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Dietmar Haltrich - One of the best experts on this subject based on the ideXlab platform.
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versatile oxidase and dehydrogenase activities of bacterial Pyranose 2 oxidase facilitate redox cycling with manganese peroxidase in vitro
Applied and Environmental Microbiology, 2019Co-Authors: Peter L Herzog, Leander Sutzl, Beate Eisenhut, Daniel Maresch, Dietmar Haltrich, Christian Obinger, Clemens K. PeterbauerAbstract:ABSTRACT Pyranose 2-oxidase (POx) has long been accredited a physiological role in lignin degradation, but evidence to provide insights into the biochemical mechanisms and interactions is insufficient. There are ample data in the literature on the oxidase and dehydrogenase activities of POx, yet the biological relevance of this duality could not be established conclusively. Here we present a comprehensive biochemical and phylogenetic characterization of a novel Pyranose 2-oxidase from the actinomycetous bacterium Kitasatospora aureofaciens (KaPOx) as well as a possible biomolecular synergism of this enzyme with peroxidases using phenolic model substrates in vitro. A phylogenetic analysis of both fungal and bacterial putative POx-encoding sequences revealed their close evolutionary relationship and supports a late horizontal gene transfer of ancestral POx sequences. We successfully expressed and characterized a novel bacterial POx gene from K. aureofaciens, one of the putative POx genes closely related to well-known fungal POx genes. Its biochemical characteristics comply with most of the classical hallmarks of known fungal Pyranose 2-oxidases, i.e., reactivity with a range of different monosaccharides as electron donors as well as activity with oxygen, various quinones, and complexed metal ions as electron acceptors. Thus, KaPOx shows the pronounced duality of oxidase and dehydrogenase similar to that of fungal POx. We further performed efficient redox cycling of aromatic lignin model compounds between KaPOx and manganese peroxidase (MnP). In addition, we found a Mn(III) reduction activity in KaPOx, which, in combination with its ability to provide H2O2, implies this and potentially other POx as complementary enzymatic tools for oxidative lignin degradation by specialized peroxidases. IMPORTANCE Establishment of a mechanistic synergism between Pyranose oxidase and (manganese) peroxidases represents a vital step in the course of elucidating microbial lignin degradation. Here, the comprehensive characterization of a bacterial Pyranose 2-oxidase from Kitasatospora aureofaciens is of particular interest for several reasons. First, the phylogenetic analysis of putative Pyranose oxidase genes reveals a widespread occurrence of highly similar enzymes in bacteria. Still, there is only a single report on a bacterial Pyranose oxidase, stressing the need of closing this gap in the scientific literature. In addition, the relatively small K. aureofaciens proteome supposedly supplies a limited set of enzymatic functions to realize lignocellulosic biomass degradation. Both enzyme and organism therefore present a viable model to study the mechanisms of bacterial lignin decomposition, elucidate physiologically relevant interactions with specialized peroxidases, and potentially realize biotechnological applications.
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the gmc superfamily of oxidoreductases revisited analysis and evolution of fungal gmc oxidoreductases
Biotechnology for Biofuels, 2019Co-Authors: Leander Sutzl, G Foley, Elizabeth M J Gillam, Mikael Boden, Dietmar HaltrichAbstract:The glucose–methanol–choline (GMC) superfamily is a large and functionally diverse family of oxidoreductases that share a common structural fold. Fungal members of this superfamily that are characterised and relevant for lignocellulose degradation include aryl-alcohol oxidoreductase, alcohol oxidase, cellobiose dehydrogenase, glucose oxidase, glucose dehydrogenase, Pyranose dehydrogenase, and Pyranose oxidase, which together form family AA3 of the auxiliary activities in the CAZy database of carbohydrate-active enzymes. Overall, little is known about the extant sequence space of these GMC oxidoreductases and their phylogenetic relations. Although some individual forms are well characterised, it is still unclear how they compare in respect of the complete enzyme class and, therefore, also how generalizable are their characteristics. To improve the understanding of the GMC superfamily as a whole, we used sequence similarity networks to cluster large numbers of fungal GMC sequences and annotate them according to functionality. Subsequently, different members of the GMC superfamily were analysed in detail with regard to their sequences and phylogeny. This allowed us to define the currently characterised sequence space and show that complete clades of some enzymes have not been studied in any detail to date. Finally, we interpret our results from an evolutionary perspective, where we could show, for example, that Pyranose dehydrogenase evolved from aryl-alcohol oxidoreductase after a change in substrate specificity and that the cytochrome domain of cellobiose dehydrogenase was regularly lost during evolution. This study offers new insights into the sequence variation and phylogenetic relationships of fungal GMC/AA3 sequences. Certain clades of these GMC enzymes identified in our phylogenetic analyses are completely uncharacterised to date, and might include enzyme activities of varying specificities and/or activities that are hitherto unstudied.
