Resolution, purification and some properties of the multiple forms of cellobiose quinone dehydrogenase from the white-rot fungus Sporotrichum pulverulentum.
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ABSTRACT:
SUBMITTER: Morpeth FF
PROVIDER: S-EPMC1146809 | biostudies-other | 1986 May
REPOSITORIES: biostudies-other
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Project description:Cellobiose oxidase from the white-rot fungus Sporotrichum pulverulentum has been purified to homogeneity by a new procedure. The carbohydrate and amino acid compositions of the enzyme have been determined. Cellobiose oxidase contains FAD and cytochrome b prosthetic groups. Mr of the enzyme has been estimated at 74400 by sedimentation equilibrium. The enzyme is a monomer. Optical, fluorescence and e.p.r. spectra of oxidized and reduced cellobiose oxidase have been determined. A preliminary investigation of the substrate specificity of cellobiose oxidase reveals that disaccharides and even some insoluble polysaccharides are substrates, but not monosaccharides. Strong substrate inhibition is seen at high concentrations of cellobiose. This effect is particularly marked when oxygen is the electron acceptor. Cellobiose oxidase is unusual among flavoproteins, since it stabilizes the red anionic flavin semiquinone and forms a sulphite adduct, yet appears to produce the superoxide anion as its primary reduced oxygen product.
| S-EPMC1145023 | biostudies-other
Project description:The reactions between cellobiose and cellobiose oxidase were investigated by stopped-flow spectrophotometry. Under anaerobic conditions rapid reduction of the associated flavin is followed by slower reduction of cytochrome b. The kinetic difference spectra are reported. The rate of flavin reduction depends on the cellobiose concentration (with an apparent second-order rate constant of approx. 10(5) M-1.s-1) but reaches a rate limit of approx. 20 s-1. In contrast, the rate of cytochrome b reduction decreases at high cellobiose concentrations. Kinetic titrations of the flavin and cytochrome b moieties yield the stoichiometries of the separate reactions, i.e. the number of moles of cellobiose needed to fully reduce 1 mole of each redox component. The rate constant for cytochrome b reduction, unlike that for flavin reduction, increased with enzyme concentration, prompting the conclusion that any given cytochrome b centre is reduced preferentially by flavin groups in different molecules rather than by its partner flavin within the same monomer. These data are discussed in the context of a scheme that rationalizes them and accounts for the overall stoichiometry in which three two-electron donors (cellobiose molecules) reduce two three-electron acceptors (the flavin-cytochrome b of cellobiose oxidase).
| S-EPMC1135474 | biostudies-other
Project description:An extracellular enzyme from culture filtrates of Sporotrichum (Chrysosporium) thermophile (A.T.C.C. 42 464) after growth on cellulose or cellobiose was shown to oxidize cellobiose to cellobionic acid in vitro. Lactose and cellodextrins were also efficiently oxidized, but the enzyme was not active against most mono- and di-saccharides. Several redox substances could act as electron acceptors, but molecular oxygen, tetrazolium salts and NAD(P) were not reduced. Activity was stimulated up to 2-fold in the presence of 0.05 M-Mg2+. The pH optimum of the enzymic reaction was acidic when the activity was tested with dichlorophenol-indophenol or Methylene Blue, but was neutral to alkaline for 3,5-di-t-butyl-1,2-benzoquinone or phenazine methosulphate as electron acceptors. As the enzyme was formed inductively in parallel with the endocellulase, its possible function in relation to cellulolysis is discussed.
| S-EPMC1158220 | biostudies-other
Project description:The pre-steady-state kinetics of inter-domain electron transfer in the extracellular flavocytochrome cellobiose dehydrogenase from Phanerochaete chrysosporium was studied using various values of pH and substrate concentration. Monitoring at the isosbestic point of each prosthetic group indicated that the reductive half-reactions of flavin and haem were biphasic and monophasic respectively. When the observed rates of the flavin and haem reactions were plotted against substrate concentration, the behaviour of the second phase of the flavin reduction was almost identical with that of haem reduction at all substrate concentrations and pH values tested, suggesting that the formation of flavin semiquinone and haem reduction involve the same electron transfer reaction. Although flavin reduction by cellobiose was observed in the range of pH 3.0-7.0, the velocity of the next electron transfer step decreased with increase of pH and was almost zero above pH 6.0. The second phase of flavin reduction and the haem reduction were inhibited similarly by high concentrations of the substrate, whereas the first phase of flavin reduction showed a hyperbolic relation to the cellobiose concentration. Increase in pH enhanced the substrate inhibition of haem reduction but not the initial flavin reduction. Moreover, the dissociation constant K(d) of flavin reduction and the substrate inhibition constant K(i) of haem reduction decreased similarly with an increase of pH. From these results, it is evident that binding of cellobiose to the active site inhibits electron transfer from flavin to haem.
