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At least 19 recordsLinked to original sources

PubMed1992

Evidence that cellobiose:quinone oxidoreductase from Phanerochaete chrysosporium is a breakdown product of cellobiose oxidase.

Phanerochaete chrysosporium releases two enzymes that oxidize cellobiose and higher cellodextrins: the flavohaemoprotein cellobiose oxidase and the flavoprotein cellobiose:quinone oxidoreductase (CBQase). Partial digestion of these enzymes with Staphylococcal V8 proteinase or cyanogen bromide yielded many identical bands on SDS-polyacrylamide gels. A polyclonal antibody to either purified protein gave cross-reaction. The purification procedure also yielded a haem protein that ran on dodecyl sulphate gels at Mr 31,000, as compared with 91,000 for cellobiose oxidase and 63,000 for CBQase. The 31 kDa haem protein cross-reacted with polyclonal antibody to cellobiose oxidase, but not with antibody to CBQase. Sulphite bleached the flavin of cellobiose oxidase, but gave no reaction with the 31 kDa haem protein, suggesting an absence of flavin. It is proposed that CBQase and the 31 kDa haem protein are formed from cellobiose oxidase by proteolytic cleavage.

Agaricales↗

PubMed1992

A comparison of the catalytic properties of cellobiose:quinone oxidoreductase and cellobiose oxidase from Phanerochaete chrysosporium.

Several catalytic properties of the FAD enzyme cellobiose:quinone oxidoreductase (CBQ) and the heme/FAD enzyme, cellobiose oxidase (CBO) have been investigated and compared. Dichlorophenol-indophenol was found to be a very good electron acceptor for cellobiose oxidation by both enzymes. The optimal pH value for this oxidation with dichlorophenol-indophenol as a co-substrate was observed around pH 4 for both enzymes. The turnover numbers of this reaction were also very similar. The Km values for cellobiose oxidation were identical, whereas the Km for CBO with dichlorophenol-indophenol is lower than that of CBQ. Atmospheric oxygen is a very poor electron acceptor for both CBO and CBQ, however, CBO can utilize cytochrome c as an effective electron acceptor, while CBQ cannot. The specific activity of CBO for cytochrome c is thus about 200-times higher than for oxygen. Thus, one way to distinguish the two enzymes is by the cytochrome-c-reducing ability of CBO. Therefore, we propose that the nomenclature for CBO is tentatively changed to cellobiose:cytochrome c oxidoreductase until a rational name can be installed. Both enzymes have radical-reducing activities. The cation radical, derived from 1,2,4,5-tetramethoxybenzene, was reduced by both enzymes at almost the same reaction rate. The phenoxyradical produced by lignin peroxidase, catalyzing the oxidation of acetosyringon, was also reduced by both enzymes. The reduction of phenoxyradicals formed by phenoloxidases (lignin peroxidases, as well as laccases) may be important in preventing repolymerization reactions which we suggest would significantly facilitate lignin degradation.

Carbohydrate Dehydrogenases↗

PubMed1991

Cellobiose uptake by the cellulolytic ruminal anaerobe Fibrobacter (Bacteroides) succinogenes.

Cellobiose transport by the cellulolytic ruminal anaerobe Fibrobacter (Bacteroides) succinogenes was measured using randomly tritiated cellobiose. When assayed at the same concentration (1 mM), total cellobiose uptake was one-fourth to one-third that of total glucose uptake. The abilities of F. succinogenes to transport cellobiose or glucose were not affected by the sugar on which the cells were grown. Aspects of the simultaneous transport of [14C(U)]glucose and [3H(G)]cellobiose, the failure of high concentrations of cold glucose to compete with hypothetical [3H(G)]glucose (derived externally from [3H(G)]cellobiose), and differential metal-ion stimulation of cellobiose transport indicate a cellobiose permease, rather than cellobiase plus glucose permease, was responsible for cellobiose transport. Glucose (10-fold molar excess) partially inhibited cellobiose transport. This was enhanced by prior incubation of the cells with glucose, suggesting subsequent metabolism of the glucose was responsible for the inhibition. Compounds interfering with electron transport or maintenance of transmembrane ion gradients inhibited cellobiose uptake, indicating that active transport rather than a phosphoenolpyruvate:phosphotransferase system catalyzed cellobiose transport. Na+, but not Li+, stimulated cellobiose transport.

