Studies of the cellulolytic system of <i>Trichoderma reesei</i> QM 9414

Tilbeurgh, Herman van; Pettersson, Göran; Bhikabhai, R.; Boeck, Hilde De; Claeyssens, Marc

doi:10.1111/j.1432-1033.1985.tb08843.x

Cited by 75 publications

(45 citation statements)

References 15 publications

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“…The individual steps in this scheme can be expressed as follows: The only simplification in the above equations is that k,, has been assumed identical for all substrates. Eqns(1a-m) can now be combined to calculate the changes in the concentrations for all species that occurs during an infinitesimal time interval dt: G, = G, + dC [1] (2 a) G, = G, + dC [2] + dC[71 + dC [8] + dC[101 + dC[12J G, = G, + dC [3] + dC [9] + dC [11] + dC [13] G, = G, + dC [4] G, = G, + dC [5] G, = G, + dC [6] G,E = G,E -dC [1] + dC [7] + dC [9] G,E = G,E -dC [2] + dC [8] …”

Section: Appendixmentioning

confidence: 99%

“…GI = G, + dC [7] + dC [9] G, = G, + dC [7] + 2 dC [8] + dC [10] + dC [11] + dC [12] G, = G, -dC [7] + dC [9] + dC [10] + dC [11] + 2 dC [13] G, = G, -dC [8] -dC [9] + dC [12] G, = G, -dC [12] -dC [13] The simulation procedure is then as follows: step 1, an equilibration period using Eqn(2) to obtain a steady state, using a short time step (dt = IO-'s); step 2, at the steady state a much larger time step (0.1 -1 s) is used to simulate Eqn(3); step 3, equilibration with 10 short time steps using Eqn(2); steps 2 and 3 are repeated for the desired time period.…”

Section: Appendixmentioning

confidence: 99%

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Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei

Harjunpää

Teleman

Koivula

et al. 1996

European Journal of Biochemistry

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The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase I1 from Trichoderma reesei was studied using 'H-NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non-reducing end were cleaved with approximately equal probability. The degradation rates of these cello-oligosaccharides, 1 -12 s-' at 27"C, are about 10-100 times faster than for the 4-methylumbelliferyl substituted analogs or for cellotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off-rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme. sites of cleavage, hydrolysis stereochemistry, kinetic and binding constants as well as in in vitro mutagenesis studies [6-81. These studies have been performed mostly using 4-methylumbelliferyl p-D-glycosides derived from cellotriose, cellotetraose and cellopentaose. The binding data have been obtained by fluorescence titration techniques, which have provided association constants also for cello-oligosaccharides by means of competition experiments [9, 101. NMR spectroscopy is well suited to follow the stereochemistry of saccharides 11 I] and we have recently used it together with progress curve analysis to study the hydrolysis of cellotriose by 7: reesei CBHII [12]. Replacing the initial-rate approximation by the complete progress curve analysis was highly advantageous, since it allows the elucidation of more complex mechanisms and reduces the amount of enzyme needed .In the present work the hydrolysis of cellotetraose, cellopentame and cellohexaose by CBHII has been studied by means of proton NMR spectroscopy and HPLC while using progress curves to analyse the kinetic data.Correspondence to V. Harjunpaa, VTT Chemical Technology, P. 0.

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Section: Appendixmentioning

confidence: 99%

Section: Appendixmentioning

confidence: 99%

Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei

Harjunpää

Teleman

Koivula

et al. 1996

European Journal of Biochemistry

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“…A study of the interaction of methylumbelliferyl p-glucoside, cellobioside and cellotrioside with cellobiohydrolase I1 from 7: reesei suggested at least four subsites for this enzyme (van Tilbeurgh et al, 1985) which also belongs to cellulase family B. The three-dimensional structure of cellobiohydrolase I1 from T. reesei shows that its active site has room to accommodate five glucose units (Jones, A., unpublished results).…”

Section: Cellulase Kinetics As Determined By the Coupled Assaymentioning

confidence: 99%

“…Cellooligosaccharides, with a chromogenic group as the aglycon, or radioactive labelled cellooligosaccharides are well-known substrates for steadystate kinetic studies of cellulases (Biely et al, 1991 ;Claeyssens et al, 1990a, b;Macarr6n et al, 1993;van Tilbeurgh et al, 1985). The cellobiose-dehydrogenase-coupled assay for the determination of cellulase activity was first described by Canevascini (1985).…”

