Bestimmung von Hexosen in Tryptophan‐haltigen Eiweisskörpern. Die Kohlenhydratkomponente des Eialbumins

The cellulose-binding properties of the highly conserved terminal region which is common to several fungai ceUulases were studied. Domains were prepared by proteolytic cleavage of Trichoderma reesei CBH 1 and the corresponding enzyme from Sporotrichum pulverulentum, and a peptide corresponding to residues 462-497 (the C-terminal part) of Trichoderma CBH 1 was synthesized. The three peptides showed similar binding behavior, whereas reduced and S-carboxymethylated T. reesei fragment was inactive. This region thus appears to serve as an independent functional domain in which the C-terminal part is responsible for the binding, which in turn requires an intact three-dimensional structure.

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“…Total sugar content was estimated using the anthrone/sulfuric acid method with mannose as standard [18].…”

Section: Methodsmentioning

confidence: 99%

Isolated fungal cellulose terminal domains and a synthetic minimum analogue bind to cellulose

Johansson¹,

Ståhlberg²,

Lindeberg³

et al. 1989

FEBS Letters

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“…The absorbance was measured at 585 nm, an isosbestic point where the absorption given by hexoses is independent of the tryptophan content of the sample [21].…”

Section: Chemical Analysesmentioning

confidence: 99%

“…The filamentous fungus Trichoderma reesei is one of the most potent cellulose degrading organisms known [l, 21. It excretes large amounts of different cellulases which, through synergistic action [3, 41, cooperate to solubilize crystalline cellulose efficiently.…”

mentioning

confidence: 99%

A binding‐site‐deficient, catalytically active, core protein of endoglucanase III from the culture filtrate of Trichoderma reesei

Ståhlberg¹,

Johansson²,

Pettersson³

1988

European Journal of Biochemistry

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From the culture filtrate of Trichoderma reesei we have isolated a novel endoglucanase (38 kDa) which was shown to be identical to endoglucanase I11 (E 111, 50 kDa), but lacking the first 61 N-terminal amino acids. This core protein, designated E 111 core, is fully active against soluble substrates, such as carboxymethylcellulose, whereas both activity against and adsorption to microcrystalline cellulose (Avicel) is markedly decreased. Sedimentation velocity experiments revealed that the intact E 111 enzyme has much higher assymmetry than the E 111 core protein, suggesting that the N-terminal region split off constitutes a protruding part of the native enzyme. These results lead to the proposal that native E 111 consists of two functionally separated domains: a catalytically active core and a protruding N-terminal domain which acts in the binding to insoluble cellulose. The N-terminal peptide missing in E I11 core corresponds to the heavily glycosylated common structural element found also in the N-terminus of cellobiohydrolase I1 and in the C-termini of cellobiohydrolase I and endoglucanase I. A similar bifunctional organization could thus be the rule for Trichoderma cellulases, endoglucanases as well as cellobiohydrolases.The filamentous fungus Trichoderma reesei is one of the most potent cellulose degrading organisms known [l, 21. It excretes large amounts of different cellulases which, through synergistic action [3, 41, cooperate to solubilize crystalline cellulose efficiently. Three different types of enzymes are involved in the process: (a) endo-1 ,CP-~-glucanases (EC 3.2.1.4), (b) exo-1,4-P-~-glucanases (EC 3.2.1.91) (also called cellobiohydralases) and (c) 1,4-P-~-ghcosidases (EC 3.2.1.21) [l]. Endoglucanases cut internal ~-1,4-~-glucosidic bonds in the cellulose chains. They are also able to degrade substituted cellulose such as carboxymethylcellulose, which is often used as substrate to measure endoglucanase activity. Cellobiohydrolases split off cellobiose units from the non-reducing ends of the cellulose chains. Finally, the cellobiose is hydrolyzed to glucose by the action of P-glucosidase.The exact number of enzymes excreted and their individual roles in the solubilization of cellulose is still far from clear. The picture is complicated due to the use of different growth conditions, which results in variable expression of secreted proteins, and to the existence of structural heterogeneity among several cellulases. As we shall show later on in this article, partial proteolysis of cellulases can produce fragments with full or partially impaired catalytic activity. The purification of several cellulases has been reported [5 -71. ApartCorrespondence to G. Pettersson, Institutionen for Naturvetenskaplig Biokemi, Biomedicinskt Centrum, Box 576, S-75123 Uppsala, SwedenAbbreviations. CM-cellulose, carboxymethylcellulose; CPB and CPY, carboxypeptidase B and Y; E I and E 111, endoglucanase I and 111; CBH I and CBH 11, cellobiohydrolase I and 11.Enzymes. Carboxypeptidase B (EC 3.4.17.2); carboxypeptidase Y (E...

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“…The specific radioactivity of the 14 C-labeled BMCC preparation was 4.5 ϫ 10 5 dpm mg Ϫ1 . Total BMCC concentration was determined using the anthrone sulfuric acid method (51). The DP of BMCC was determined from the reducing end groups on BMCC, as measured using the modified bicinchoninic acid method (50,52).…”

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confidence: 99%

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes

Kuusk

Sørlie

Väljamaë

2015

Journal of Biological Chemistry

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Background: Rate-limiting steps in the hydrolysis of recalcitrant polysaccharides by processive enzymes are not known. Results:The predominant molecular state of bound enzyme was revealed by the type and strength of product inhibition. Conclusion: Complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation. Significance: Knowledge of rate-limiting steps aids the engineering of better catalysts.

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Bestimmung von Hexosen in Tryptophan‐haltigen Eiweisskörpern. Die Kohlenhydratkomponente des Eialbumins

Cited by 32 publications

References 42 publications

Isolated fungal cellulose terminal domains and a synthetic minimum analogue bind to cellulose

Isolated fungal cellulose terminal domains and a synthetic minimum analogue bind to cellulose

A binding‐site‐deficient, catalytically active, core protein of endoglucanase III from the culture filtrate of Trichoderma reesei

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes

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