| silver stained polyacrylamide gels and fluorescence-based automated capillary electrophoresis for detection of amplified fragment length polymorphism patterns obtained from white-rot fungi in the genus trametes. | silver stained denaturing polyacrylamide gels (pages) and fluorescent denaturing automated capillary electrophoresis (ce) were used to detect amplified fragment length polymorphism (aflp) patterns obtained from white-rot fungi belonging to the genus trametes. aflp fingerprinting detected by the fluorescence-based method as well as by silver staining showed a high discriminatory power in differentiating nine strains of trametes ochracea, nine strains of trametes hirsuta and ten isolates of tramet ... | 2000 | 10889313 |
| temperature-induced changes in copper centers and protein conformation of two fungal laccases from coriolus hirsutus and coriolus zonatus. | the paper reports on two fungal laccases from coriolus hirsutus and coriolus zonatus and their type-2 copper-depleted derivatives. temperature-induced changes of the copper centers were characterized by optical and electron paramagnetic resonance (epr) spectroscopy, and the overall protein stability by differential scanning microcalorimetry. the intact enzymes showed highly cooperative thermal unfolding transitions at about 90 degrees c. type-2 copper depletion led to uncoupling of the domains c ... | 2001 | 11410296 |
| comparative characterization of methods for removal of cu(ii) from the active sites of fungal laccases. | copper-containing sites of laccases isolated from the basidiomycetes coriolus hirsutus and coriolus zonatus were characterized by optical methods and epr spectroscopy. methods for preparation of fungal laccase derivatives free from type 2 copper ions were compared. the data of epr spectroscopy and spectrophotometric titration of copper sites showed that only a modified method based on the use of bathocuproine as a chelator for type 2 copper yielded laccase derivatives completely free from type 2 ... | 2001 | 11703175 |
| comparative analysis of laccase-isozymes patterns of several related polyporaceae species under different culture conditions. | the study of isozymes has to date been successfully applied in the identification of a number of different microorganisms, particularly fungi. trametes sp. i-62 (cect20197) is a white-rot fungus with potential biotechnological applications. it has been identified as trametes on the basis of the mycelium morphology, but the features observed are not sufficient for its classification at the species level. given that this strain produces a very characteristic laccase isozyme pattern, in the present ... | 2004 | 15069666 |
| expression of the pyranose 2-oxidase from trametes pubescens in escherichia coli and characterization of the recombinant enzyme. | cdna-encoding pyranose 2-oxidase (p2o) from trametes pubescens was sequenced and cloned into escherichia coli strain bl21/de3 on a multicopy plasmid under the control of trc promoter. the synthesis of p2o was studied in a batch culture in m9-based mineral medium: the enzyme was synthesized constitutively at 28 degrees c in amount corresponding to 8% of the cell soluble protein (0.6 umg(-1)). only small portion of p2o (11%) was in the form of non-active inclusion bodies. purified recombinant enzy ... | 2005 | 16105703 |
| a ph switch affects the steady-state kinetic mechanism of pyranose 2-oxidase from trametes ochracea. | the flavin-dependent pyranose 2-oxidase catalyzes the oxidation of d-glucose and other pyranoses at the c2 atom to yield 2-keto-sugars and hydrogen peroxide. here, the steady-state kinetic mechanism of the enzyme from trametes ochracea was investigated as a function of ph. our findings show that the enzyme follows a bi-bi ping-pong kinetic mechanism at ph values <7.0, and a bi-bi ordered mechanism at ph values >7.0. thus, at low ph the reactivity of the reduced enzyme with oxygen is controlled a ... | 2009 | 19146821 |
| kinetic mechanism of pyranose 2-oxidase from trametes multicolor. | pyranose 2-oxidase (p2o) from trametes multicolor is a flavoprotein oxidase that catalyzes the oxidation of aldopyranoses by molecular oxygen to yield the corresponding 2-keto-aldoses and hydrogen peroxide. p2o is the first enzyme in the class of flavoprotein oxidases, for which a c4a-hydroperoxy-flavin adenine dinucleotide (fad) intermediate has been detected during the oxidative half-reaction. in this study, the reduction kinetics of p2o by d-glucose and 2-d-d-glucose at ph 7.0 was investigate ... | 2009 | 19317444 |
