Separation of endo‐ and exo‐type cellulases using a new affinity chromatography method

Tilbeurgh, Herman van; Bhikhabhai, Ramagauri; Pettersson, Linda; Claeyssens, Marc

doi:10.1016/0014-5793(84)80321-x

Cited by 70 publications

(32 citation statements)

References 9 publications

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“…Homogeneous exo-cellobiohydrolase I and cellobiohydrolase II were obtained by affinity chromatography [6]. Endoglucanase II and III from T. reesei QM 9414, purified by ion-exchange chromatography, were kindly donated by Dr G. Pettersson (Uppsala, Sweden).…”

Section: Methodsmentioning

confidence: 99%

“…Under these conditions partial separations of the enzymes present are observed: ,&glucosidase activities are eluted before the bulk of the fractions containing enzymes active on the test substrate MeUmb &Gal ,8(1-+4)Glc and 2 main fractions are characterised by their differential inhibition by cellobiose (figs 1 and 2). They can further be purified by affinity chromatography [6].…”

Section: Fractionation Of the Cellulase Complex Bymentioning

confidence: 99%

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Detection and differentiation of cellulase components using low molecular mass fluorogenic substrates

Tilbeurgh

Claeyssens

1985

FEBS Letters

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The 4-methylumbelliferyl /I-D-glycosides of glucose, cellobiose, cellotriose and lactose are used to differentiate several exo-cellobiohydrolase, endocellulase and /I-glucosidase activities in crude cellulase from Trichoderma reesei. Spectrophotometric or fluorimetric assays allow simple detection and quantitative measurements of eluate activities from ion-exchange chromatography and after analytical gel electrofocusing. Using the fluoropho~c glucoside several /?-glucosidases can be visual&d after isoelectric focusing on polyacrylamide gels. The use of the lactoside and of the same substrate supplemented with cellobiose as inhibitor allows a clearcut distinction to be made between endocellulase II and exo-cellobiohydrolase I. Both enzymes are present as 'iso-enzyme' mixtures. With the cellotrioside only one fraction is detectable (endocellulase III). The same methods could be used in culture growth experiments.

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Section: Methodsmentioning

confidence: 99%

Section: Fractionation Of the Cellulase Complex Bymentioning

confidence: 99%

Detection and differentiation of cellulase components using low molecular mass fluorogenic substrates

Tilbeurgh

Claeyssens

1985

FEBS Letters

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“…Endoglucanase I (component with pl 4.6), cellobiohydrolase I and cellobiohydrolase II from Trichoderma reesei QM 9414 were isolated as described van Tilbeurgh et al, 1984)) and their purity was checked by SDS/PAGE (Laemmli, 1970) and gel isoelectric focusing (van Tilbeurgh & Claeyssens, 1985).…”

Section: Enzymesmentioning

confidence: 99%

Studies of the cellulolytic system of the filamentous fungus Trichoderma reesei QM 9414. Substrate specificity and transfer activity of endoglucanase I

Claeyssens

Tilbeurgh

Kamerling

et al. 1990

Biochemical Journal

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The Netherlands, and $Chemicky ustav Slovenskej akademie vied, Dubravska cesta, 9, CS-8423 Bratislava, Czechoslovakia Endoglucanase I from the filamentous fungus Trichoderma reesei catalyses hydrolysis and glycosyl-transfer reactions of cello-oligosaccharides. Initial bond-cleaving frequencies determined with 1-3H-labelled cello-oligosaccharides proved to be substrate-concentration-dependent. Using chromophoric glycosides and analysing the reaction products by h.p.l.c., kinetic data are obtained and, as typical for an endo-type depolymerase, apparent hydrolytic parameters (kct., kcat /Km) increase steadily as a function of the number of glucose residues. At high substrate concentrations, and for both free cellodextrins and their aromatic glycosides, complex patterns (transfer reactions) are, however, evident. In contrast with the corresponding lactosides and 1-thiocellobiosides, and in conflict with the expected specificity, aromatic 1-0-,6-cellobiosides are apparently hydrolysed at both scissile bonds, yielding the glucoside as one of the main reaction products. Its formation rate is clearly non-hyperbolically related to the substrate concentration and, since the rate of Dglucose formation is substantially lower, strong indications for dismutation reactions (self-transfer) are again obtained. Evidence for transfer reactions catalysed by endoglucanase I further results from experiments using different acceptor and donor substrates. A main transfer product accumulating in a digest containing a chromophoric 1-thioxyloside was isolated and its structure elucidated by proton n.m.r. spectrometry (500 MHz). The fll-4 configuration of the newly formed bond was proved. INTRODUCTIONThe filamentous fungus Trichoderma reesei secretes a very efficient cellulolytic system which contains several components.

