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Kinetic and Thermodynamic Investigation on Ascorbate Oxidase Activity and Stability of a Cucurbita maxima Extract
Author(s) -
Porto Tatiana S.,
Porto Camila S.,
Cavalcanti Maria T. H.,
Filho José L. Lima,
Perego Patrizia,
Porto Ana L. F.,
Converti Attilio,
Pessoa Adalberto
Publication year - 2006
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0602350
Subject(s) - cucurbita maxima , ascorbic acid , chemistry , thermostability , substrate (aquarium) , enzyme assay , reaction rate constant , michaelis–menten kinetics , chemical stability , enzyme kinetics , yield (engineering) , kinetics , chromatography , nuclear chemistry , enzyme , thermodynamics , biochemistry , organic chemistry , active site , botany , food science , oceanography , physics , quantum mechanics , biology , geology
The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 °C using initial ascorbic acid concentration in the range 50–750 μM allowed estimating the Michaelis constant for this substrate ( K m = 126 μM) and the maximum initial rate of ascorbic acid oxidation ( A 0,max = 1.57 mM min −1 ). The main thermodynamic parameters of the enzyme reaction (Δ H* = 10.3 kJ mol −1 ; Δ G* = 87.2 kJ mol −1 ; Δ S* = –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 °C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (Δ H* D = 51.7 kJ mol −1 ; Δ G* D = 103 kJ mol −1 ; Δ 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 °C and 21.2 min at 70 °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.