| direct electron transfer reactions of laccases from different origins on carbon electrodes. | electrochemical studies of laccases from basidiomycetes, i.e., trametes hirsuta, trametes ochracea, coriolopsis fulvocinerea, cerrena maxima, and cerrena unicolor, have been performed. direct (mediatorless) electrochemistry of laccases on graphite electrodes has been investigated with cyclic voltammetry, square wave voltammetry as well as potentiometry. for all mentioned high potential laccases direct electron transfer (det) has been registered at spectrographic graphite and highly ordered pyrol ... | 2005 | 15941673 |
| comparison of physico-chemical characteristics of four laccases from different basidiomycetes. | new strains of basidiomycetes producing extracellular laccases (trametes ochracea 92-78, and trametes hirsuta 56) have been found by screening of isolates of trametes fungi. the laccases from t. hirsuta 56 and t. ochracea 92-78 as well as two laccases from previously found and described strains of basidiomycetes, namely cerrena maxima and coriolopsis fulvocinerea, were purified to homogeneity. the standard redox potentials of type 1 copper in the enzymes were determined and found to be 780, 790, ... | 2004 | 15556280 |
| laccase of coriolus zonatus: isolation, purification, and some physicochemical properties. | laccase is one of the lignolytic enzymes found in liquid cultures of the fungus coriolus zonatus in defined medium. the enzyme was isolated from culture liquid and characterized. laccase from c. zonatus is a single-chain protein with a molecular mass of 60 kda. carbohydrate moiety of enzyme consisted of mannose, galactose and n-acetyl-glucosamine in a ratio of 6:2:0.6, respectively, and comprised 10% of the entire molecule. isoelectric point was detected at ph 4.6. laccase was found to have a ph ... | 1999 | 15304730 |
| crystal structure of the 270 kda homotetrameric lignin-degrading enzyme pyranose 2-oxidase. | pyranose 2-oxidase (p2ox) is a 270 kda homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. p2ox oxidizes d-glucose and other aldopyranoses regioselectively at c2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at c3. the crystal structure of p2ox fro ... | 2004 | 15288786 |
| high-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of escherichia coli using lactose as inducer. | expression of a recombinant pyranose oxidase (p2o) from the basidiomycete trametes ochracea has been increased 10-fold in shaking flask cultures of escherichia coli bl21(de3) harboring plasmid pse33 by optimizing the composition of the culture medium using an experimental design approach. inexpensive lactose was used as a medium component and inducer of expression of the p2o gene, which is under the control of a trc promoter. the expression system was studied in detail in batch and fed-batch cul ... | 2004 | 15177285 |
| molecular cloning and expression of the pyranose 2-oxidase cdna from trametes ochracea mb49 in escherichia coli. | a cdna of a structural gene encoding pyranose 2-oxidase (p2o) from trametes ochracea strain mb49 was cloned into escherichia coli strain bl21(de3) on a multicopy plasmid under the control of the trc promoter. synthesis of p2o was studied in batch cultures in lb or m9-based mineral medium at 28 degrees c. while there was a low specific activity of p2o in lb medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 u mg(-1)). the effect ... | 2004 | 14689250 |
| crystallization and preliminary x-ray diffraction analysis of pyranose 2-oxidase from the white-rot fungus trametes multicolor. | pyranose 2-oxidase (p2ox) is a 270 kda homotetrameric flavoenzyme that catalyzes the oxidation of d-glucose to 2-keto-d-glucose. p2ox participates in lignin degradation by white-rot fungi and a tentative role of the enzyme is the production of h(2)o(2) for lignin peroxidases. crystals of trametes multicolor p2ox were grown from monomethylether peg 2000, sodium acetate, mgcl(2) and ta(6)br(12). they belong to space group p2(1), with unit-cell parameters a = 99.9, b = 101.7, c = 135.6 a, beta = 90 ... | 2004 | 14684929 |
| combination of biological pretreatment with liquid hot water pretreatment to enhance enzymatic hydrolysis of populus tomentosa. | a novel stepwise pretreatment of combination of fungal treatment with liquid hot water (lhw) treatment was conducted to enhance the enzymatic hydrolysis of populus tomentosa. the results showed that lignin and cellulose increased with the elevating temperature, while significant amount of hemicellulose was degraded during the lhw pretreatment. a highest hemicellulose removal of 92.33% was observed by combination of lenzites betulina c5617 with lhw treatment at 200°c, which was almost 2 times hig ... | 2011 | 22244900 |