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“…CBH I and CBH II were purified from the culture filtrate of T. reesei QM 9414, as described by Bhikhabhai et al [12]. Final purification of CBH I and CBH II was performed by affinity chromatography according to Tilbeurgh et al [13] using a cellobiose-based (p-aminobenzyl-1-thio-β-Dcellobioside) affinity column. The enzymes from P. chrysosporium [1,4-β-D-glucan-cellobiohydrolase 58 (CBH 58) and 1,4-β-D-glucan-cellobiohydrolase 50 (CBH 50)] were purified according to Uzcategui et al [7].…”

Section: Methodsmentioning

confidence: 99%

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Nutt

Sild²,

Pettersson³

et al. 1998

European Journal of Biochemistry

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The chain-end preference and processivity of the cellulases 1,4-β-D-glucan-cellobiohydrolase I (CBH I) and 1,4-β-D-glucan-cellobiohydrolase II (CBH II) from Trichoderma reesei and 1,4-β-D-glucancellobiohydrolase 50 (CBH 50) and 1,4-β-D-glucan-cellobiohydrolase 58 (CBH 58) from Phanerochaete chrysosporium were studied by comparing experimental degradation data on reducing end-labelled bacterial microcrystalline cellulose with computer simulations of different models. Our results with T. reesei and P. chrysosporium cellulases show that there is a common pattern of hydrolysis for CBH-I-type enzymes and another clearly distinguishable pattern for CBH-II-type enzymes, especially in the initial part of the progress curve.Keywords : cellulase ; cellulose; hydrolysis ; simulation ; mode of action.In nature, the breakdown of cellulose is carried out mainly by fungi and bacteria. These organisms degrade crystalline cellulose by means of several enzymes acting at the ends (exocellulases) or randomly (endoglucanases) on the cellulose chains. Cellulases, like many other enzymes attacking insoluble or 'larger-than-enzyme' polymeric substrates, have a characteristic two-domain organisation : a catalytic domain connected by a flexible linker to a separate cellulose-binding domain [1]. Effective degradation of native cellulose requires cooperation between different functional types of cellulases and displays two distinctive modes of synergism; namely, cooperation among endoglucanases and cellobiohydrolases (CBHs) (endo/exo synergism), where the endoglucanases are supposed to create a large number of free ends susceptible to exoglucanase action [2Ϫ4], and cooperation among different CBHs [5Ϫ7]. The molecular basis for this latter synergism is not yet understood, mainly because the modes of action of the individual enzymes on crystalline cellulose are not known. By definition, CBHs attack the cellulose chain from the non-reducing end [8]. Recent data indicate, however, that the exhaustively studied Trichoderma reesei 1,4-β-D-glucan-cellobiohydrolase I (CBH I) preferentially attacks the reducing end of cellooligosaccharides, whereas the similarly well-characterised 1,4-β-D-glucan-cellobiohydrolase II (CBH II) from the same organism indeed prefers the non-reducing end [9,10].

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Separation of endo‐ and exo‐type cellulases using a new affinity chromatography method

Cited by 70 publications

References 9 publications

Detection and differentiation of cellulase components using low molecular mass fluorogenic substrates

Detection and differentiation of cellulase components using low molecular mass fluorogenic substrates

Studies of the cellulolytic system of the filamentous fungus Trichoderma reesei QM 9414. Substrate specificity and transfer activity of endoglucanase I

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