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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.
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.
Coniglio, Romina Olga - One of the best experts on this subject based on the ideXlab platform.
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Aspectos bioquímicos y moleculares de celobiohidolasas fúngicas con potencial aplicación en el proceso de sacarificación
2020Co-Authors: Coniglio, Romina OlgaAbstract:Las prácticas agrícolas generan desperdicios lignocelulósicos compuestos por celulosa, hemicelulosas y lignina que pueden ser bioconvertidos por hongos para generar productos de valor agregado como los biocombustibles. En este sentido, las enzimas celulolíticas como las endoglucanasas, celobiohidrolasas y β-glucosidasas se presentan como una alternativa para su utilización en la hidrólisis de celulosa a azúcares que pueden ser fermentados a etanol.El objetivo general de esta tesis fue obtener un medio enriquecido en celobiohidrolasas a partir del cultivo de hongos políporos nativos de Misiones (Argentina) con características propicias para ser utilizados en el proceso de sacarificación, un paso clave en la producción de bioetanol.En un screening inicial, se evaluó el potencial celulolítico de 14 políporos mediante ensayos cualitativos seleccionándose 11 aislamientos que mostraron actividad endoglucanasa, celobiohidrolasa y β-glucosidasa.Luego, se realizó un screening cuantitativo en cultivos sumergidos y se seleccionó el aislamiento LBM 033 por presentar los mayores títulos de actividad enzimática para las tres celulasas. Además, los sobrenadantes de cultivo de cada aislamiento mostraron diferentes perfiles isoenzimáticos, correspondiendo la mayoría de las bandas a 45 kDa para las celobiohidrolasas y a un rango entre 69 y 88 kDa para las endoglucanasas.El aislamiento LBM 033 fue identificado como Trametes villosa mediante el uso de los marcadores ITS y rbp2 y la construcción de árboles.La celulosa cristalina, la peptona y el sulfato de amonio afectaron (P≤0,01) a la actividad celobiohidrolasa, obteniéndose la mayor actividad enzimática luego de 12 días de cultivo. Las condiciones del medio se estandarizaron, obteniéndose el valor óptimo de 49±5 U/l en las siguientes condiciones: 15 g/l de celulosa cristalina y 3 g/l de peptona y sulfato de amonio.El sobrenadante de cultivo estandarizado de T. villosa LBM 033 presentó el mayor valor de actividad celobiohidrolasa a 60°C y pH 4, 8. La termoestabilidad fue mayor a 30ºC seguida de 50ºC, disminuyendo a temperaturas más elevadas, mientras que la actividad enzimática fue más estable a los pHs 4, 8 y 10.El sobrenadante presentó además títulos elevados de otras actividades enzimáticas necesarias para la hidrólisis de los sustratos holocelulósicos, hidrolizando los sustratos artificiales celulósicos hasta celobiosa y glucosa. En la sacarificación del residuo de cebada pretratatado por extrusión, el porcentaje de sacarificación de la holocelulosa fue del 30 %, representado probablemente por oligosacáridos reductores.En el análisis del secretoma se logró identificar una CBHII. Además, el 66 % de las proteínas estuvieron relacionadas con la bioconversión de la biomasa lignocelulósica.Estos resultados indican que los macrohongos de Misiones, como T. villosa LBM 033 pueden constituir una valiosa fuente de celulasas, especialmente celobiohidrolasas que pueden aplicarse en la etapa de sacarificación lo que podría reducir el costo total de la producción de bioetanol.The agricultural practices generate lignocellulosic waste composed of cellulose, hemicelluloses and lignin that can be bioconverted by fungi to generate value-added products such as biofuels. In this context, cellulolytic enzymes such as endoglucanases, Cellobiohydrolases and β-glucosidases are presented as an alternative for their use in the hydrolysis of cellulose to sugars that can be fermented to ethanol. The general objective of this thesis was to obtain a medium enriched in Cellobiohydrolases from the cultivation of native polypore fungi from Misiones (Argentina) with favorable characteristics to be used in the saccharification process, a key step in the production of bioethanol. In an initial screening, the cellulolytic potential of 14 polypores was evaluated by qualitative assays, selecting 11 isolates that showed endoglucanase, Cellobiohydrolase and β-glucosidase activity. Then, a