation and biological function particularly at high temperatures is important for applications in biocatalysis and biotechnology. With this study we also suggest that nature may employ adsorption as a complementary mode to maintain structural integrity of essential biomolecules at extreme conditions of temperature.
    Impact of phenolic compounds and related enzymes in Sorghum varieties for resistance and susceptibility to biotic and abiotic stresses
    Dicko, M.H. ; Gruppen, H. ; Barro, C. ; Traore, A.S. ; Berkel, W.J.H. van; Voragen, A.G.J. - \ 2005
    Journal of Chemical Ecology 31 (2005)11. - ISSN 0098-0331 - p. 2671 - 2688.
    grain mold resistance - 3-deoxyanthocyanidin phytoalexins - polyphenol oxidase - burkina-faso - peroxidase - genotypes - apigeninidin - accumulation - mechanisms - sorghicola
    Contents of phenolic compounds and related enzymes before and after sorghum grain germination were compared between varieties either resistant or susceptible to biotic (sooty stripe, sorghum midge, leaf anthracnose, striga, and grain molds) and abiotic (lodging, drought resistance, and photoperiod sensitivity) stresses. Independent of grain germination, sorghum varieties resistant to biotic and abiotic stresses had on average higher contents of proanthocyanidins (PAs), 3-deoxyanthocyanidins (3-DAs), and flavan-4-ols than susceptible varieties. Results show that content of 3-DAs is a good marker for sorghum resistance to both biotic and abiotic stresses because it correlates with resistance to all stresses except for photoperiod sensitivity. The second good marker for stress resistance is content of PAs. Total phenolic compounds and the activities of related enzymes are not good markers for stress resistance in sorghum grains
    Wound-induced and bacteria-induced xylem blockage in roses, Astilbe and Viburnum
    Loubaud, M. ; Doorn, W.G. van - \ 2004
    Postharvest Biology and Technology 32 (2004)3. - ISSN 0925-5214 - p. 281 - 288.
    cut chrysanthemum flowers - polyphenol oxidase - cathechol oxidase - peroxidase - occlusion - tissue - stems
    We previously concluded that the xylem blockage that prevents water uptake into several cut flowers is mainly due to the presence of bacteria, whilst in chrysanthemum and Bouvardia we observed a xylem occlusion that was mainly due to a wound-reaction of the plant. We have further tested which of these two mechanisms was dominant in Astilbe,Viburnum and rose flowers. Astilbe x arendsii (cvs. Erica and Glut) flowers were stored dry in plastic bags (24 h at 5 degreesC, 100% RH) and placed in water at 20 degreesC without recutting the steins. The dry storage treatment considerably hastened a wounding-induced xylem occlusion in the stems. A 5 h pulse treatment with inhibitors of peroxidase (hydroquinone) and catechol oxidase (tropolone and 2,3-dihydroxynaphtalene), prior to dry storage, considerably delayed the xylem blockage. The 24 h dry storage treatment had no effect in rose (Rosa x hybrida cv. Red One), and Viburnum opulus (cv. Roseum). These flowers were therefore directly placed in water, with and without enzyme inhibitors. Except hydroquinone, all tested enzyme inhibitors reduced bacterial growth in the vase water. The latter chemicals could therefore not be used to distinguish between a plant-induced and a bacterial occlusion of the xylem. Hydroquinone had no effect on the time to wilting in roses, nor in Viburnum. It considerably delayed wilting in Astilbe flowers that were directly placed in water after harvest. It is concluded that the blockage in Astilbe is mainly due to the plant-induced xylem occlusion. The xylem occlusion in the tested rose and Viburnum cultivar was apparently not due to this mechanism. (C) 2004 Elsevier B.V. All rights reserved.
    Relationship between browning and the activities of polyphenol oxidase and phenylalanine ammonia lyase in banana peel during low temperature storage
    Nguyen, T.B.T. ; Ketsa, S. ; Doorn, W.G. van - \ 2003
    Postharvest Biology and Technology 30 (2003)2. - ISSN 0925-5214 - p. 187 - 193.
    cut lettuce - inhibitors - fruits - peroxidase - ethylene
    Kluai Khai (Musa AA Group) and Kluai Hom Thong (Musa AAA Group) bananas were stored at 6 and 10 °C. Visible chilling injury (CI) in the peel, mainly browning, occurred at both temperatures, but more so at 6 °C, and without significant differences between the cultivars. At the time of harvest, total free phenolics in the peel were three times lower in Kluai Khai than in Kluai Hom Thong fruit, the polyphenol oxidase (PPO) activity in Kluai Khai being considerably higher and phenylalanine ammonia lyase (PAL) activity much lower. As CI developed, PAL and PPO activities in the peel increased, and total free phenolics decreased. The decrease in total free phenolic compounds and the increase in PAL and PPO activities occurred more rapidly at 6 °C than at 10 °C, in both banana cultivars. Correlations between visible CI and the level of total free phenolics, and between CI and the activities of PPO and PAL, were all highly significant. The results indicate that low temperature stress induced concerted activities of PAL and PPO, which resulted in browning. Since the concentrations of free phenolic compounds and the rate of PAL and PPO activities varied considerably between the two cultivars, but browning did not, the changes in the biochemical parameters rather than their absolute levels were correlated with peel browning.
    Effect of water unextractable solids on gluten formation and properties: mechanistic consideration.
    Wang, M. ; Hamer, R.J. ; Vliet, T. van; Gruppen, H. ; Marseille, H. ; Weegels, P.L. - \ 2003
    Journal of Cereal Science 37 (2003)1. - ISSN 0733-5210 - p. 55 - 64.
    non-starch polysaccharides - wheat-flour - pentosans - fractions - quality - protein - bread - dough - peroxidase - separation
    A miniaturised set-up for gluten-starch separation was used to systematically study the effect of water unextractable solids (WUS) on the formation and properties of gluten. The results showed that WUS not only have a negative effect on gluten yield, but also affect gluten and glutenin macropolymer (GMP) composition and rheological properties. The negative effect of WUS on gluten yield could be compensated for to a large extent, but not completely, by increasing mixing time and mixing water. Adding xylanase can effectively counteract the effect of WUS. On the basis of these results we hypothesize that WUS interfere with gluten formation in both a direct and an indirect way. WUS interfere indirectly by competing for water and thus changing conditions for gluten development. This effect can be corrected for by the combination of adding more 0·2% NaCl solution during dough mixing and a longer mixing time. The particulate nature of WUS requires that the direct effect occurs through an interaction between WUS particles and gluten particles. Both effects of WUS can be counteracted through the use of xylanase.
    Glutathione protects Lactococcus lactis against oxidative stress
    Li, Y. ; Hugenholtz, J. ; Abee, T. ; Molenaar, D. - \ 2003
    Applied and Environmental Microbiology 69 (2003)10. - ISSN 0099-2240 - p. 5739 - 5745.
    escherichia-coli - saccharomyces-cerevisiae - oxidized glutathione - growth-conditions - acid bacteria - reductase - thioredoxin - purification - peroxidase - metabolism
    Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to similar to60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H2O2 compared to the resistance of cells without intracellular glutathione. The resistance to H2O2 treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H2O2. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.
    Lignin peroxidase mediated biotransformations useful in the biocatalytic production of vanillin
    Have, R. ten - \ 2000
    Agricultural University. Promotor(en): J.A.M. de Bont; J.A. Field. - S.l. : S.n. - ISBN 9789058081698 - 90
    vanilline - lignine - peroxidase - vanillin - lignin - peroxidase

