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Giorgio Canevascini - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of two exo Cellobiohydrolases from the brown rot fungus coniophora puteana schum ex fr karst
Archives of Biochemistry and Biophysics, 1993Co-Authors: Diego R. Schmidhalter, Giorgio CanevasciniAbstract:Abstract Two extracellular exo-Cellobiohydrolases (EC 3.2.1.9 1) were purified to homogeneity from the culture filtrate of the brown-rot fungus Coniophora puteana (Schum ex Fr) Karsten, strain EMPA 62. The purification scheme involved three successive chromatographic steps, namely Q Sepharose fast flow, Superose 12, and Fractogel TSK DEAE-650S. The two enzymes, named cellobiohydrolase (CBH) I and CBH II, were purified by a factor of 4.6 and 3.9, respectively, with an activity recovery of 9 and 19% of total, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both enzymes migrated as single bands according to a Mr of 52,000 for CBH I and 50,000 for CBH II; by FPLC gel filtration (TSK G3000 SW) the Mr′s were higher (65,000 and 60,000). Both enzymes were glycosylated, had similar isoelectric points (pI 3.6 and 3.55) and nearly identical pH optima for activity close to 5. Endoglycosidase H digestion gave two distinct polypeptides where the molecular weight was lowered by 6.5 kDa for CBH I and by 2.5 kDa for CRH II. The specific activities for the hydrolysis of p-nitrophenyllactoside (pNPL) were nearly identical for both enzymes (0.46 versus 0.40 μmol mgμ1 minμ1 at 40°C) and the Km values (6.8 and 4.3 mM at 30°C) were also very close. Both enzymes were competitively inhibited by cellobiose: with pNPL as substrate, Km values of 1.2 mM for CBH I and 2.4 mM for CBH II were determined. The two enzymes acted in an identical fashion on cellulose (either amorphous or crystalline) and on cellodextrins, liberating mainly cellobiose, but were inactive on dyed carboxymethylcellulose. Cellobiose was not hydrolyzed whereas cellotriitol was hydrolyzed to equimolar amounts of cellobiose and glucitol: these results support the interpretation that these enzymes are exo-Cellobiohydrolases. Their presence in a brown-rot fungus is a new fact.
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Characterization of the cellulolytic enzyme system from the brown-rot fungus Coniophora puteana
Applied Microbiology and Biotechnology, 1992Co-Authors: Diego R. Schmidhalter, Giorgio CanevasciniAbstract:The cellulolytic enzymes of various strains of the brown-rot fungus Coniophora puteana were studied. The organism was grown in an air-lift fermentor in mineral medium containing glucose, cellobiose or amorphous cellulose. The specific growth rate varied between 0.082 and 0.062 h^−1. On amorphous cellulose as sole carbon source, the organism secreted various proteins, some of which were characterized. The mixture contained inter alia four endocellulases, two exo-Cellobiohydrolases and a cellobiose dehydrogenase. Three endocellulases (named type I) were active on soluble cellulose derivatives but inactive on p -nitrophenyllactoside ( p -NPL), whereas a fourth endocellulase (named type II) was active on both. The two exo-Cellobiohydrolases released cellobiose from amorphous cellulose; they were inactive on soluble cellulose derivatives but hydrolyzed p -NPL with strong cellobiose inhibition. A cellobiose dehydrogenase having spectral characteristics compatible with a flavo b-cytochrome was also identified. Neither the exo-cellobiohydrolase nor the type II endocellulase were secreted during growth on cellobiose whereas type I endocellulases and cellobiose dehydrogenase were formed at a reduced rate. No formation of cellulolytic enzymes was observed during growth on glucose alone.
Maria G Tuohy - One of the best experts on this subject based on the ideXlab platform.
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three dimensional structure of a thermostable native cellobiohydrolase cbh ib and molecular characterization of the cel7 gene from the filamentous fungus talaromyces emersonii
FEBS Journal, 2004Co-Authors: Alice Grassick, Patrick Murray, Roisin Thompson, Catherine Majella Collins, Lucy Byrnes, Gabriel Birrane, Tim Higgins, Maria G TuohyAbstract:The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungus Talaromyces emersonii, PDB 1Q9H, was solved to 2.4 A by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of ≈ 60 A × 40 A × 50 A and an overall β-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A from Trichoderma reesei is conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties of T. emersonii 1Q9H are different. The gene (cel7) encoding CBH IB was isolated from T. emersonii and expressed heterologously with an N-terminal polyHis-tag, in Escherichia coli. The deduced amino acid sequence of cel7 is homologous to fungal Cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles of cel7 expression in T. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of the cel7 genomic sequence.