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importance of the gating segment in the substrate recognition loop of Pyranose 2 oxidase
FEBS Journal, 2010Co-Authors: Oliver Spadiut, Dietmar Haltrich, Christina Divne, Tienchye Tan, Ines PisanelliAbstract:Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldoPyranoses present in lignocellulose to 2-ketoaldoses. The active site in Pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment ((454)FSY(456)) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp(452), Phe(454) and Tyr(456)) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of Pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of Pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.
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evaluation of different expression systems for the heterologous expression of Pyranose 2 oxidase from trametes multicolor in e coli
Microbial Cell Factories, 2010Co-Authors: Oliver Spadiut, Dietmar Haltrich, Gerald Posch, Roland Ludwig, Clemens K. PeterbauerAbstract:The heterologous production of the industrially relevant fungal enzyme Pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d+, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d+ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active Pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d+ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d+ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h-1resulted in the highest productivity rate of active Pyranose oxidase with 206 U/L·h.
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Pyranose 2 oxidase from phanerochaete chrysosporium expression in e coli and biochemical characterization
Journal of Biotechnology, 2009Co-Authors: Ines Pisanelli, Dietmar Haltrich, Roman Kittl, Petr Halada, Jindrich Volc, Oliver Spadiut, Magdalena Kujawa, Michael D Mozuch, Philip J Kersten, Clemens K. PeterbauerAbstract: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.
Clemens K. Peterbauer - One of the best experts on this subject based on the ideXlab platform.
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versatile oxidase and dehydrogenase activities of bacterial Pyranose 2 oxidase facilitate redox cycling with manganese peroxidase in vitro
Applied and Environmental Microbiology, 2019Co-Authors: Peter L Herzog, Leander Sutzl, Beate Eisenhut, Daniel Maresch, Dietmar Haltrich, Christian Obinger, Clemens K. PeterbauerAbstract:ABSTRACT Pyranose 2-oxidase (POx) has long been accredited a physiological role in lignin degradation, but evidence to provide insights into the biochemical mechanisms and interactions is insufficient. There are ample data in the literature on the oxidase and dehydrogenase activities of POx, yet the biological relevance of this duality could not be established conclusively. Here we present a comprehensive biochemical and phylogenetic characterization of a novel Pyranose 2-oxidase from the actinomycetous bacterium Kitasatospora aureofaciens (KaPOx) as well as a possible biomolecular synergism of this enzyme with peroxidases using phenolic model substrates in vitro. A phylogenetic analysis of both fungal and bacterial putative POx-encoding sequences revealed their close evolutionary relationship and supports a late horizontal gene transfer of ancestral POx sequences. We successfully expressed and characterized a novel bacterial POx gene from K. aureofaciens, one of the putative POx genes closely related to well-known fungal POx genes. Its biochemical characteristics comply with most of the classical hallmarks of known fungal Pyranose 2-oxidases, i.e., reactivity with a range of different monosaccharides as electron donors as well as activity with oxygen, various quinones, and complexed metal ions as electron acceptors. Thus, KaPOx shows the pronounced duality of oxidase and dehydrogenase similar to that of fungal POx. We further performed efficient redox cycling of aromatic lignin model compounds between KaPOx and manganese peroxidase (MnP). In addition, we found a Mn(III) reduction activity in KaPOx, which, in combination with its ability to provide H2O2, implies this and potentially other POx as complementary enzymatic tools for oxidative lignin degradation by specialized peroxidases. IMPORTANCE Establishment of a mechanistic synergism between Pyranose oxidase and (manganese) peroxidases represents a vital step in the course of elucidating microbial lignin degradation. Here, the comprehensive characterization of a bacterial Pyranose 2-oxidase from Kitasatospora aureofaciens is of particular interest for several reasons. First, the phylogenetic analysis of putative Pyranose oxidase genes reveals a widespread occurrence of highly similar enzymes in bacteria. Still, there is only a single report on a bacterial Pyranose oxidase, stressing the need of closing this gap in the scientific literature. In addition, the relatively small K. aureofaciens proteome supposedly supplies a limited set of enzymatic functions to realize lignocellulosic biomass degradation. Both enzyme and organism therefore present a viable model to study the mechanisms of bacterial lignin decomposition, elucidate physiologically relevant interactions with specialized peroxidases, and potentially realize biotechnological applications.
