Jindrich Volc

Purification and Characterization of Pyranose Oxidase from the White Rot Fungus Trametes multicolor

ABSTRACT We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are d-glucose,d-xylose, and l-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (kcat/Km), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H2O2. An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.

Properties of pyranose dehydrogenase purified from the litter‐degrading fungus Agaricus xanthoderma

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion‐exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose–methanol–choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi‐covalent flavinylation in PDH, 9‐S‐cysteinyl, 8‐α‐N3‐histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are d‐glucose, d‐galactose, l‐arabinose, and d‐xylose. As shown by in situ NMR analysis, d‐glucose and d‐galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino‐bis‐(3‐ethylbenzthiazolin‐6‐sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (kcat/Km). The enzyme may play a role in lignocellulose degradation.

The Cetus Process Revisited: A Novel Enzymatic Alternative for the Production of Aldose-Free D-Fructose

In the Cetus process crystalline D-fructose is produced from D-glucose via the intermediate 2-keto-D-glucose. Whereas the first step in the traditional process is catalyzed by the immobilized enzyme pyranose 2-oxidase, the ensuing reduction is performed by catalytic hydrogenation. In an entirely enzymatic variation of this process, soluble pyranose 2-oxidase from Trametes multicolor was employed. This biocatalyst could be efficiently stabilized under operational conditions by the addition of bovine serum albumin (BSA) together with catalase which decomposes hydrogen peroxide formed as a by-product. D-Glucose could be converted into 2-keto-D-glucose in yields above 98%. When the biocatalyst together with both stabilizing agents was separated from the product solution by ultrafiltration, it could be reutilized for several subsequent batch operation cycles. 2-Keto-D-glucose thus obtained was quantitatively reduced to D-fructose by NAD(P)-dependent aldose reductase from Candida tenuis. Two different enzymatic...

Pyranose Oxidase Modified Carbon Paste Electrodes for Monosaccharide Determination

An amperometric biosensor for the detection of some monosaccharides was developed with co-immobilized pyranose oxidase (purified from the basidiomycete fungus Phanerochaete chrysosporium) and horseradish peroxidase in carbon paste. Due to rather low selectivity of pyranose oxidase, the biosensor could detect both predominating anomers of D-glucose as well as the substrates D-xylose, D-galactose, and δ-gluconolactone. The best sensor performance was obtained from a carbon paste with covalently immobilized enzymes together with the additives lactitol and polyethylenimine, resulting in a sensitivity for glucose of 30.2 μA cm−2 mM−1. The enzyme modified electrode was investigated in an automated flow injection system at the operating potential of −50 mV (vs. Ag/AgCl) with an injection frequency of 40 h−1. The biosensor was also integrated as the detection unit in a liquid chromatographic system for the detection of monosaccharides to show its potential use in real applications.

Pyranose 2-oxidase from Phanerochaete chrysosporium—Expression in E. coli and biochemical characterization

The presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in yields of approximately 270 mg/l medium. The recombinant enzyme was provided with an N-terminal T7-tag and a C-terminal His(6)-tag to facilitate simple one-step purification. We obtained an apparently homogenous enzyme preparation with a specific activity of 16.5 U/mg. Recombinant P2Ox from P. chrysosporium was characterized in some detail with respect to its physical and catalytic properties, both for electron donor (sugar substrates) and - for the first time - alternative electron acceptors (1,4-benzoquinone, substituted quinones, 2,6-dichloroindophenol and ferricenium ion). As judged from the catalytic efficiencies k(cat)/K(m), some of these alternative electron acceptors are better substrates than oxygen, which might have implications for the proposed in vivo function of pyranose 2-oxidase.