    This research concentrates on lignin peroxidase (LiP) mediated biotrans-formations that are useful in producing vanillin.

    In order to obtain this extracellular enzyme, the white-rot fungus Bjerkandera sp. strain BOS55 was cultivated on nitrogen rich medium. This procedure resulted in a successful LiP production of 600 U/L. Peptone in the culture medium was shown to interfere with the standard LiP assay in which the formation of veratraldehyde (VAD) from veratryl alcohol (VA) is monitored. Removal of peptone by (NH 4 ) 2 SO 4 precipitation minimized the interference.

    BOS55 excreted at least seven LiP isozymes of which two were purified and characterized. The predominant LiP isozyme (LiP-2) oxidized VA to VAD in the pH range from 2.5 to 6.5. The VA oxidizing activity was optimal at the lowest pH. The K m for H 2 O 2 was strongly depended on the pH. At pH 5.0, a physiological pH, the Km for H 2 O 2 was similar to the extracellular H 2 O 2 concentration measured in cultures of BOS55.

    A model based on calculated ionisation potentials (IP) was developed to predict which potential vanillin precursor would be oxidized by LiP. By testing a series of non-phenolic aromatic compounds, of which the IP was calculated, an IP-threshold value of 9.0 eV was determined. This value was used to select compounds with a lower IP like O -acetyl coniferyl alcohol, and O -acetyl isoeugenol (isoeugenyl acetate, IEA). Indeed, these compounds were consumed and in part converted into vanillyl acetate, the acetyl ester of vanillin.

    IEA was studied to elucidate mechanisms of its oxidation andCalpha - Cbetacleavage of the propenyl side chain in IEA into vanillyl acetate. It was found that IEA was consumed via redox mediation. IEA was only oxidized in the presence of the redox mediator VA. The latter was first oxidized by LiP to the radical cation (VA · +) which in its turn oxidized IEA to its corresponding radical cation IEA · +. The followingCalpha - Cbetacleavage reaction was the result of O 2 -dependent chemical reactions which resulted in vanillyl acetate and ethanal.

    The isoeugenol methyl ether (DMPP) was also used to investigate the consumption and theCalpha - Cbetacleavage mechanism. DMPP was not only consumed enzymatically, but also by O 2 -dependent self-propagating reactions. Mn 2+inhibited this chemical consumption. Since Mn 2+also inhibited the molar yield of the predominantCalpha - Cbetacleavage product, VAD, it was concluded that this product was formed also during these chemical reactions. An other VAD producing route was discovered by incubating a DMPP oxidation side product, 1-(3',4'-dimethoxyphenyl)-2-propanone (DMPA) with LiP. Interestingly, VAD was only formed in the presence of O 2 . Without O 2 , solely DMPA dimers were detected by GC-MS.

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