quantitative screening was performed in submerged cultures and the LBM033 isolate was selected because it had the highest enzymatic activity titers for the three cellulases. In addition, the culture supernatants of each isolate showed different isoenzyme profiles, most of the bands corresponding to 45 kDa for the Cellobiohydrolases and to a range between 69 and 88 kDa for the endoglucanases. The LBM 033 isolate was identified as Trametes villosa through the use of the ITS and rbp2 markers and the construction of trees. Crystalline cellulose, peptone and ammonium sulfate affected (P≤0.01) Cellobiohydrolase activity, obtaining the highest enzymatic activity after 12 days of culture. The conditions of the medium were standardized, obtaining the optimum value of 49 ± 5 U / l under the following conditions: 15 g/l of crystalline cellulose and 3 g/l of peptone and ammonium sulfate. The standardized culture supernatant of T. villosa LBM 033 showed the highest value of Cellobiohydrolase activity at 60° C and pH 4.8. The thermostability was higher at 30ºC followed by 50ºC, decreasing at higher temperatures, while the enzymatic activity was more stable at pHs 4, 8 and 10. The supernatant also showed high titers of other enzymatic activities necessary for the hydrolysis of the holocellulosic substrates, hydrolyzing the artificial cellulosic substrates up to cellobiose and glucose. In the saccharification of the pre-treated barley residue, the saccharification percentage of the holocellulose was 30 %, probably represented by reducing oligosaccharides. In the analysis of the secretome, a CBHII was identified. In addition, 66 % of the proteins were related to the bioconversion of the lignocellulosic biomass. These results indicate that macrofungi from Misiones, such as T. villosa LBM 033 can be a valuable source of cellulases, especially Cellobiohydrolases that can be applied in the saccharification stage, which could reduce the total cost of bioethanol production
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Aspectos bioquímicos y moleculares de celobiohidolasas fúngicas con potencial aplicación en el proceso de sacarificación
2020Co-Authors: Coniglio, Romina OlgaAbstract:Las prácticas agrícolas generan desperdicios lignocelulósicos compuestos por celulosa, hemicelulosas y lignina que pueden ser bioconvertidos por hongos para generar productos de valor agregado como los biocombustibles. En este sentido, las enzimas celulolíticas como las endoglucanasas, celobiohidrolasas y β-glucosidasas se presentan como una alternativa para su utilización en la hidrólisis de celulosa a azúcares que pueden ser fermentados a etanol.El objetivo general de esta tesis fue obtener un medio enriquecido en celobiohidrolasas a partir del cultivo de hongos políporos nativos de Misiones (Argentina) con características propicias para ser utilizados en el proceso de sacarificación, un paso clave en la producción de bioetanol.En un screening inicial, se evaluó el potencial celulolítico de 14 políporos mediante ensayos cualitativos seleccionándose 11 aislamientos que mostraron actividad endoglucanasa, celobiohidrolasa y β-glucosidasa.Luego, se realizó un screening cuantitativo en cultivos sumergidos y se seleccionó el aislamiento LBM 033 por presentar los mayores títulos de actividad enzimática para las tres celulasas. Además, los sobrenadantes de cultivo de cada aislamiento mostraron diferentes perfiles isoenzimáticos, correspondiendo la mayoría de las bandas a 45 kDa para las celobiohidrolasas y a un rango entre 69 y 88 kDa para las endoglucanasas.El aislamiento LBM 033 fue identificado como Trametes villosa mediante el uso de los marcadores ITS y rbp2 y la construcción de árboles.La celulosa cristalina, la peptona y el sulfato de amonio afectaron (P≤0,01) a la actividad celobiohidrolasa, obteniéndose la mayor actividad enzimática luego de 12 días de cultivo. Las condiciones del medio se estandarizaron, obteniéndose el valor óptimo de 49±5 U/l en las siguientes condiciones: 15 g/l de celulosa cristalina y 3 g/l de peptona y sulfato de amonio.El sobrenadante de cultivo estandarizado de T. villosa LBM 033 presentó el mayor valor de actividad celobiohidrolasa a 60°C y pH 4, 8. La termoestabilidad fue mayor a 30ºC seguida de 50ºC, disminuyendo a temperaturas más elevadas, mientras que la actividad enzimática fue más estable a los pHs 4, 8 y 10.El sobrenadante presentó además títulos elevados de