Acriflavine↗

PubMed1991

Cellobiose uptake and metabolism by Ruminococcus flavefaciens.

The cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 utilizes cellobiose but not glucose as a substrate for growth. Cellobiose uptake by R. flavefaciens FD-1 was measured under anaerobic conditions (N2), using [G-3H]cellobiose. The rate of cellobiose uptake for early- or late-log-phase cellobiose-grown cells was 9 nmol/min per mg of whole-cell protein. Cellobiose uptake was inhibited by electron transport inhibitors, iron-reactive compounds, proton ionophores, sulfhydryl inhibitors, N,N-dicyclohexylcarbodiimide, and NaF, as well as lasalocid and monensin. The results support the existence of an active transport system for cellobiose. Transport of [U-14C]glucose was not detected with this system. Phosphorylation of cellobiose was not by a phosphoenolpyruvate-dependent system. Cellobiose phosphorylase activity was detected by both a coupled spectrophotometric assay and a discontinuous assay. The enzyme was produced constitutively in cellobiose-grown cells at a specific activity of 329 nmol/min per mg of cell-free extract protein.

Antimetabolites↗

PubMed1990

Transport of glucose and cellobiose by Candida wickerhamii and Clavispora lusitaniae.

The cellular location of beta-1,4-glucosidase activity from, as well as the transport of glucose and cellobiose into, cells of Clavispora lusitaniae NRRL Y-5394 and Candida wickerhamii NRRL Y-2563 was investigated. The beta-glucosidase from Cl. lusitaniae appeared to be a soluble cytoplasmic enzyme. This yeast transported both glucose and cellobiose when grown in medium containing cellobiose as the sole carbon source. Glucose, but not cellobiose, uptake was observed for cells grown on glucose. The Ks and Vmax values for cellobiose transport were different when Cl. lusitaniae was cultured either aerobically (0.11 mM, 6.28 nmol.min-1.mg-1) or anaerobically (0.25 mM, 3.88 nmol-1.min-1.mg-1). The Ks and Vmax values for glucose transport (0.23-1.10 mM and 17.2-33.9 nmol.min-1.mg-1) also differed with the various growth conditions. The beta-glucosidase from C. wickerhamii was extracytoplasmically located. This yeast transported glucose, but not cellobiose, under all growth conditions tested. The Ks for glucose uptake was 0.13-0.28 mM when C. wickerhamii was cultured on cellobiose and 0.25-0.30 mM when cultured on glucose. The Vmax values for glucose uptake were greater for cells cultured on cellobiose (35.0-37.9 nmol.min-1.mg-1) than for cells cultured on glucose (15.6-21.4 nmol.min-1.mg-1). Cellobiose did not inhibit glucose uptake in either yeast. Glucose partially inhibited cellobiose transport in C. lusitaniae, but only if the yeast was grown aerobically. In both yeasts, sugar transport was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and 1799, but insensitive to valinomycin.

Aerobiosis↗

PubMed1992

Cellobiose chemotaxis by the cellulolytic bacterium Cellulomonas gelida.

In the course of a study on the bacterial degradation of plant cell wall polysaccharides, we observed that growing cells of motile cellulolytic bacteria accumulated, without attachment, near cellulose fibers present in the cultures. Because it seemed likely that the accumulation was due to chemotactic behavior, we investigated the chemotactic responses of one of the above-mentioned bacteria (Cellulomonas gelida ATCC 488). We studied primarily the responses toward cellobiose, which is the major product of cellulose hydrolysis by microorganisms, and toward hemicellulose hydrolysis products. We found that cellobiose, cellotriose, D-glucose, xylobiose, and D-xylose, as well as other sugars that are hemicellulose components, served as chemoattractants for C. gelida, as determined by a modification of Adler's capillary assay. Competition and inducibility experiments indicated that C. gelida possesses at least two types of separately regulated cellobiose chemoreceptors (Cb1 and cellobiose, cellotriose, xylobiose, and D-glucose, and it is constitutively synthesized. The presence in C. gelida of a constitutive response toward cellobiose and of at least two distinct cellobiose chemoreceptors has implications for the survival of this cellulolytic bacterium in nature. A possible mechanism for cellobiose-mediated bacterial chemotaxis toward cellulose is proposed. We suggest that, in natural environments, motile cellulolytic bacteria migrate toward plant materials that contain cellulose and hemicellulose by swimming up cellobiose concentration gradients and/or concentration gradients of other sugars (e.g., xylobiose, D-xylose, and D-glucose) formed by enzymatic hydrolysis of plant cell wall polysaccharides.

