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 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 Polarimetry and 13C n.m.r. show that the hydrolyses of β-d-glucopyranosyl fluoride by β(1→3)-glucanases from Phanerochaete chrysosporium and Sporotrichum dimorphosporum have opposite stereochemistries

1993 ◽  
Vol 293 (2) ◽  
pp. 591-594 ◽  
Author(s):  
J L Copa-Patiño ◽  
Y Zhang ◽  
B Padmaperuma ◽  
I Marsden ◽  
P Broda ◽  
...  
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Optical Rotation ◽  
Fluoride Ion ◽  
Ion Release ◽  
Unpublished Work ◽  
Time Courses ◽  
Hydrolysis Of ◽  
Proton Decoupling

The time courses of optical rotation and fluoride ion release during hydrolysis of beta-D-glucopyranosyl fluoride by the beta(1-->3)-glucanase of Phanerochaete chrysosporium (J. L. Copa-Patiño and P. Broda, unpublished work) indicated that the initial sugar product was beta-D-glucopyranose. This was confirmed by monitoring the hydrolysis of 1-[13C]beta-D-glucopyranosyl fluoride by this enzyme with 13C n.m.r. (without proton decoupling). The same two techniques were used to confirm that hydrolysis of beta-D-glucopyranosyl fluoride by the exo beta(-->3)-glucanase of ‘Basidiomycete QM 806’ (identified as Sporotrichum dimorphosporum) yielded alpha-glucopyranose as first sugar product, in accordance with previous results using laminarin as substrate [Parrish and Reese (1963) Carbohydr. Res. 3, 424-429; Nelson (1970) J. Biol. Chem. 245, 869-872].

scholarly journals The cellulase of Penicillium pinophilum. Synergism between enzyme components in solubilizing cellulose with special reference to the involvement of two immunologically distinct cellobiohydrolases

1986 ◽  
Vol 234 (1) ◽  
pp. 93-99 ◽  
Author(s):  
T M Wood ◽  
S I McCrae
Keyword(s):  
Fusarium Solani ◽  
Maximum Rate ◽  
Crystalline Cellulose ◽  
Cellobiohydrolase I ◽  
Beta Glucanase ◽  
Trichoderma Koningii ◽  
End Groups ◽  
Penicillium Pinophilum ◽  
Cellobiohydrolase Ii ◽  
Hydrolysis Of

Two immunologically unrelated cellobiohydrolases (I and II), isolated from the extracellular cellulase system elaborated by the fungus Penicillum pinophilum, acted in synergism to solubilize the microcrystalline cellulose Avicel; the ratio of the two enzymes for maximum rate of attack was approx. 1:1. A hypothesis to explain the phenomenon of synergism between two endwise-acting cellobiohydrolases is presented. It is suggested that the cellobiohydrolases may be two stereospecific enzymes concerned with the hydrolysis of the two different configurations of non-reducing end groups that would exist in cellulose. Only one type of cellobiohydrolase has been isolated so far from the cellulases of the fungi Fusarium solani and Trichoderma koningii. Only cellobiohydrolase II of P. pinophilum acted synergistically with the cellobiohydrolase of the fungi T. koningii or F. solani to solubilize Avicel. Cellobiohydrolase II showed no capacity for co-operating with the endo-1,4-beta-glucanase of T. koningii or F. solani to solubilize crystalline cellulose, but cellobiohydrolase I did. These results are discussed in the context of the hypothesis presented.

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