otras actividades enzimáticas necesarias para la hidrólisis de los sustratos holocelulósicos, hidrolizando los sustratos artificiales celulósicos hasta celobiosa y glucosa. En la sacarificación del residuo de cebada pretratatado por extrusión, el porcentaje de sacarificación de la holocelulosa fue del 30 %, representado probablemente por oligosacáridos reductores.En el análisis del secretoma se logró identificar una CBHII. Además, el 66 % de las proteínas estuvieron relacionadas con la bioconversión de la biomasa lignocelulósica.Estos resultados indican que los macrohongos de Misiones, como T. villosa LBM 033 pueden constituir una valiosa fuente de celulasas, especialmente celobiohidrolasas que pueden aplicarse en la etapa de sacarificación lo que podría reducir el costo total de la producción de bioetanol.The agricultural practices generate lignocellulosic waste composed of cellulose, hemicelluloses and lignin that can be bioconverted by fungi to generate value-added products such as biofuels. In this context, cellulolytic enzymes such as endoglucanases, Cellobiohydrolases and β-glucosidases are presented as an alternative for their use in the hydrolysis of cellulose to sugars that can be fermented to ethanol. The general objective of this thesis was to obtain a medium enriched in Cellobiohydrolases from the cultivation of native polypore fungi from Misiones (Argentina) with favorable characteristics to be used in the saccharification process, a key step in the production of bioethanol. In an initial screening, the cellulolytic potential of 14 polypores was evaluated by qualitative assays, selecting 11 isolates that showed endoglucanase, Cellobiohydrolase and β-glucosidase activity. Then, a quantitative screening was performed in submerged cultures and the LBM033 isolate was selected because it had the highest enzymatic activity titers for the three cellulases. In addition, the culture supernatants of each isolate showed different isoenzyme profiles, most of the bands corresponding to 45 kDa for the Cellobiohydrolases and to a range between 69 and 88 kDa for the endoglucanases. The LBM 033 isolate was identified as Trametes villosa through the use of the ITS and rbp2 markers and the construction of trees. Crystalline cellulose, peptone and ammonium sulfate affected (P≤0.01) Cellobiohydrolase activity, obtaining the highest enzymatic activity after 12 days of culture. The conditions of the medium were standardized, obtaining the optimum value of 49 ± 5 U / l under the following conditions: 15 g/l of crystalline cellulose and 3 g/l of peptone and ammonium sulfate. The standardized culture supernatant of T. villosa LBM 033 showed the highest value of Cellobiohydrolase activity at 60° C and pH 4.8. The thermostability was higher at 30ºC followed by 50ºC, decreasing at higher temperatures, while the enzymatic activity was more stable at pHs 4, 8 and 10. The supernatant also showed high titers of other enzymatic activities necessary for the hydrolysis of the holocellulosic substrates, hydrolyzing the artificial cellulosic substrates up to cellobiose and glucose. In the saccharification of the pre-treated barley residue, the saccharification percentage of the holocellulose was 30 %, probably represented by reducing oligosaccharides. In the analysis of the secretome, a CBHII was identified. In addition, 66 % of the proteins were related to the bioconversion of the lignocellulosic biomass. These results indicate that macrofungi from Misiones, such as T. villosa LBM 033 can be a valuable source of cellulases, especially Cellobiohydrolases that can be applied in the saccharification stage, which could reduce the total cost of bioethanol production.Fil: Coniglio, Romina Olga. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Autor
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Optimization of Cellobiohydrolase production and secretome analysis of Trametes villosa LBM 033 suitable for lignocellulosic bioconversion