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molecular cloning transcriptional and expression analysis of the first cellulase gene cbh2 encoding cellobiohydrolase ii from the moderately thermophilic fungus talaromyces emersonii and structure prediction of the gene product
Biochemical and Biophysical Research Communications, 2003Co-Authors: Patrick Murray, Alice Grassick, Catherine Majella Collins, Maria G TuohyAbstract:Abstract A gene (cbh2) encoding cellobiohydrolase II was isolated from the fungus Talaromyces emersonii by rapid amplification of cDNA ends techniques and the equivalent genomic sequence was subsequently cloned. This represents the first report of a key component of the cellulase regulon from this organism. DNA sequencing revealed that cbh2 has an open reading frame of 1377 bp, which encodes a putative polypeptide of 459 amino acids, and is interrupted by seven introns. The deduced amino acid sequence revealed that cbh2 has a modular structure with a predicted molecular mass of 47 kDa and consisting of a fungal type carbohydrate binding module separated from a catalytic domain by a proline/serine/threonine rich linker region. The deduced protein is homologous to fungal Cellobiohydrolases in Family 6A of the glycosyl hydrolases. Profiles of cbh2 expression in T. emersonii investigated by Northern blot analysis revealed that expression is regulated at the transcriptional level. Expression of the T. emersonii cbh2 gene is induced by cellulose, xylan, xylose, and gentiobiose and clearly repressed by glucose. Putative regulatory element consensus sequences have been identified in the upstream regulatory sequence of the cbh2 gene including the catabolite repressor element and the activator of cellulase expression (Ace) binding sites. High sequence identity (67%) between the catalytic domain of Cel 6A from Trichoderma reesei and the T. emersonii cbh2 gene product allowed structure prediction for the 3D model of the T. emersonii catalytic domain to be a variant of the classical TIM α/β fold.
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kinetic parameters and mode of action of the Cellobiohydrolases produced by talaromyces emersonii
Biochimica et Biophysica Acta, 2002Co-Authors: Maria G Tuohy, Patrick Murray, Marc Claeyssens, Daniel J Walsh, Michelle M Cuffe, Angela V Savage, Michael P CoughlanAbstract:Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal Cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-β-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (Ki) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-β-cellobioside as substrate), while a Ki of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-β-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of β-cellobioside and β-cellotrioside (MeUmbGn). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.
Diego R. Schmidhalter - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of two exo Cellobiohydrolases from the brown rot fungus coniophora puteana schum ex fr karst
Archives of Biochemistry and Biophysics, 1993Co-Authors: Diego R. Schmidhalter, Giorgio CanevasciniAbstract:Abstract Two extracellular exo-Cellobiohydrolases (EC 3.2.1.9 1) were purified to homogeneity from the culture filtrate of the brown-rot fungus Coniophora puteana (Schum ex Fr) Karsten, strain EMPA 62. The purification scheme involved three successive chromatographic steps, namely Q Sepharose fast flow, Superose 12, and Fractogel TSK DEAE-650S. The two enzymes, named cellobiohydrolase (CBH) I and CBH II, were purified by a factor of 4.6 and 3.9, respectively, with an activity recovery of 9 and 19% of total, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both enzymes migrated as single bands according to a Mr of 52,000 for CBH I and 50,000 for CBH II; by FPLC gel filtration (TSK G3000 SW) the Mr′s were higher (65,000 and 60,000). Both enzymes were glycosylated, had similar isoelectric points (pI 3.6 and 3.55) and nearly identical pH optima for activity close to 5. Endoglycosidase H digestion gave two distinct polypeptides where the molecular weight was lowered by 6.5 kDa for CBH I and by 2.5 kDa for CRH II. The specific activities for the hydrolysis of p-nitrophenyllactoside (pNPL) were nearly identical for both enzymes (0.46 versus 0.40 μmol mgμ1 minμ1 at 40°C) and the Km values (6.8 and 4.3 mM at 30°C) were also very close. Both enzymes were competitively inhibited by cellobiose: with pNPL as substrate, Km values of 1.2 mM for CBH I and 2.4 mM for CBH II were determined. The two enzymes acted in an identical fashion on cellulose (either amorphous or crystalline) and on cellodextrins, liberating mainly cellobiose, but were inactive on dyed carboxymethylcellulose. Cellobiose was not hydrolyzed whereas cellotriitol was hydrolyzed to equimolar amounts of cellobiose and glucitol: these results support the interpretation that these enzymes are exo-Cellobiohydrolases. Their presence in a brown-rot fungus is a new fact.