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confidence: 99%

Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases

Schou

Rasmussen

Kaltoft

et al. 1993

European Journal of Biochemistry

101

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The catalytic activity of nine enzymes (endoglucanases 1-111, V, VI and cellobiohydrolases I and I1 from Humicola insolens; endoglucanases A and C from Bacillus lautus), representative of cellulase families A-C, H, J and K, has been investigated using a series of reduced cellooligosaccharides (cellotriitol to cellohexaitol) as substrates. For each enzyme, the specificity of cleavage was determined by analytical HPLC while the kinetic constants were obtained from a kinetic assay involving a cellobiose dehydrogenase purified from H . insotens as a coupled enzyme using 2,6-dichloroindophenol as the electron acceptor. These data were used to estimate the number of subsites in the enzymes. The stereochemical course of hydrolysis by seven enzymes, representing the six different families, was assessed using 'H-NMR. The enzymes belonging to families which had already been investigated (A-C), showed results in agreement with previous studies. The three other families (H, J and K), for which no mechanistic data was previously available, gave results which indicated that enzymes in group H had retaining-type activity and enzymes in groups J and K had inverting-type activity. The retaining endoglucanases I and I11 displayed a high glycosyltransferase activity under the conditions used during the NMR experiments resulting in precipitates of higher oligomers.

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“…Van Tilbeurgh et al 31) reported that the higher rate of hydrolysis with increasing degree of polymerization of cellooligosaccharides is probably due to the binding requirements of the enzyme, i.e., the need for more than two adjacent glucose units. They showed that 4-methylumbelliferyl-(glucose)3 (MeUmb(Glc)3) and MeUmb(Glc)4 were hydrolyzed by EG Ib at the second holosidic bond from the non-reducing end, and that MeUmbGlc and MeUmb(Glc)2 were produced from these substrates, respectively.…”

Section: Hydrolysis Of Cellooligosaccharidesmentioning

confidence: 99%

Purification and some properties of a microcrystalline cellulose-hydrolyzing enzyme (avicelase II) from fungal strain Y-94.

Yamanobe

Mitsuishi

Yagisawa

1988

Agricultural and Biological Chemistry

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A component of the microcrystalline cellulose (Avicel)-hydrolyzing enzyme produced by a fungal strain, Y-94, tentatively called Avicelase II, was purified by several kinds of column chromatography followed by chromatofocusing with Polybuffer anion exchanger (PBE 94), BioGel A0.5 m gel filtration, electrophoresis on polyacrylamide gel and disc isoelectric focusing (IEF). The molecular weight of the enzyme was estimated to be 68,000 by the SDS-polyacrylamide gel method and 56,000 by Bio-Gel A0.5 m gel filtration, and its isoelectric point was found to be around 4.4 by the chromatofocusing and disc IEF methods. The optimum pH and temperature for Avicel hydrolysis were 5.3 and 62°C, respectively. The enzyme was stable between pH 4.1 and 6.0 at 4°C for 24hr, and up to 61°C for lOmin. Heavy metal ions such as Cu2+ and Hg2+ strongly inactivated the enzyme. A highly purified enzyme preparation showed hydrolyzing activities towards Avicel, xylan, carboxylmethyl cellulose (CMC) (activity ratio of 100: 50 : 20, respectively) and acidswollen cellulose. By the PAS method, staining glycoprotein with Shiffs reagent and periodic acid, the enzyme seemed to be a glycoprotein containing some carbohydrate in its structure. Avicelase II tended to show a higher affinity towards cellooligosaccharides of high molecular weight. The enzyme hydrolyzed Avicel and acid-swollen cellulose, and produced trace amounts of cellotriose and significant quantities of glucose. In addition, the enzyme is able to act on the internal glycosidic bonds of CMCand xylan. Judging from the results, it appears that Avicelase II should be classified as a member of the specific endoglucanases rather than as an exoglucanase.As described in the previous paper,1} a mesophilic fungus (Y-94) isolated from soil produces a great amount of a cellulolytic enzyme in the culture filtrate, and the enzyme exhibits higher thermostability and hydrolysis limitation towards cellulosic substances than those of other mesophiles belonging to the genera, Trichoderma, Aspergillus, Penicillium and so on. This indicates that the cellulolytic enzyme system produced by this fungus will exhibit high potential when it is applied to the hydrolysis of the vegetable biomass.

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Studies of the cellulolytic system of Trichoderma reesei QM 9414

Cited by 75 publications

References 15 publications

Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei

Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei

Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases

Purification and some properties of a microcrystalline cellulose-hydrolyzing enzyme (avicelase II) from fungal strain Y-94.

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