'Informa UK Limited', 2019Co-Authors: Coniglio, Romina Olga, Ontañon, Ornella Mailén, Ghio Silvina, Campos Eleonora, Burgos Fonseca, María Isabel, Díaz, Gabriela Verónica, Zapata, Pedro DaríoAbstract:The production of bioethanol from lignocellulosic biomass comprises the enzymatic hydrolysis of lignocellulosic structures by three major cellulases. Among them, Cellobiohydrolases are considered to be key enzymes playing a significant role on cellulose degradation. The ability to produce lignocellulolytic enzymes by fungi such as Trametes villosa makes them appropriate degraders for large-scale applications. In this context, the aim of this study was to obtain and characterize a Cellobiohydrolase-enriched extracellular extract of T. villosa LBM 033 (Misiones, Argentina), which is suitable for the enzymatic hydrolysis of lignocellulosic residues. The effect of carbon and nitrogen sources on Cellobiohydrolase activity was evaluated using experimental designs and a culture medium was optimized to obtain a Cellobiohydrolase-enriched extract suitable for the hydrolysis of lignocellulosic biomass. Moreover, by secretome analysis, nine enzymes involved in lignocellulosic biomass degradation were identified under the optimized conditions; among them is a Cellobiohydrolase II from the glycosil-hydrolase 6 family.Fil: Coniglio, Romina Olga. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Burgos Fonseca, María Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; ArgentinaFil: Díaz, Gabriela Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; ArgentinaFil: Ontañon, Ornella Mailén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Ghio, Silvina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zapata, Pedro Dario. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentin
Michael E. Himmel - One of the best experts on this subject based on the ideXlab platform.
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a constitutive expression system for glycosyl hydrolase family 7 Cellobiohydrolases in hypocrea jecorina
Biotechnology for Biofuels, 2015Co-Authors: Jeffrey G Linger, Michael E. Himmel, Larry E Taylor, John O Baker, Todd Vander Wall, Sarah E Hobdey, Kara Podkaminer, Stephen R DeckerAbstract:One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is Cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous Cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence. The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity. The elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse Cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.
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structural characterization of a unique marine animal family 7 Cellobiohydrolase suggests a mechanism of cellulase salt tolerance
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Marcelo Kern, Christina M Payne, John Mcgeehan, Simon Streeter, Richard N A Martin, Katrin Besser, Luisa Elias, William Eborall, Graham P Malyon, Michael E. HimmelAbstract:Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) Cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a Cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 Cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.
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computational simulations of the trichoderma reesei Cellobiohydrolase i acting on microcrystalline cellulose iβ the enzyme substrate complex
Carbohydrate Research, 2009Co-Authors: Linghao Zhong, Peter Ibsen Hansen, Joseph M Cleary, William S Adney, James F. Matthews, Mark R. Nimlos, Ross C Walker, Charles L. Brooks, Michael F Crowley, Michael E. HimmelAbstract:Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei cellulase system. Although natural cellulases can totally hydrolyze crystalline cellulose to soluble sugars, the current enzyme loadings and long digestion times required render these enzymes less than cost effective for biomass conversion processes. It is clear that Cellobiohydrolases must be improved via protein engineering to reduce processing costs. To better understand Cellobiohydrolase function, new simulations have been conducted using CHARMM of Cellobiohydrolase I (CBH I) from T. reesei interacting with a model segment (cellodextrin) of a cellulose microfibril in which one chain from the substrate has been placed into the active site tunnel mimicking the hypothesized configuration prior to final substrate docking (i.e., the +1 and +2 sites are unoccupied), which is also the structure following a catalytic bond scission. No tendency was found for the protein to dissociate from or translate along the substrate surface during this initial simulation, nor to align with the direction of the cellulose chains. However, a tendency for the decrystallized cellodextrin to partially re-anneal into the cellulose surface hints that the arbitrary starting configuration selected was not ideal.