| novel laccase redox mediators: spectral, electrochemical, and kinetic properties. | the screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from coriolus hirsutus and coriolus zonatus. it was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. the characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4- methylamino-pyrazolone-5-n(4)-methanesulfonate and 1-(3'-sulfophenyl)-3- methylpyrazolone-5, in the reaction catalyzed by laccase ... | 2003 | 14665736 |
| [comparative stability assessment of laccases from the basidiomycetes coriolus hirsutus and coriolus zonatus in the presence of effectors]. | stability characteristics of the laccases of the basidiomycetes coriolus hirsutus and coriolus zonatus were measured comparatively at temperatures 25 and 40 degrees c in the presence of various effectors (proteins, salts, polyalcohols, polyacids, and polyelectrolytes). stabilization effects of cations on the laccases from c. hirsutus and c. zonatus decreased in the descending series cu2+ > mg2+ > ca2+ and ca2+ > mg2+ > mn2+, respectively. tween 20 caused insignificant stabilization of the two en ... | 2003 | 14593868 |
| identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from trametes multicolor. | we present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. pyranose 2-oxidase from the basidiomycete fungus trametes multicolor, catalyzing c-2/c-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. no release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. the flavopeptide fragment resulting from tryptic/chymotryptic digestio ... | 2003 | 12654310 |
| [fungal decomposition of oat straw during liquid and solid state fermentation]. | white rot fungi (coriolus hirsutus, coriolus zonatus, and cerrena maxima from the collection of the komarov botanical institute of the russian academy of sciences) and filamentous fungi (mycelia sterilia inbi 2-26 and trichoderma reesei 6/16) were grown on oat straw-based liquid and solid media, as well as in a bench-scale reactor, either individually or as co-cultures. all fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. under these conditions ... | 2003 | 12625046 |
| purification and characterization of a laccase from the white-rot fungus trametes multicolor. | the wood-degrading fungus trametes multicolor secretes several laccase isoforms when grown on a simple medium containing copper in the millimolar range for stimulating laccase synthesis. the main isoenzyme laccase ii was purified to apparent homogeneity from the culture supernatant by using anion-exchange chromatography and gel filtration. laccase ii is a monomeric glycoprotein with a molecular mass of 63 kda as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, contains 18% ... | 2002 | 12018276 |
| enhanced formation of extracellular laccase activity by the white-rot fungus trametes multicolor. | the white-rot fungus trametes multicolor mb 49 has been identified as an excellent producer of the industrially important enzyme laccase. the formation of extracellular laccase could be considerably stimulated by the addition of cu(ii) to a simple, glycerol-based culture medium. in this study, optimal concentrations of copper were found to be 0.5-1 mm, which were added during the growth phase of the fungus. other medium components important for laccase production are the carbon and nitrogen sour ... | 2002 | 12018250 |
| thermostable variants of pyranose 2-oxidase showing altered substrate selectivity for glucose and galactose. | the homotetrameric flavoprotein pyranose 2-oxidase (p2ox) has several proposed biotechnological applications, among others as a biocatalyst for carbohydrate transformations toward higher-value products. to improve some of the catalytic properties of p2ox from trametes multicolor, we selected a semirational enzyme engineering approach, namely, saturation mutagenesis of the amino acid his450 located at a pivotal point of the active site loop and subsequent screening of the libraries thus obtained ... | 2010 | 20158200 |
