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Kinetic and Thermodynamic Investigation on Ascorbate Oxidase Activity and Stability of a Cucurbita maxima Extract.

Porto TS, Porto CS, Cavalcanti MT, Filho JL, Perego P, Porto AL, Converti A, Jr AP

Department of Biochemical and Pharmaceutical Technology, University of São Paulo, Av. Prof. Lineu Prestes, 580, B. 16., 05508-000 São Paulo-SP, Brazil, Laboratory of Immunopathology Keizo Asami (LIKA), 50670-901 Recife-PE, Brazil, Department of Chemical and Process Engineering, Genoa University, I-16145 Genoa, Italy, and Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco, 52171-900 Recife-PE, Brazil.

The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 degrees C using initial ascorbic acid concentration in the range 50-750 &mgr;M allowed estimating the Michaelis constant for this substrate (Km = 126 &mgr;M) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min-1). The main thermodynamic parameters of the enzyme reaction (&Dgr;H* = 10.3 kJ mol-1; &Dgr;G* = 87.2 kJ mol-1; &Dgr;S* = -258 J mol-1 K-1) were estimated through activity tests performed at 25-48 degrees C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long-term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10-70 degrees C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (&Dgr;H*D = 51.7 kJ mol-1; &Dgr;G*D = 103 kJ mol-1; &Dgr;S*D = -160 J mol-1 K-1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half-life (1047 min at 10 degrees C and 21.2 min at 70 degrees C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid-liquid extraction techniques.

Published 1 December 2006 in Biotechnol Prog, 22(6): 1637-1642.
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