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Computational simulations of the Trichoderma reesei Cellobiohydrolase I acting on microcrystalline cellulose Iβ: the enzyme–substrate complex
Carbohydrate research, 2009Co-Authors: Linghao Zhong, Peter Ibsen Hansen, Joseph M Cleary, William S Adney, James F. Matthews, Mark R. Nimlos, Ross C Walker, Charles L. Brooks, Michael F Crowley, Michael E. HimmelAbstract:Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei cellulase system. Although natural cellulases can totally hydrolyze crystalline cellulose to soluble sugars, the current enzyme loadings and long digestion times required render these enzymes less than cost effective for biomass conversion processes. It is clear that Cellobiohydrolases must be improved via protein engineering to reduce processing costs. To better understand Cellobiohydrolase function, new simulations have been conducted using CHARMM of Cellobiohydrolase I (CBH I) from T. reesei interacting with a model segment (cellodextrin) of a cellulose microfibril in which one chain from the substrate has been placed into the active site tunnel mimicking the hypothesized configuration prior to final substrate docking (i.e., the +1 and +2 sites are unoccupied), which is also the structure following a catalytic bond scission. No tendency was found for the protein to dissociate from or translate along the substrate surface during this initial simulation, nor to align with the direction of the cellulose chains. However, a tendency for the decrystallized cellodextrin to partially re-anneal into the cellulose surface hints that the arbitrary starting configuration selected was not ideal.
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probing the role of n linked glycans in the stability and activity of fungal Cellobiohydrolases by mutational analysis
Cellulose, 2009Co-Authors: William S Adney, Gregg T Beckham, John O Baker, Tina Jeoh, Yatchen Chou, William E Michener, Roman Brunecky, Michael E. HimmelAbstract:The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many fungal species produce a glycoside hydrolase family 7 (Cel7A) Cellobiohydrolase, a class of enzymes that catalytically process from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10, 2008) that expression of T. reesei Cellobiohydrolase I in a commonly used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A Cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120 h compared to the heterologously expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after 120 h compared to the wild type P. funiculosum enzyme expressed in A. niger var. awamori. Overall, this study demonstrates that “tuning” enzyme glycosylation for expression from heterologous expression hosts is essential for generating engineered enzymes with optimal stability and activity.
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.
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the crystal structure of the catalytic core domain of endoglucanase i from trichoderma reesei at 3 6 a resolution and a comparison with related enzymes
Journal of Molecular Biology, 1997Co-Authors: Gerard J Kleywegt, Jerry Stahlberg, Gideon J. Davies, J Y Zou, Christina Divne, I Sinning, Tapani Reinikainen, M Srisodsuk, Tuula T Teeri, T A JonesAbstract:Cellulose is the most abundant polymer in the biosphere. Although generally resistant to degradation, it may be hydrolysed by cellulolytic organisms that have evolved a variety of structurally distinct enzymes, Cellobiohydrolases and endoglucanases, for this purpose. Endoglucanase I (EG I) is the major endoglucanase produced by the cellulolytic fungus Trichoderma reesei, accounting for 5 to 10% of the total amount of cellulases produced by this organism. Together with EG I from Humicola insolens and T. reesei Cellobiohydrolase I (CBH I), the enzyme is classified into family 7 of the glycosyl hydrolases, and it catalyses hydrolysis with a net retention of the anomeric configuration.The structure of the catalytic core domain (residues 1 to 371) of EG I from T. reesei has been determined at 3.6 A resolution by the molecular replacement method using the structures of T. reesei CBH I and H. insolens EG I as search models. By employing the 2-fold non-crystallographic symmetry (NCS), the structure was refined successfully, despite the limited resolution. The final model has an R-factor of 0.201 (Rfree 0.258).The structure of EG I reveals an extended, open substrate-binding cleft, rather than a tunnel as found in the homologous Cellobiohydrolase CBH I. This confirms the earlier proposal that the tunnel-forming loops in CBH I have been deleted in EG I, which has resulted in an open active site in EG I, enabling it to function as an endoglucanase. Comparison of the structure of EG I with several related enzymes reveals structural similarities, and differences that relate to their biological function in degrading particular substrates. A possible structural explanation of the drastically different pH profiles of T. reesei and H. insolens EG I is proposed.