Kinetic and thermodynamic investigation on ascorbate oxidase activity and stability of a Cucurbita maxima extract.
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 M allowed estimating the Michaelis constant for this substrate (Km = 126 microM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min-1). The main thermodynamic parameters of the enzyme reaction (DeltaH* = 10.3 kJ mol-1; DeltaG* = 87.2 kJ mol-1; DeltaS* = -258 J mol-1 K-1) were estimated through activity tests performed at 25-48 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 (DeltaH*D = 51.7 kJ mol-1; DeltaG*D = 103 kJ mol-1; 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.
Porto
Tatiana S
TS
Department of Biochemical and Pharmaceutical Technology, University of São Paulo, Av. Prof. Lineu Prestes, 580, B. 16., 05508-000 São Paulo-SP, Brazil. portots@usp.br
Porto
Camila S
CS
Cavalcanti
Maria T H
MT
Filho
José L Lima
JL
Perego
Patrizia
P
Porto
Ana L F
AL
Converti
Attilio
A
Pessoa
Adalberto
A
D001204:Ascorbate Oxidase / Q000737:chemistry*; D028464:Cucurbita / Q000201:enzymology*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005638:Fruit / Q000201:enzymology*; D007700:Kinetics; D010936:Plant Extracts / Q000737:chemistry*; D013816:Thermodynamics
D010936:Plant Extracts; D001204:Ascorbate Oxidase