| probing active-site residues of pyranose 2-oxidase from trametes multicolor by semi-rational protein design. | d-tagatose is a sweetener with low caloric and non-glycemic characteristics. it can be produced by an enzymatic oxidation of d-galactose specifically at c2 followed by chemical hydrogenation. pyranose 2-oxidase (p2ox) from trametes multicolor catalyzes the oxidation of many aldopyranoses to their corresponding 2-keto derivatives. since d-galactose is not the preferred substrate of p2ox, semi-rational design was employed to improve the catalytic efficiency with this poor substrate. saturation mut ... | 2009 | 19370721 |
| a thermostable triple mutant of pyranose 2-oxidase from trametes multicolor with improved properties for biotechnological applications. | in order to increase the thermal stability and the catalytic properties of pyranose oxidase (p2ox) from trametes multicolor toward its poor substrate d-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-bq), we designed the triple-mutant t169g/e542k/v546c. whereas the wild-type enzyme clearly favors d-glucose as its substrate over d-galactose [substrate selectivity (k(cat)/k(m))(glc)/(k(cat)/k(m))(gal) = 172], the variant oxidizes both sugars equally well [(k(cat)/k(m))(glc)/( ... | 2009 | 19291706 |
| improving thermostability and catalytic activity of pyranose 2-oxidase from trametes multicolor by rational and semi-rational design. | the fungal homotetrameric flavoprotein pyranose 2-oxidase (p2ox; ec 1.1.3.10) catalyses the oxidation of various sugars at position c2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (e.g. oxidized ferrocenes). these properties make p2ox an interesting enzyme for various biotechnological applications. random mutagenesis has previously been used to identify variant e542k, which shows increased thermostability. in the present study, we select ... | 2009 | 19143837 |
| engineering of pyranose 2-oxidase: improvement for biofuel cell and food applications through semi-rational protein design. | pyranose 2-oxidase (p2ox) has several proposed biotechnological applications such as a bio-component in biofuel cells or for carbohydrate transformations. to improve some of the catalytic properties of p2ox from trametes multicolor, we selected a semi-rational approach of enzyme engineering, saturation mutagenesis of active-site residues and subsequent screening of mutant libraries for improved activity. one of the active-site mutants with improved catalytic characteristics identified was v546c, ... | 2009 | 19095017 |
| detection of a c4a-hydroperoxyflavin intermediate in the reaction of a flavoprotein oxidase. | this work describes for the first time the identification of a reaction intermediate, c4a-hydroperoxyflavin, during the oxidative half-reaction of a flavoprotein oxidase, pyranose 2-oxidase (p2o) from trametes multicolor, by using rapid kinetics. the reduced p2o reacted with oxygen with a forward rate constant of 5.8 x 10 (4) m (-1) s (-1) and a reverse rate constant of 2 s (-1), resulting in the formation of a c4a-hydroperoxyflavin intermediate which decayed with a rate constant of 18 s (-1). t ... | 2008 | 18652479 |
| the c-terminal region controls correct folding of genus trametes pyranose 2-oxidases. | the pyranose 2-oxidases from trametes ochracea and trametes pubescens share markedly similar amino acid sequences with identity of 93.4%. when expressed from the recombinant plasmids based on the same vector in the escherichia coli host strain bl21(de3) at higher growth temperatures, they differ strikingly in the formation of the inclusion bodies. upon overexpression in the cultures performed at 28 degrees c, the specific activity of pyranose 2-oxidase from t. pubescens was eight times higher th ... | 2007 | 17566580 |
| autoreduction and aggregation of fungal laccase in solution phase: possible correlation with a resting form of laccase. | this paper reports results of a reexamination of some poorly understood peculiarities of laccases, an enzyme family which has been extensively studied in our laboratories as well as by others for some years. the issue that is reconsidered here is the previously proposed existence of "active" and "resting" forms of laccases. the presence of fungal laccases with partly reduced active sites is demonstrated. of further interest is that an aggregated state in solution, not to our knowledge previously ... | 2006 | 16581176 |
| evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from trametes multicolor in e. coli. | 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 degrees c and optimized induction conditions to compare the productivity levels of the different expression systems. the pet21d+ and the pcold system gave 29 ... | 2010 | 20214772 |