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bioelectrochemical behavior of the composite pvp os chitosan as a mediator with different types of enzymes at graphite electrode
Insights in Analytical Electrochemistry, 2015Co-Authors: Najat Beden, Clemens K. Peterbauer, Ra Digambar Jirimali, Woonsup Shin, Lo GortonAbstract:Chitosan was cross-linked to an osmium redox polymer, poly(4-vinylpyridine) osmium bipyridyl [PVP-Os-(bpy)2-Cl], to form PVP-Os-(bpy)2-Cl/chitosan composite known to make a porous and hydrophilic film with an enzyme. In this work we demonstrate such a composite is a useful trestle, for hosting various sugars oxidizing enzymes to construct biosensors. Glucose sensing ability has been proven with the following glucose oxidizing redox enzymes; Aspergillus niger glucose oxidase (AnGOX), Myriococcum thermophilum cellobiose dehydrogenase, (MtCDH), glycosylated Agaricus meleagris Pyranose dehydrogenase (gAmPDH), fragmented deglycosylated Agaricus meleagris Pyranose dehydrogenase (fdgAmPDH), and Aspergillus sp. glucose dehydrogenase (AspGDH), as well as recombinant Glomerella cingulata glucose dehydrogenase (rGcGDH).
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evaluation of different expression systems for the heterologous expression of Pyranose 2 oxidase from trametes multicolor in e coli
Microbial Cell Factories, 2010Co-Authors: Oliver Spadiut, Dietmar Haltrich, Gerald Posch, Roland Ludwig, Clemens K. PeterbauerAbstract:The heterologous production of the industrially relevant fungal enzyme Pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d+, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d+ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active Pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d+ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d+ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h-1resulted in the highest productivity rate of active Pyranose oxidase with 206 U/L·h.
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Pyranose dehydrogenases: biochemical features and perspectives of technological applications
Applied Microbiology and Biotechnology, 2010Co-Authors: Clemens K. Peterbauer, Jindřich VolcAbstract:Pyranose dehydrogenase is a fungal flavin-dependent sugar oxidoreductase which is structurally and catalytically related to fungal Pyranose oxidase and cellobiose dehydrogenase and probably fulfills similar biological functions in lignocellulose breakdown. It is a monomeric secretory glycoprotein and is limited to a rather small group of litter-decomposing basidiomycetes. Compared with Pyranose oxidase, it displays broader substrate specificity and a variable regioselectivity and is unable to utilize oxygen as electron acceptor using substituted benzoquinones and (organo) metallic ions instead. Depending on the structure of the sugar in Pyranose form (mono/di/oligosaccharide or glycoside) and the enzyme source, selective monooxidations at C-1, C-2, C-3, or dioxidations at C-2,3 or C-3,4 of the molecule to the corresponding aldonolactones (C-1), or (di)dehydrosugars (aldos(di)uloses) can be performed. These features make Pyranose dehydrogenase a promising and versatile biocatalyst for production of highly reactive, sometimes unique, di- and tri-carbonyl sugar derivatives that may serve as interesting chiral intermediates for the synthesis of rare sugars, novel drugs, and fine chemicals.