Enzymatic redox isomerization of 1,6-disaccharides by pyranose oxidase and NADH-dependent aldose reductase

Abstract Pyranose 2-oxidase, a homotetrameric FAD-flavoprotein from the basidiomycete Trametes multicolor, catalyzes regioselectively the oxidation of the 1→6 disaccharides allolactose [β- d -Galp-(1→6)- d -Glc], gentiobiose [β- d -Glcp-(1→6)- d -Glc], melibiose [α- d -Galp-(1→6)- d -Glc], and isomaltose [α- d -Glcp-(1→6)- d -Glc] at position C-2 of their reducing moiety. The resulting glycosyl d -arabino-hexos-2-uloses can be reduced specifically at C-1 by NAD(P)H-dependent aldose reductase from the yeast Candida tenuis. By this novel, two-step redox isomerization process the four disaccharide substrates could be converted to the corresponding keto-disaccharides allolactulose [β- d -Galp-(1→6)- d -Fru], gentiobiulose [β- d -Glcp-(1→6)- d -Fru], melibiulose [α- d -Galp-(1→6)- d -Fru], and isomaltulose (palatinose, [α- d -Glcp-(1→6)- d -Fru]) in high yields. These products could find application in food technology as alternative sweeteners.

Distribution of γ‐tubulin in cellular compartments of higher plant cells

Higher plants represent an important group of eukaryotes in which discrete sites for microtubule nucleation are absent in all mitotic and meiotic cells, and a concept of dispersed microtubule-nucleation sites has been generally accepted. Current models of mitotic spindle formation in the absence of centrosomes are based on chromatin-mediated microtubule organization. Moreover, even in cells equipped with centrosomes, microtubules can nucleate on cytoplasmic factors, which are so far poorly characterized (Vorobjev et al., 1997). To understand how microtubules are nucleated and organized without the centrosome, the first step is to determine the molecular composition of the dispersed cytoplasmic or chromatin-associated microtubulenucleation sites. -Tubulin, a distantly related member of the tubulin superfamily, plays a pivotal role in microtubule nucleation. It is organized into the large w2000 kDa open-ring complexes ( -TuRC), located at the centrosomes in animal cells and at the spindle pole bodies in yeast (Moritz et al., 1995). The complete genome of Arabidopsis contains two expressed genes encoding -tubulins. Using affinity purified polyclonal and monoclonal antibodies, directed against different antigenic determinants on the -tubulin molecule, we studied the subcellular distribution of -tubulin in six plant species (Arabidopsis thaliana, Vicia faba, Pisum sativum, Medicago sativa, Hordeum vulgare and Zea mays). Biochemical and immunolocalization studies revealed that -tubulin is highly abundant in different cellular compartments. -Tubulin was localized along all microtubular arrays, discrete staining was found in some interphase nuclei. The size of nuclear spots gradually increased until late G2 when they often occurred as double spots. A similar labelling pattern for -tubulin was found in prekinetochore region of isolated G2 nuclei. -Tubulin was also localized on the nuclear surface, decorating perinuclear microtubules focused to the poles. At the onset of mitosis, when nuclear envelope broke down, -tubulin was found on short kinetochore microtubule fibres organized from kinetochores. A strong signal for -tubulin was associated with kinetochore microtubules along their whole length including the close vicinity of the kinetochores. In cells recovering from anti-microtubular drugs, -tubulin was localized with the re-growing kinetochore microtubules nucleated or captured by kinetochore/centromeric regions. Similarly, on isolated chromosomes, -tubulin co-localized with -tubulin in the kinetochore/centromeric region. Flow cytometry was employed to sort isolated plant nuclei in the G1 and G2 stages of the cell cycle. They were thereafter subjected to quantitative immunoblot analysis. An increase of about 50% of -tubulin in G2 compared to G1 nuclei was found in V. faba. In contrast, when supernatants of centrifuged cell extracts from synchronized cells were analyzed by immunoblotting, there were no differences in the amount of -tubulin between cells in the G1 and G2 stages of the cell cycle (Binarova et al., 2000). Circular configurations of chromosomes with the kinetochore region in the centre and chromosome arms pointing outwards were induced transiently by treatment of root meristems of V. faba, M. sativa and A. thaliana with specific cyclin-dependent protein kinase inhibitors (Binarova et al., 1998a). Kinetochore microtubules, * Corresponding author. Tel. +42-2-4106-2130; fax: +42-4-4106-2384. Cell Biology International 27 (2003) 167–169 Cell Biology International

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.