Actinomycetales↗

PubMed1992

Production of Fenton's reagent by cellobiose oxidase from cellulolytic cultures of Phanerochaete chrysosporium.

The reduction of dioxygen by cellobiose oxidase leads to accumulation of H2O2, with either cellobiose or microcrystalline cellulose as electron donor. Cellobiose oxidase will also reduce many Fe(III) complexes, including Fe(III) acetate. Many Fe(II) complexes react with H2O2 to produce hydroxyl radicals or a similarly reactive species in the Fenton reaction as shown: H2O2 + Fe2+----HO. + HO- + Fe3+. The hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid is a standard test for hydroxyl radicals. Hydroxylation was observed in acetate buffer (pH 4.0), both with Fe(II) plus H2O2 and with cellobiose oxidase plus cellobiose, O2 and Fe(III). The hydroxylation was suppressed by addition of catalase or the absence of iron [Fe(II) or Fe(III) as appropriate]. Another test for hydroxyl radicals is the conversion of deoxyribose to malondialdehyde; this gave positive results under similar conditions. Further experiments used an O2 electrode. Addition of H2O2 to Fe(II) acetate (pH 4.0) or Fe(II) phosphate (pH 2.8) in the absence of enzyme led to a pulse of O2 uptake, as expected from production of hydroxyl radicals as shown: RH+HO.----R. + H2O; R. + O2----RO2.----products. With phosphate (pH 2.8) or 10 mM acetate (pH 4.0), the O2 uptake pulse was increased by Avicel, suggesting that the Avicel was being damaged. Oxygen uptake was monitored for mixtures of Avicel (5 g.1-1), cellobiose oxidase, O2 and Fe(III) (30 microM). An addition of catalase after 20-30 min indicated very little accumulation of H2O2, but caused a 70% inhibition of the O2 uptake rate. This was observed with either phosphate (pH 2.8) or 10 mM acetate (pH 4.0) as buffer, and is further evidence that oxidative damage had been taking place, until the Fenton reaction was suppressed by catalase. A separate binding study established that with 10 mM acetate as buffer, almost all (98%) of the Fe(III) would have been bound to the Avicel. In the presence of Fe(III), cellobiose oxidase could provide a biological method for disrupting the crystalline structure of cellulose.

Carbohydrate Dehydrogenases↗

PubMed1979

[Cellobiose as a regulator of endoglucanase activity of cellulase complexes. Mechanism of the regulation].

Cellobiose may exert different effects on the activities of various endoglucanases. The endoglucanases of T. reesei and Rapidase are noticeably suppressed by cellobiose at concentrations above 3 mM. On the other hand, a low molecular weight endoglucanase from T. koningii is activated by cellobiose, whereas high molecular weight endoglucanases from the same source are inhibited by cellobiose. A detailed kinetic analysis of the effects showed that the low molecular weight endoglucanase is activated by a transglycosylation mechanism, in which cellobiose acts as an additional nucleophile. At saturating concentrations of cellobiose (Ks = 15 mM) the enzyme activity is increased 6-fold. Such a specific mechanism of activation manifests itself in an acceleration of random cleavage of CM-cellulose by the low molecular weight endoglucanase, which can be recorded by a viscosimetric technique. However, its action does not accelerate the production of soluble reducing sugars.

Cellobiose↗

PubMed1992

Evidence that cellobiose oxidase from Phanerochaete chrysosporium is primarily an Fe(III) reductase. Kinetic comparison with neutrophil NADPH oxidase and yeast flavocytochrome b2.

Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30 degrees C, with excess cellobiose as substrate and O2 or Fe(III) as acceptor. With O2 at 230 microM as sole electron acceptor, the O2 uptake rate corresponded to a one-electron turnover number of 0.13 +/- 0.01 s-1. Measurements at different O2 concentrations indicated Km(O2) greater than 120 microM. In separate experiments, the reduction of Fe(III) acetate was monitored at 340 nm in the absence of oxygen. The maximum velocity of Fe(III)-acetate reduction (Vmax) was 4.5 +/- 0.7 s-1, while Km[Fe(III) acetate] was 34 +/- 12 microM. With ferricyanide in place of Fe(III) acetate, the corresponding values were 6.9 +/- 0.7 s-1 and 23 +/- 5 microM. Redox titrations established the potential of the haem prosthetic group of the oxidase at pH 4.0 as +165 mV. The midpoint potential for Fe(III)/Fe(II) acetate at pH 4.0 is much higher, a value of +535 mV being obtained with 200 microM Fe. Cellobiose oxidase resembles yeast flavocytochrome b2 and differs from the neutrophil NADPH oxidase in having the potential of its haem group far above the potential for one-electron reduction of O2 to superoxide (Em,4 = -110 mV). A kinetic comparison led to the conclusion that the role of cellobiose oxidase is as an Fe(III) reductase. Fe(II) may have a biological importance as a component of Fenton's reagent [Fe(II)/H2O2]. The concentration of cellobiose oxidase in the growth medium at harvest (0.3 microM) can provide a far higher flux of Fe(II) than a non-enzymic proposal in the literature.

Basidiomycota↗

PubMed1990

[Use of glucose and cellobiose by 3 strains of Fibrobacter succinogenes].

F succinogenes strains S85, 128 and 095 were compared with respect to their growth using glucose and/or cellobiose as the carbon and energy substrate(s) and their capacities to degrade cellulose. The growth rate of F succinogenes strain S85 was the same using glucose or cellobiose, whereas the growth rates of strains 128 and 095 were about thrice the rate when using cellobiose. Strain S85 could simultaneously use glucose and cellobiose, while strains 128 and 095 tended to use preferentially glucose then cellobiose in a mixture of the 2 sugars. Their capacities to degrade cellulose were equivalent.

Animals↗

PubMed1991

Cellobiose oxidase from Phanerochaete chrysosporium can be cleaved by papain into two domains.

Cellobiose oxidase from the white rot fungus Phanerochaete chrysosporium has been purified to homogeneity by a new method. The enzyme has been cleaved by papain into two fragments: one containing the heme group and one containing the flavin group. The flavin fragment can oxidize cellobiose and is reoxidized by oxygen. Cellobiose oxidase binds to cellulose to approximately the same extent as cellobiohydrolase I. The cellulose-binding site is located on the flavin domain. The enzyme cannot be totally displaced from cellulose by cellobiose, and it is still active when adsorbed to cellulose. The possible role of the enzyme in lignocellulose degradation is discussed.

Amino Acids↗

PubMed1991

Construction of a Saccharomyces cerevisiae strain able to ferment cellobiose.

The bglA gene, encoding a beta-glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the CYC-GAL promoter inducible by galactose. The expression of bglA-encoded activity in the strain used as a host was not sufficient to allow its growth with cellobiose as a carbon source. However, a recessive mutation in a gene designated cem1 has been obtained which, combined with the expression of beta-glucosidase activity, allows the growth of S. cerevisiae on cellobiose. The expression of the blgA gene in a cem1 strain confers on S. cerevisiae the capability for an efficient fermentation of cellobiose, as detected by the formation of CO2.

Bacillus↗

PubMed1991

Triiodide reduction by cellobiose:quinone oxidoreductase of Phanerochaete chrysosporium.

Cellobiose:quinone oxidoreductase (CBQase) in the presence of cellobiose inhibits peroxidase-catalyzed oxidation of iodide to triiodide (I3). This inhibition is due to the two-electron reduction of I3- by CBQase. The apparent Km of I3- for this reaction is 120 microM and the specific activity is 57 mumol.min-1.mg-1. A proposed mechanism for I3- reduction by CBQase involves initial reduction of the flavin moiety by cellobiose to produce a dihydroflavin. This is followed by the substitution of one of the iodine atoms of I3- at the C(4a)-position of dihydroflavin to generate C(4a)-iododihydroflavin and two iodide ions. The C(4a)-iododihydroflavin eliminates HI to regenerate the oxidized CBQase.

Basidiomycota↗

PubMed1992

Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.

Cellobiose↗

PubMed1991

Mechanisms of redox interactions between lignin peroxidase and cellobiose:quinone oxidoreductase.