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Pyranose 2 oxidase from phanerochaete chrysosporium expression in e coli and biochemical characterization
Journal of Biotechnology, 2009Co-Authors: Ines Pisanelli, Dietmar Haltrich, Roman Kittl, Petr Halada, Jindrich Volc, Oliver Spadiut, Magdalena Kujawa, Michael D Mozuch, Philip J Kersten, Clemens K. PeterbauerAbstract: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.
Friedrich Giffhorn - One of the best experts on this subject based on the ideXlab platform.
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fungal Pyranose oxidases occurrence properties and biotechnical applications in carbohydrate chemistry
Applied Microbiology and Biotechnology, 2000Co-Authors: Friedrich GiffhornAbstract:Pyranose oxidases are widespread among lignin-degrading white rot fungi and are localized in the hyphal periplasmic space. They are relatively large flavoproteins which oxidize a number of common monosaccharides on carbon-2 in the presence of oxygen to yield the corresponding 2-keto sugars and hydrogen peroxide. The preferred substrate of Pyranose oxidases is d-glucose which is converted to 2-keto-d-glucose. While hydrogen peroxide is a cosubstrate in ligninolytic reactions, 2-keto-d-glucose is the key intermediate of a secondary metabolic pathway leading to the antibiotic cortalcerone. The finding that 2-keto-d-glucose can serve as an intermediate in an industrial process for the conversion of d-glucose into d-fructose has stimulated research on the use of Pyranose oxidases in biotechnical applications. Unique catalytic potentials of Pyranose oxidases have been discovered which make these enzymes efficient tools in carbohydrate chemistry. Converting common sugars and sugar derivatives with Pyranose oxidases provides a pool of sugar-derived intermediates for the synthesis of a variety of rare sugars, fine chemicals and drugs.
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laboratory procedures for producing 2 keto d glucose 2 keto d xylose and 5 keto d fructose from d glucose d xylose and l sorbose with immobilized Pyranose oxidase of peniophora gigantea
Journal of Biotechnology, 1994Co-Authors: Alexander Huwig, Hansjurgen Danneel, Friedrich GiffhornAbstract:Abstract The carbonyl sugars 2-keto- d -glucose ( d -arabino-hexos-2-ulose; d -glucosone), 2-keto- d -xylose ( d -threo-pentos-2-ulose; d -xylosone), and 5-keto- d -fructose ( d -threo-hexos-2,5-diulose) were prepared from d -glucose, d -xylose and l -sorbose by bioconversion with Pyranose oxidase (EC 1.1.3.10) from Peniophora gigantea immobilized to Eupergit C 250 L. The specific binding capacity was found to be 4 mg enzyme protein per g matrix (dry mass) with an activity yield of about 50%. Immobilization of Pyranose oxidase resulted in an increased storage and operational stability, but kinetic parameters of the enzyme ( K m and v max values) remained almost unchanged. Bioconversions of the sugars into the corresponding carbonyl derivatives were performed at 22°C in batch procedures, using water as solvent. The hydrogen peroxide which was generated during the process was decomposed with catalase. The products 2-keto- d -glucose and 5-keto- d -fructose were recovered with yields ranging from 95% to 98% by ultrafiltration and lyophilization, whereas the yield of 5-keto- d -xylose was 85%.
