Kinetic study of a cellobiase purified from Neocallimastix frontalis EB188.
A cellobiase was purified from the culture supernatant of Neocallimastix frontalis EB188. This enzyme possessed a molecular weight of 85,000 and an isoelectric point of 6.95. The enzyme rapidly hydrolyzed cellobiose, p-nitrophenyl (pNP) beta-D-glucopyranoside (pNPG) and cellotriose and slowly hydrolyzed cellopentaose and salicin. The enzyme did not hydrolyze pNP alpha-D-glucopyranoside or pNP beta-D-cellobioside. Substrate inhibition was observed when cellobiose or pNPG were used as the substrates and glucose production was measured. The kinetic parameters were: K = 0.053 mM, V = 5.88 U/mg of protein and Ki = 0.95 mM for cellobiose; K = 0.36 mM, V = 1.05 U/mg and Ki = 8.86 mM for pNPG. Substrate inhibition was not detected during the hydrolysis of pNPG when pNP production was measured. The kinetic parameters for pNPG were: K = 0.67 mM and V = 1.49 U/mg of protein. The presence of an enzyme.glucose.substrate complex and transglucosylation was evident during the catalysis. Glucose, cellobiose, glucono-delta-lactone, galactose, lactose, maltose and salicin acted as competitive inhibitors during the hydrolysis of pNPG with the apparent inhibition constants (Kis) of 4.8 mM, 0.035 mM, 0.062 mM, 28.5 mM, 0.38 mM, 15.0 mm and 31.0 mM, respectively.
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Department of Animal Sciences, Washington State University, Pullman 99164-633.
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D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology; D002417:Cattle; D002475:Cellobiose; D043366:Cellulose 1,4-beta-Cellobiosidase; D008411:Chytridiomycota / Q000201:enzymology* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D012417:Rumen / Q000382:microbiology; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000276:immunology* / Q000302:isolation & purification
D002241:Carbohydrates; D002475:Cellobiose; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; D043366:Cellulose 1,4-beta-Cellobiosidase