| kinetic isotope effects on the noncovalent flavin mutant protein of pyranose 2-oxidase reveal insights into the flavin reduction mechanism. | pyranose 2-oxidase (p2o) from trametes multicolor contains a flavin adenine dinucleotide (fad) cofactor covalently linked to the n(3) atom of his167. the enzyme catalyzes the oxidation of aldopyranoses by molecular oxygen to generate 2-keto-aldoses and h(2)o(2) as products. in this study, the transient kinetics and primary and solvent kinetic isotope effects of the mutant in which his167 has been replaced with ala (h167a) were investigated, to elucidate the functional role of the 8a-n(3)-histidy ... | 2010 | 20359206 |
| importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase. | 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 (oxyge ... | 2010 | 20528921 |
| laccases from basidiomycetes: physicochemical characteristics and substrate specificity towards methoxyphenolic compounds. | laccases from the basidiomycetes coriolus hirsutus, coriolus zonatus, cerrena maxima, and coriolisimus fulvocinerea have been isolated and purified to homogeneity and partially characterized. the kinetics of oxidation of different methoxyphenolic compounds by the fungal laccases has been studied. as laccase substrates, such methoxyphenolic compounds as 4-hydroxy-3,5-dimethoxycinnamic acid (sinapinic acid), 4-hydroxy-3-methoxycinnamic acid (ferulic acid), and 2-methoxyphenol (guaiacol) were used. ... | 2001 | 11563958 |
| purification and characterization of pyranose oxidase from the white rot fungus trametes multicolor. | 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 ... | 2001 | 11472941 |
| peroxidative degradation of selected pcb: a mechanistic study. | the enzyme-induced decomposition and biodegradation of pcb were investigated. 2,5-dichlorobiphenyl (pcb 9) and 2,2',5,5'-tetrachlorobiphenyl (pcb 52) were used as example compounds to study efficiency and mechanism of the degradation processes. it was found that the application of horseradish peroxidase (hrp) together with defined amounts of hydrogen peroxide removed 90%, of the initial concentration of pcb 9 and 55% of the initial concentration of pcb 52 from an aqueous solution after a reactio ... | 2000 | 11061304 |
| production of a novel pyranose 2-oxidase by basidiomycete trametes multicolor. | during a screening for the enzyme pyranose 2-oxidase (p2o) which has a great potential as a biocatalyst for carbohydrate transformations, trametes multicolor was identified as a promising, not-yet-described producer of this particular enzyme activity. furthermore, it was found in this screening that the enzyme frequently occurs in basidiomycetes. intracellular p2o was produced in a growth-associated manner by t. multicolor during growth on various substrates, including mono-, oligo-, and polysac ... | 1998 | 9627385 |
| characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete lyophyllum shimeji (hon-shimeji). | the flavin-dependent enzyme pyranose 2-oxidase (p2ox) has gained increased attention during the last years because of a number of attractive applications for this enzyme. p2ox is a unique biocatalyst with high potential for biotransformations of carbohydrates and in synthetic carbohydrate chemistry. recently, it was shown that p2ox is useful as bioelement in biofuel cells, replacing glucose oxidase (gox), which traditionally is used in these applications. p2ox offers several advantages over gox ... | 2010 | 20630076 |
| h-bonding and positive charge at the n5/o4 locus are critical for covalent flavin attachment in trametes pyranose 2-oxidase. | flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. the enzyme-flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. the formation of the enzyme-flavin covalent bond--the flavinylation reaction--has been studied for the past 40 years. for the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of deve ... | 2010 | 20708626 |
| regioselective control of β-d-glucose oxidation by pyranose 2-oxidase is intimately coupled to conformational degeneracy. | trametes multicolor pyranose 2-oxidase (p2o) is a flavoprotein oxidase that oxidizes d-glucose at c2 to 2-keto-d-glucose by a highly regioselective mechanism. in this work, fluorinated sugar substrates were used as mechanistic probes to investigate the basis of regioselectivity in p2o. although frequently used to study the mechanisms of glycoside hydrolases, our work provides the first example of applying these probes to sugar oxidoreductases. our previous structure of the p2o mutant h167a in co ... | 2011 | 21515286 |