Hydrolysis of barley (1→3), (1→4)-β-d-glucan by a cellobiohydrolase II preparation from Trichoderma reesei

1995 ◽  
Vol 26 (2) ◽  
pp. 109-119 ◽  
Author(s):  
Kirsti Henriksson ◽  
Anita Teleman ◽  
Tapani Suortti ◽  
Tapani Reinikainen ◽  
Johanna Jaskari ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Cellobiohydrolase Ii ◽  
Hydrolysis Of

scholarly journals Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Dennis J. Díaz-Rincón ◽  
Ivonne Duque ◽  
Erika Osorio ◽  
Alexander Rodríguez-López ◽  
Angela Espejo-Mojica ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Recombinant Expression ◽  
Expression System ◽  
Enzymatic Extract ◽  
Native Strain ◽  
Wickerhamomyces Anomalus ◽  
Optimum Ph ◽  
Hydrolysis Of Cellulose ◽  
Cellobiohydrolase Ii ◽  
Hydrolysis Of

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.

scholarly journals Hydrolyses of α- and β-cellobiosyl fluorides by cellobiohydrolases of Trichoderma reesei

1993 ◽  
Vol 291 (3) ◽  
pp. 883-888 ◽  
Author(s):  
A K Konstantinidis ◽  
I Marsden ◽  
M L Sinnott
Keyword(s):  
Trichoderma Reesei ◽  
Biological Function ◽  
Fluoride Ion ◽  
Cellobiohydrolase I ◽  
Ion Release ◽  
Cellulase Complex ◽  
Cellobiohydrolase Ii ◽  
Glycoside Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

Cellobiohydrolase II hydrolyses alpha- and beta-D-cellobiosyl fluorides to alpha-cellobiose at comparable rates, according to Michaelis-Menten kinetics. The stereochemistry, absence of transfer products and strict hyperbolic kinetics of the hydrolysis of alpha-cellobiosyl fluoride suggest that the mechanism for the alpha-fluoride may be the enzymic counterpart of the SNi reaction observed in the trifluoroethanolysis of alpha-glucopyranosyl fluoride [Sinnott and Jencks (1980) J. Am. Chem. Soc. 102, 2026-2032]. The absolute factors by which this enzyme accelerates fluoride ion release are small and greater for the alpha-fluoride than for the beta, suggesting that its biological function may not be just glycoside hydrolysis. Cellobiohydrolase I hydrolyses only beta-cellobiosyl fluoride, which is, however, an approx. 1-3% contaminant in alpha-cellobiosyl fluoride as prepared and purified by conventional methods. Instrumental assays for the various components of the cellulase complex are discussed.

scholarly journals Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens

2000 ◽  
Vol 345 (2) ◽  
pp. 315-319 ◽  
Author(s):  
Dieter BECKER ◽  
Karin S. H. JOHNSON ◽  
Anu KOIVULA ◽  
Martin SCHÜLEIN ◽  
Michael L. SINNOTT
Keyword(s):  
Trichoderma Reesei ◽  
Active Site ◽  
Substrate Concentration ◽  
Initial Rate ◽  
Enzyme Active Site ◽  
Humicola Insolens ◽  
Cellobiohydrolase Ii ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Identical Crystal

We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.

scholarly journals Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as ‘burst’ kinetics on fluorescent polymeric model substrates

2005 ◽  
Vol 385 (2) ◽  
pp. 527-535 ◽  
Author(s):  
Kalle KIPPER ◽  
Priit VÄLJAMÄE ◽  
Gunnar JOHANSSON
Keyword(s):  
Bacterial Cellulose ◽  
Trichoderma Reesei ◽  
Anthranilic Acid ◽  
Degree Of Polymerization ◽  
Cellulose Substrates ◽  
Quantitative Monitoring ◽  
End Groups ◽  
Alternative Means ◽  
Burst Kinetics ◽  
Hydrolysis Of

Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88±10, 42±10 and 34±2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.

scholarly journals On-site integrated production of cellulases and β-glucosidases by Trichoderma reesei Rut C30 using steam-pretreated sugarcane bagasse

2018 ◽  
Author(s):  
Marcella Fernandes de Souza ◽  
Elba Pinto da Silva Bon ◽  
Ayla Sant’ Ana da Silvab
Keyword(s):  
Carbon Source ◽  
Lignocellulosic Biomass ◽  
Trichoderma Reesei ◽  
Sugarcane Bagasse ◽  
Enzyme Preparation ◽  
Integrated Approach ◽  
Aspergillus Awamori ◽  
Pretreated Sugarcane Bagasse ◽  
Hydrolysis Of ◽  
Rut C30

AbstractThe high cost of commercial cellulases still hampers the economic competitiveness of the production of fuels and chemicals from lignocellulosic biomasses. This cost may be decreased by the on-site production of cellulases with the integrated use of the lignocellulosic biomass as carbon source. This integrated approach was evaluated in the present study whereby steam-pretreated sugarcane bagasse (SPSB) was used as carbon source for the production of cellulases by Trichoderma reesei Rut C30 and the produced enzymes were subsequently used for SPSB hydrolysis. An enzyme preparation with a high cellulase activity, of 1.93 FPU/mL, was obtained, and a significant β-glucosidase activity was achieved in buffered media, indicating the importance of pH control during enzyme production. The hydrolysis of SPSB with the laboratory-made mixture resulted in a glucose yield of 80%, which was equivalent to those observed for control experiments using commercial enzymes. Even though the supplementation of this mixture with external β-glucosidase from Aspergillus awamori was found to increase the initial hydrolysis rates, it had no impact on the final hydrolysis yield. It was shown that SPSB is a promising carbon source for the production of cellulases and β-glucosidases by T. reesei Rut C30 and that the enzyme preparation obtained is effective for the hydrolysis of SPSB, supporting the on-site integrated approach to decrease the cost of the enzymatic hydrolysis of lignocellulosic biomass.

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