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

Tilbeurgh, Herman van; Claeyssens, Marc

doi:10.1016/0014-5793(85)81260-6

Cited by 96 publications

(49 citation statements)

References 13 publications

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“…From our previous work on the bifunctional organisation of CBH I [8], it becomes clear that the modification with WRK is directed against the catalytic domain present in the core fragment and not against the cellulose-binding region (BA glycopeptide domain) which is seemingly left intact. An enzyme sample with 40070 residual activity is bound in approximately the same amount onto an affinity column carrying p-aminobenzyl 1-thio cellobioside as ligand and the retained fraction is completely desorbed by lactose [16]. This proves the homogeneity of the modified enzyme preparations and again the active site-directed character of the modification.…”

Section: Properties Of the Modified Enzymementioning

confidence: 68%

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Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Tomme

Claeyssens

1989

FEBS Letters

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Several aspects of the specific modification of cellobiohydrolase I (CBH I) from Trichoderma reesei with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-Y-sulfonate) are presented. The pH dependence of the resulting inactivation points to the implication of an ionising group with a pKa of approx. 5.5. The rapid inactivation kinetics, the specific protection and the stoichiometry of modification (3 versus 2 residues), together with the isolation and amino acid sequencing of the putative active site peptide, provide a large body of evidence for the presence of a catalytically important carboxyl residue in the 125-135 region of the CBH I amino acid sequence. From the striking homology between this peptide sequence and those of the active site regions of different lysozymes, glutamic acid 126 is retained as the most plausible catalytic residue (proton donor) in CBH I, equivalent to glutamic acid 35 in hen egg white lysozyme. Glutamic acid 127 is proposed as a potential active site residue to the homologous endoglucanase I (EG I) isolated from the same Trichoderma species.

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Section: Properties Of the Modified Enzymementioning

confidence: 68%

“…Knowles, VTT, Espoo, Finland) by affinity chromatography [16]. Core I protein was obtained by papaln digestion and purified as previously described [8].…”

Section: Enzymes and Enzymic Assaysmentioning

confidence: 99%

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Tomme

Claeyssens

1989

FEBS Letters

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“…One unit of CDH was defined as the amount of the enzyme that reduced 1 mmol of DCPIP min À1 ml À1 . Degradation of 4-methylumbelliferyl--cellobioside (4-MUC) 28) was determined with glucono-1,5-lactone 29) at pH 5.0. -Glucosidase activity was determined with 4-methylumbelliferyl--glucoside (4-MUG) at pH 5.0.…”

Section: Methodsmentioning

confidence: 99%

“…The amounts of 4-methylumbelliferon (4-MU) were measured by fluorescence. 28) One unit of 4-MUC degrading activity and -Glucosidase was defined as the amount of the enzyme that released 1 mmol of 4-MU min À1 ml À1 from 4-MUC and 4-MUG respectively. CMCase activity was determined by release of reducing sugars 30) from carboxymethylcellulose (CMC, 1.0% [w/v]) at pH 5.0 for 30 min at 40 C. Avicelase activity was determined by release of reducing sugars 30) from Avicel Ò SF (Asahi Kasei, Osaka, Japan, 1.0% [w/v]) at pH 5.0 for 2 h at 40 C. One unit of Avicelase and CMCase was defined as the amount of the enzyme which released 1 mmol of reducing sugars min À1 ml À1 from Avicel Ò SF and CMC respectively.…”

Section: Methodsmentioning

confidence: 99%

Purification and Characterization of Cellobiose Dehydrogenase from White-Rot BasidiomyceteTrametes hirsuta

Nakagame¹,

Furujyo²,

Sugiura³

2006

Bioscience, Biotechnology, and Biochemistry

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In order to save energy during the pulp making process, we tried to use white-rot basidiomycete, Trametes hirsuta, which degrades lignin efficiently. But a decrease in paper strength caused by cellulolytic activity ruled this out for practical application. Since the cellulolytic activity of the fungus must be decreased, we purified and characterized a cellobiose dehydrogenase (CDH) that was reported to damage pulp fiber. The CDH in the culture filtrate of C. hirsutus was purified by freeze-thawing and chromatographic methods. The pI of the enzyme was 4.2 and its molecular weight was 92 kDa. The optimal temperature and pH of the enzyme were 60-70 C and 5.0 respectively. Since the purified CDH decreased the viscosity of pulp in the presence of Fe(III) and cellobiose, it was shown that the suppression of CDH should be an effective way to reduce cellulose damage.

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“…Mixtures of isoenzymes were obtained and were further fractionated by ion-exchange chromatography [7]. The homogeneity was tested both by SDS-PAGE and IEF-PAG [19]. The PI 3.9 (CBH I) and PI 5.9 component (CBH 11) were used in most of the experiments, but the results with isoenzyme mixtures were essentially identical.…”

Section: Enzyme Purificationmentioning

confidence: 99%

Studies of the cellulolytic system of Trichoderma reesei QM 9414

Tomme¹,

Tilbeurgh²,

Pettersson³

et al. 1988

European Journal of Biochemistry

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537

279

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Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II,58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel).By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH I1 core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from ~r~c~o d e r m a reesei [Teeri et al. (1987) Gene 51,43 -521. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH TI in terms of adsorption and catalytic properties, Distinct functions can be attributed to the terminal peptides: for intact CBH I1 the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH I1 synergism experiments.The filamentous fungus Trichoderma reesei presents an ideal system for the study of cellulose hydrolysis since it produces a variety of enzymes including endocellulase and exocellulase (cellobiohydrolases) and b-glucosidases (cellobiases) [l]. The primary structure of several of these proteins is presently known [2 -61 but no structure/function relationships have yet been established. The two cellobiohydrolases, CBH I and CBH I1 [7 -111, lack any apparent homology in their amino acid sequence except for a conserved region present at their C and N termini respectively [4].Their action is similar both with regard to their strong adsorption onto insoluble substrates and lack of carboxymethylcellulase activity. However, studies both with microcrystalline cellulose [9] and small, soluble substrates [I 21 reveal some differences in specificity. In addition, the two cellobiohydrolases interact synergistically both with each other and with endoglucanases on microcrystalline substrates In order to differentiate these enzymes further by their function and to investigate possible domain structure, proteolysis studies were performed. Preliminary results on the limited action of papain on native CBH I have been reported by us [16]. Two fragments were isolated: first, a heavily glycosylated peptide, tentatively identified with the C terminus; secondly, a core protein containing the active (hydrolytic) site. The terminal peptide was characterised as an independent structural and functional dom...

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

Cited by 96 publications

References 13 publications

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Purification and Characterization of Cellobiose Dehydrogenase from White-Rot BasidiomyceteTrametes hirsuta

Studies of the cellulolytic system of Trichoderma reesei QM 9414

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