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

Abstract

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.

Oxidative Enzyme Effects in Malt for Brewing

“…Actually, AAO II has much greater affinity (lower Km) for both substrates than AAO I does, although the latter displays a higher V max value. The Km value for AA displayed by AAO II is comparable with that reported for AAO from other organisms [32][33][34][35][36][37][38]. Few other papers report a Km value for oxygen, but the value we have measured for AAO II is comparable to that reported for Acremonium spp.…”

“…Molecular weight estimates for the two enzymes are, respectively, 25-27 and 6-9 kDa. Two AAO enzymes were isolated from barley grains that are extremely different from any previously reported AAO [32][33][34][35][36]. Both enzymes are of much lower molecular size than previously Steeping in water Air rest reported activities.…”

“…ϕ2/ϕ0 is the Km for the secondary substrate (hydrogen peroxide). AAO has been widely observed in various plant and fungal tissues [31][32][33][34][35][36]. Tamas et al [37] reported AAO in germinating barley seed with the agent causing substantial inhibition of rootlet growth.…”

Effect of microwave assisted blanching on the ascorbic acid oxidase inactivation and vitamin C degradation in frozen mangoes

“…Although there are available data in the literature regarding thermal inactivation kinetics of AAO, the residual activity of this enzyme after thermal treatment of broccoli, pumpkin (Cucurbita maxima), carrots (Daucus carota subsp. sativus) and others indicate that inactivation kinetics can be different with respect to the origin of the matrix (Leong & Oey, 2012;Munyaka et al, 2010;Porto et al, 2006). To our knowledge, there is not available information in literature regarding the thermal inactivation kinetics AAO present in mango.…”

O efeito da proximidade do fragmento florestal de Mata Atlântica sobre a área de cultivo no amadurecimento de bananas (Musa acuminata AAA cv. Nanicão) e nos compostos fenólicos das folhas de bananeiras

“…Quanto a AOx, estudos visando o seu isolamento e purificação são desenvolvidos desde a década de 1950 (DUNN;DAWSON, 1951;DAWSON, 1973). Atualmente esta enzima, já identificada em diversas espécies de fungos (DUPLESSIS et al, 2011;O'CONNELL et al, 2012) e vegetais (ESAKA et al, 1990KATO;ESAKA, 1996;OHKAWA et al, 1989), se encontra comercialmente disponível, permitindo a utilização da mesma em ensaios ou técnicas que Nesta etapa, antes da adição da AOx, utilizou-se fita de pH para verificar se as amostras se encontravam com valores de pH na faixa considerada para a atividade da enzima (pH 5,5-7,0) (CARVALHO et al, 1981;PORTO, 2008;PORTO et al, 2006).Todas as amostras apresentaram valores de pH próximo a 6, não sendo necessário o ajuste de pH.…”