Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
Volume 1545, Issue 1-2, Pages 349-356Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S0167-4838(00)00296-X
Keywords
psychrophile; extremophile; microcalorimetry; isothermal titration calorimetry; chitinase; insoluble chitin
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Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E-a) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart. (C) 2001 Elsevier Science B.V. All rights reserved.
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