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Project description:One oxidase (EC 1.10.3.2) and three lignin peroxidases (EC 1.11.1.-) were purified from the culture liquid of the white-rot fungus Phlebia radiata Fr. All the enzymes were glycoproteins. The oxidase had Mr 64,000 and the lignin peroxidases I, II and III had Mr values 42,000, 45,000 and 44,000 respectively. The lignin peroxidases were found to share common antigenic determinants: lignin peroxidases II and III were serologically indistinguishable and lignin peroxidase I was related but distinguishable. The oxidase did not share any immunological properties with the lignin peroxidases. Lignin peroxidases of Phlebia contain protoporphyrin IX as a prosthetic group. In the presence of H2O2 and an electron donor, veratryl alcohol, lignin peroxidases exhibit spectral shifts analogous to those of animal catalase (EC 1.11.1.6). Phlebia enzymes show optimal activity at pH 3-4.5 at 40 degrees C and are stable in the pH range 5-6. They modify Kraft lignin and phenolic compounds containing hydroxy and methoxy groups.
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Project description:A glutathione S-transferase (GST) was purified to homogeneity from the white-rot fungus, Phanerochaete chrysosporium, by affinity chromatography on glutathione-agarose followed by Mono-Q ion-exchange FPLC. This protein immunoblotted with antisera to rat Theta class GST 5-5 and also showed N-terminal sequence similarity to the Theta class, including the presence of a conserved serine residue that has been specifically implicated in catalysis in this class [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133-2143] and other residues conserved in plant sequences. Catalytic activity was found to be highly labile in the purified protein, although preliminary evidence for activity (approx. 120 m-units/mg) with 1,2-epoxy-3-(p-nitrophenoxy)propane was obtained in some preparations. The enzyme seems to be a dimer with a subunit molecular mass of 25 kDa by SDS/PAGE. The native molecular masses estimated by non-denaturing electrophoresis and by Superose-12 gel filtration were 58 and 45 kDa respectively. A second protein purified in this study also gave low level of activity with 1,2-epoxy-3-(p-nitrophenoxy)propane and had a subunit molecular mass of 28 kDa (native size 62-63 kDa), but did not immunoblot with any GST class and seemed to be N-terminally blocked.
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Project description:Fungi can decompose plant biomass into small oligo- and monosaccharides to be used as carbon sources. Some of these small molecules may induce metabolic pathways and the production of extracellular enzymes targeted for degradation of plant cell wall polymers. Despite extensive studies in ascomycete fungi, little is known about the nature of inducers for the lignocellulolytic systems of basidiomycetes. In this study, we analyzed six sugars known to induce the expression of lignocellulolytic genes in ascomycetes for their role as inducers in the basidiomycete white-rot fungus Dichomitus squalens using a transcriptomic approach. This identified cellobiose and l-rhamnose as the main inducers of cellulolytic and pectinolytic genes, respectively, of D. squalens Our results also identified differences in gene expression patterns between dikaryotic and monokaryotic strains of D. squalens cultivated on plant biomass-derived monosaccharides and the disaccharide cellobiose. This suggests that despite conservation of the induction between these two genetic forms of D. squalens, the fine-tuning in the gene regulation of lignocellulose conversion is differently organized in these strains.IMPORTANCE Wood-decomposing basidiomycete fungi have a major role in the global carbon cycle and are promising candidates for lignocellulosic biorefinery applications. However, information on which components trigger enzyme production is currently lacking, which is crucial for the efficient use of these fungi in biotechnology. In this study, transcriptomes of the white-rot fungus Dichomitus squalens from plant biomass-derived monosaccharide and cellobiose cultures were studied to identify compounds that induce the expression of genes involved in plant biomass degradation.
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Project description:The white rot fungus Pleurotus ostreatus is an edible basidiomycete with increasing agricultural and biotechnological importance. Genetic manipulation and breeding of this organism are restricted because of the lack of knowledge about its genomic structure. In this study, we analyzed the genomic constitution of P. ostreatus by using pulsed-field gel electrophoresis optimized for the separation of its chromosomes. We have determined that it contains 11 pairs of chromosomes with sizes ranging from 1.4 to 4.7 Mbp. In addition to chromosome separation, the use of single-copy DNA probes allowed us to resolve the ambiguities caused by chromosome comigration. When the two nuclei present in the dikaryon were separated by protoplasting, analysis of their karyotypes revealed length polymorphisms affecting various chromosomes. This is, to our knowledge, the clearest chromosome separation available for this species.
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Project description:Phanerochaete chrysosporium, the model white rot fungus, has been the focus of research for the past about four decades for understanding the mechanisms and processes of biodegradation of the natural aromatic polymer lignin and a broad range of environmental toxic chemicals. The ability to degrade this vast array of xenobiotic compounds was originally attributed to its lignin-degrading enzyme system, mainly the extracellular peroxidases. However, subsequent physiological, biochemical, and/or genetic studies by us and others identified the involvement of a peroxidase-independent oxidoreductase system, the cytochrome P450 monooxygenase system. The whole genome sequence revealed an extraordinarily large P450 contingent (P450ome) with an estimated 149 P450s in this organism. This review focuses on the current status of understanding on the P450 monooxygenase system of P. chrysosproium in terms of pre-genomic and post-genomic identification, structural and evolutionary analysis, transcriptional regulation, redox partners, and functional characterization for its biodegradative potential. Future research on this catalytically diverse oxidoreductase enzyme system and its major role as a newly emerged player in xenobiotic metabolism/degradation is discussed.
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