The mechanism of redox interactions between the heme-enzyme, lignin peroxidase (LiP), and the FAD-enzyme, cellobiose:quinone oxidoreductase (CBQ) (EC 1.1.5.1), was investigated under various conditions. Veratryl alcohol oxidation by LiP was inhibited by CBQ in the presence of cellobiose. Lineweaver-Burk plots at various CBQ concentrations suggest that this inhibition is non-competitive. The oxidation rate of the reduced CBQ (FADH2) by LiP plus H2O2 increased significantly only in the presence of veratryl alcohol. Furthermore, the cation radical derived from 1,2,4,5-tetramethoxybenzene was reduced by CBQ in the presence of cellobiose. It is concluded from these results that CBQ can reduce aromatic cation radicals and that veratryl alcohol acts as a radical mediator of the redox interactions between LiP and CBQ.

Carbohydrate Dehydrogenases↗

PubMed1992

Cellobiose oxidase of Phanerochaete chrysosporium enhances crystalline cellulose degradation by cellulases.

The effect of Phanerochaete chrysosporium cellobiose oxidase (CBO) on microcrystalline cellulose hydrolysis by Trichoderma cellulases was determined. Addition of 10 micrograms.ml-1 CBO to a reaction mixture containing T. viride cellulase increased glucose and cellobiose production by 10% and 48%, respectively. Cellulose weight loss was also enhanced by 19%. At higher concentrations (20-80 micrograms.ml-1), CBO decreased glucose and cellobiose production. Cellulose weight loss at 60 micrograms.ml-1 CBO was 76% compared to control cellulase reactions. This decrease appears to be due to inactivation of cellulase by H2O2 produced via CBO reaction, because addition of catalase enhances sugar production and cellulose weight loss. These findings suggest that at low, perhaps physiologically relevant concentrations, CBO enhances crystalline cellulose degradation by cellulases.

Basidiomycota↗

PubMed1992

Cellobiose oxidase from Phanerochaete chrysosporium. Stopped-flow spectrophotometric analysis of pH-dependent reduction.

Cellobiose oxidase (CBO) from Phanerochaete chrysosporium can utilize dichlorphenol-indophenol (Cl2Ind) and cytochrome c as effective electron acceptors for the oxidation of cellobiose. However, the pH dependencies of activity for these electron acceptors are significantly different. Both compounds act as effective electron acceptors at pH 4.2, whereas only dichlorophenol-indophenol is active at pH 5.9. To explain this discrepancy, the pH dependencies of the reduction rates of FAD and heme, respectively, in CBO by cellobiose have been investigated by stopped-flow spectrophotometry. Both FAD and heme are reduced with a high rate constant at pH 4.2. In contrast, at pH 5.9, only FAD reduction is fast, while the reduction of the heme is extremely slow. As a conclusion, the reduction of cytochrome c by CBO is dependent on heme, which functions at a lower pH range compared to reduction of FAD.

Carbohydrate Dehydrogenases↗

PubMed1991

Cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile.

Both cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile, ATCC 42464, obtained after fractionation with DEAE-Trisacryl chromatography and named cellobiose dehydrogenase I and II have been purified to homogeneity by different chromatographic techniques. Both enzymes are slightly glycosylated flavocytochrome-b proteins with similar catalytic properties but with distinct molecular masses (91 kDa and 192 kDa for enzymes I and II, respectively) and isoelectric point (4.1 versus 3.45). Examination by SDS/PAGE clearly showed that the larger enzyme II is a homodimer, whose subunit is close to, but different from dehydrogenase I which is homogeneous by this technique. After limited digestion of both enzymes with papain, two main fractions with residual activity are formed, one carrying the heme, the other being the flavin component; each fraction is characterized by its particular chromatographic behaviour. The flavin carrying component shows an atypical (for flavoprotein) three-banded spectrum indicative of the presence of a flavin derivative. Both enzymes react very slowly with oxygen clearly forming some superoxide radicals and possibly hydrogen peroxide. Cellobiose and other cellodextrins are oxidized at their reducing glycosyl moiety to the corresponding aldonic acid. With the use of the autooxidable phenazinemethosulphate, cellulose (either in a hydrated form or crystalline) is also oxidized at free reducing ends so that appreciable amounts of cellobionic acid are released upon enzymatic hydrolysis.

Amino Acids↗