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Characterization of the cellulolytic enzyme system from the brown-rot fungus Coniophora puteana
Applied Microbiology and Biotechnology, 1992Co-Authors: Diego R. Schmidhalter, Giorgio CanevasciniAbstract:The cellulolytic enzymes of various strains of the brown-rot fungus Coniophora puteana were studied. The organism was grown in an air-lift fermentor in mineral medium containing glucose, cellobiose or amorphous cellulose. The specific growth rate varied between 0.082 and 0.062 h^−1. On amorphous cellulose as sole carbon source, the organism secreted various proteins, some of which were characterized. The mixture contained inter alia four endocellulases, two exo-Cellobiohydrolases and a cellobiose dehydrogenase. Three endocellulases (named type I) were active on soluble cellulose derivatives but inactive on p -nitrophenyllactoside ( p -NPL), whereas a fourth endocellulase (named type II) was active on both. The two exo-Cellobiohydrolases released cellobiose from amorphous cellulose; they were inactive on soluble cellulose derivatives but hydrolyzed p -NPL with strong cellobiose inhibition. A cellobiose dehydrogenase having spectral characteristics compatible with a flavo b-cytochrome was also identified. Neither the exo-cellobiohydrolase nor the type II endocellulase were secreted during growth on cellobiose whereas type I endocellulases and cellobiose dehydrogenase were formed at a reduced rate. No formation of cellulolytic enzymes was observed during growth on glucose alone.
Johanna Buchert - One of the best experts on this subject based on the ideXlab platform.
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modification of hardwood dissolving pulp with purifiedtrichoderma reesei cellulases
Cellulose, 1996Co-Authors: Leena Rahkamo, Marianna Vehvilainen, Matti Dolk, Pertti Nousiainen, Matti Siikaaho, Liisa Viikari, Johanna BuchertAbstract:Hardwood dissolving pulp was treated with purifiedTrichoderma reeseiendoglucanases and Cellobiohydrolases. Endoglucanases were more efficient in hydrolysing pulp carbohydrates than were the Cellobiohydrolases at the same protein dosage. Endoglucanases also lowered the viscosity and improved the alkaline solubility more dramatically. There was a clear correlation between the alkaline solubility and viscosity, and therefore the solubility could only be improved by lowering the viscosity of the pulp. At the same degree of cellulose degradation, endoglucanase II was found to be most effective in reducing the viscosity and thus improving the solubility. Cellobiohydrolases had a less pronounced effect on the viscosity or solubility.
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modification of hardwood dissolving pulp with purifiedtrichoderma reesei cellulases
Cellulose, 1996Co-Authors: Leena Rahkamo, Marianna Vehvilainen, Matti Dolk, Pertti Nousiainen, Matti Siikaaho, Liisa Viikari, Johanna BuchertAbstract:Hardwood dissolving pulp was treated with purifiedTrichoderma reeseiendoglucanases and Cellobiohydrolases. Endoglucanases were more efficient in hydrolysing pulp carbohydrates than were the Cellobiohydrolases at the same protein dosage. Endoglucanases also lowered the viscosity and improved the alkaline solubility more dramatically. There was a clear correlation between the alkaline solubility and viscosity, and therefore the solubility could only be improved by lowering the viscosity of the pulp. At the same degree of cellulose degradation, endoglucanase II was found to be most effective in reducing the viscosity and thus improving the solubility. Cellobiohydrolases had a less pronounced effect on the viscosity or solubility.
Gideon J. Davies - One of the best experts on this subject based on the ideXlab platform.