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purification and characterization of a Pyranose oxidase from the basidiomycete peniophora gigantea and chemical analyses of its reaction products
FEBS Journal, 1993Co-Authors: Hansjurgen Danneel, Ellen Rossner, Axel Zeeck, Friedrich GiffhornAbstract:A Pyranose oxidase was isolated from mycelium extracts of the basidiomycete Peniophora gigantea. This enzyme was purified 104-fold to apparent homogeneity with a yield of about 75% by steps involving fractionated ammonium sulphate precipitation, chromatography on DEAE-Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native Pyranose oxidase has a relative molecular mass (Mr) of 322800 ± 18300 as determined on the basis of its Stokes' radius (rs= 6.2 nm) and sedimentation coefficient (s20,w= 10.6), dynamic light-scattering experiments, gradient-gel electrophoresis and cross-linking studies. SDS/PAGE resulted in one single polypeptide band of Mr76000 indicating that the enzyme consists of four subunits of identical size. The Pyranose oxidase was shown to be an extremely stable glycoprotein with an isoelectric point of pH 5.3. It contains covalently bound FAD with an estimated stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates d-glucose, d-xylose, l-sorbose, d-galactose, methyl β-d-glucoside, maltose and d-fucose. Regioselective oxidation of d-glucose, l-sorbose and d-xylose to 2-keto-d-glucose, 5-keto-d-fructose and 2-keto-d-xylose, was demonstrated by identifying the reaction products by mass spectroscopy 13C-NMR spectroscopy and 1H-NMR spectroscopy after purification and derivatization. The pH optimum of the Pyranose oxidase was in the range pH 6.0–6.5 in 0.1 M potassium phosphate, and its activation energy ( H°) for the conversion of d-glucose was 34.6 kJ/mol. The reactions with the sugars exhibited Michaelis-Menten kinetics, and the Km values determined for d-glucose, l-sorbose, d-xylose and oxygen were 1.1 mM, 50.0 mM, 29.4 mM and 0.65 mM, respectively. The activity of Pyranose oxidase was only slightly affected by chelating reagents, thiol reagents, reducing reagents and bivalent cations each at 1 mM.
Huanfeng Jiang - One of the best experts on this subject based on the ideXlab platform.
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highly facialselective synthesis of Pyranose 1 3 oxazines and their ring opening with nucleophiles a novel entry to 2 c branched glycosides
ChemInform, 2012Co-Authors: Biaolin Yin, Zeren Zhang, Huanfeng JiangAbstract:The reaction of various glycals with N-benzoyl-O-alkylacetals, promoted by TmsOTf, leads to Pyranose 1,3-oxazines structures with high diastereoselectivity.
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highly facialselective synthesis of Pyranose 1 3 oxazines and their ring opening with nucleophiles a novel entry to 2 c branched glycosides
Organic Letters, 2011Co-Authors: Biaolin Yin, Zeren Zhang, Huanfeng JiangAbstract:A TMSOTf-promoted cycloaddition of N-benzoyl-N,O-acetals with various glycals and 3-deoxy glycals affords Pyranose 1,3-oxazines with high facial selectivity. In addition, a highly diastereoselective ring opening of the resulting Pyranose 1,3-oxazines is reported. With diverse nucleophiles, these reactions take place upon heating at 80 °C. This novel ring-opening reaction affords structurally diversified 2-C-branched glycosides with three newly formed contiguous stereocenters.
Oliver Spadiut - One of the best experts on this subject based on the ideXlab platform.
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importance of the gating segment in the substrate recognition loop of Pyranose 2 oxidase
FEBS Journal, 2010Co-Authors: Oliver Spadiut, Dietmar Haltrich, Christina Divne, Tienchye Tan, Ines PisanelliAbstract:Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldoPyranoses present in lignocellulose to 2-ketoaldoses. The active site in Pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment ((454)FSY(456)) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp(452), Phe(454) and Tyr(456)) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of Pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of Pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.
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evaluation of different expression systems for the heterologous expression of Pyranose 2 oxidase from trametes multicolor in e coli
Microbial Cell Factories, 2010Co-Authors: Oliver Spadiut, Dietmar Haltrich, Gerald Posch, Roland Ludwig, Clemens K. PeterbauerAbstract:The heterologous production of the industrially relevant fungal enzyme Pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d+, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d+ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active Pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d+ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d+ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h-1resulted in the highest productivity rate of active Pyranose oxidase with 206 U/L·h.
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Pyranose 2 oxidase from phanerochaete chrysosporium expression in e coli and biochemical characterization
Journal of Biotechnology, 2009Co-Authors: Ines Pisanelli, Dietmar Haltrich, Roman Kittl, Petr Halada, Jindrich Volc, Oliver Spadiut, Magdalena Kujawa, Michael D Mozuch, Philip J Kersten, Clemens K. PeterbauerAbstract: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.