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structural basis for ligand binding and processivity in cellobiohydrolase cel6a from humicola insolens
Structure, 2003Co-Authors: Annabelle Varrot, Martin Schülein, Torben Peter Frandsen, Ingemar Von Ossowski, Viviane Boyer, Sylvain Cottaz, Hugues Driguez, Gideon J. DaviesAbstract:Abstract The enzymatic digestion of cellulose entails intimate involvement of Cellobiohydrolases, whose characteristic active-center tunnel contributes to a processive degradation of the polysaccharide. The cellobiohydrolase Cel6A displays an active site within a tunnel formed by two extended loops, which are known to open and close in response to ligand binding. Here we present five structures of wild-type and mutant forms of Cel6A from Humicola insolens in complex with nonhydrolyzable thio-oligosaccharides, at resolutions from 1.7–1.1 A, dissecting the structural accommodation of a processing substrate chain through the active center during hydrolysis. Movement of ligand is facilitated by extensive solvent-mediated interactions and through flexibility in the hydrophobic surfaces provided by a sheath of tryptophan residues.
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structure and function of humicola insolens family 6 cellulases structure of the endoglucanase cel6b at 1 6 a resolution
Biochemical Journal, 2000Co-Authors: Gideon J. Davies, Annabelle Varrot, Andrzej M Brzozowski, Miroslawa Dauter, Martin SchüleinAbstract:Cellulases are traditionally classified as either endoglucanases or Cellobiohydrolases on the basis of their respective catalytic activities on crystalline cellulose, which is generally hydrolysed more efficiently only by the Cellobiohydrolases. On the basis of the Trichoderma reesei cellobiohydrolase II structure, it was proposed that the active-site tunnel of Cellobiohydrolases permitted the processive hydrolysis of cellulose, whereas the corresponding endoglucanases would display open active-site clefts [Rouvinen, Bergfors, Teeri, Knowles and Jones (1990) Science 249, 380-386]. Glycoside hydrolase family 6 contains both Cellobiohydrolases and endoglucanases. The structure of the catalytic core of the family 6 endoglucanase Cel6B from Humicola insolens has been solved by molecular replacement with the known T. reesei cellobiohydrolase II as the search model. Strangely, at the sequence level, this enzyme exhibits the highest sequence similarity to family 6 Cellobiohydrolases and displays just one of the loop deletions traditionally associated with endoglucanases in this family. However, this enzyme shows no activity on crystalline substrates but a high activity on soluble substrates, which is typical of an endoglucanase. The three-dimensional structure reveals that the deletion of just a single loop of the active site, coupled with the resultant conformational change in a second 'cellobiohydrolase-specific' loop, peels open the active-site tunnel to reveal a substrate-binding groove.
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Structural changes of the active site tunnel of Humicola insolens cellobiohydrolase, Cel6A, upon oligosaccharide binding.
Biochemistry, 1999Co-Authors: Annabelle Varrot, Martin Schülein, Gideon J. DaviesAbstract:The mechanisms of crystalline cellulose degradation by cellulases are of paramount importance for the exploitation of these enzymes in applied processes, such as biomass conversion. Cellulases have traditionally been classified into Cellobiohydrolases, which are effective in the degradation of crystalline materials, and endoglucanases, which appear to act on “soluble” regions of the substrate. Humicola insolens Cel6A (CBH II) is a cellobiohydrolase from glycoside hydrolase family 6 whose native structure has been determined at 1.9 A resolution [Varrot, A., Hastrup, S., Schulein, M., and Davies, G. J. (1999) Biochem. J. 337, 297−304]. Here we present the structure of the catalytic core domain of Humicola insolens cellobiohydrolase II Cel6A in complex with glucose/cellotetraose at 1.7 A resolution. Crystals of Cel6A, grown in the presence of cellobiose, reveal six binding subsites, with a single glucose moiety bound in the −2 subsite and cellotetraose in the +1 to +4 subsites. The complex structure is stron...
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crystal structure of the catalytic core domain of the family 6 cellobiohydrolase ii cel6a from humicola insolens at 1 92 a resolution
Biochemical Journal, 1999Co-Authors: Annabelle Varrot, Martin Schülein, Sven Hastrup, Gideon J. DaviesAbstract:The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 A. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reesei equivalent. The structure features both O- (alpha-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of Cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme.