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Kinetic and Thermodynamic Studies of Glucose Oxidase Catalysed Oxidation Reaction of Glucose

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Kinetic and Thermodynamic Studies of Glucose Oxidase Catalysed Oxidation Reaction of Glucose JASEM ISSN 1119-8362 Full-text Available Online at J. Appl. Sci. Environ. Manage. December, 2007 All rights reserved www.bioline.org.br/ja Vol. 11(4) 95 - 100 Kinetic and thermodynamic studies of glucose oxidase catalysed oxidation reaction of glucose ODEBUNMI, E O; *OWALUDE, S O Chemistry Department, University of Ilorin, P. M. B. 1515, Ilorin, Nigeria. Telephone: 08066105199 E-mail: [email protected] ABSTRACT : The kinetics of oxidation of D-glucose catalysed by the enzyme glucose oxidase has been studied over a wide range of experimental conditions. The reaction velocities increased with increase in the concentrations of the glucose oxidase and glucose, as well as increase in temperature and ionic strength of the solution. The reaction velocity initially increased with increase in pH, reaching a maximum at pH 6.5 and then decreased with further increase in pH. The reaction exhibited saturation kinetics and experimental data were analysed using the Michaelis- Menten equation. Arrhenius activation energy and thermodynamic activation parameters were measured and are ≠ -1 -1 reported. The large negative value of entropy of activation ∆S , -148.8JK mol , and positive value of the enthalpy of ≠ -1 activation ∆H , 26.3kJmol , give further support to the proposed mechanism. The results are interpreted in terms of a mechanism involving both an oxidative half reaction and a reductive half reaction. @ JASEM Glucose oxidase is a flavo protein consisting of 2 for the removal of glucose from powdered eggs, for moles of flavin adenine dinucleotide (FAD) per gluconic acid production, and as a source of mole enzyme and catalyses the oxidation of β-D- hydrogen peroxide in food preservation (Witt et al, glucose by molecular oxygen to D-glucose-1, 5- 1998). Glucose oxidase is also used extensively for lactone and hydrogen peroxide (Kalisz, et al 1997). the quantitative determination of D-glucose in It has been isolated from various sources, however, samples such as blood, food and fermentation only the enzyme from Aspergillus niger and products (Wilson and Turner, 1992). The overall Penicillium amagasakiense has been studied in oxidation reaction catalysed by glucose oxidase can detail (Schomburg et al 2000, Gibson and Bright be represented as follows: 1967). Glucose oxidase is used in the food industry Gluconic acid β - D - glucose gluconolactone gluconic o 6 o o 5 o o o o o 1 o o GOX H2O 4 o o 3 2 o o o o o o o H O O I 5 N N NH NH 1 10 N N O N N O I I I I R H R H E-FADH2 E - FAD The hydrogen peroxide, H2O2, which is generated agent such as o-dianisidine, it is catalytically as a by-product in the above equation is a good reduced to water by the enzyme Horseradish oxidizing agent and in the presence of a reducing peroxidase. Horseradish H2O2 + o-dianisidine oxidised o-dianisidine + H2O (2) Peroxidase The oxidised form of o-dianisidine absorbs readily studied. (Dulley and Holmes, 1975). The strongly at 430nm, thus, by monitoring the kinetics and mechanism of glucose oxidase absorbance at 430nm, the kinetics of glucose reactions have been studied from a number of oxidase catalysed oxidation of D-glucose can be steady – state and transient state kinetic analysis, * Corresponding author: Owalude, S O Kinetic and thermodynamic studies of glucose oxidase 96 (Weibel and Bright, 1971; Madson and Feather, appropriate volume ratios as described in the 1983; Ryabow and Firsova, 1997), and in our literature(Weast, 1977) while maintaining constant laboratory we have been investigating the kinetics the concentrations of the glucose, glucose oxidase, and mechanism of oxidation of sugars by a variety ionic strength and temperature. The average initial of oxidising agents (Owalude, 2004; Odebunmi and rates, kobs, were then estimated at different pH Owalude, 2005; Odebunmi et al 2006). We now values from the slopes of the log(absorbance) present the thermodynamic activation parameters in versus time plots. support of the earlier proposed glucose oxidase mechanism. Effect of ionic strength: The ionic strength of the reaction medium was always kept constant using MATERIALS AND METHODS sodium perchlorate of known strength. The ionic Materials: All the chemicals were of analytical strength effect on the reaction rates was then grade and were used without further purification. carried out at different initial concentrations of All the solutions were prepared using doubly sodium perchlorate while keeping constant the distilled water. concentrations of the glucose at 0.04M, glucose -9 oxidase at 6.24 x 10 M, pH at 7.0 and temperature o Kinetic Measurements: The kinetic procedure at 28 C. As above, the pseudo-first-order rate was adapted from that used by Dulley and Holmes, constants were estimated from the slopes of the 1975. The method involved the coupling of the plots of log(absorbance) versus time. hydrogen peroxide produced from the oxidase reaction to the reduction of a chromogen, o- Effect of temperature: The oxidation reactions dianisidine dihydrochloride, by Horseradish were carried out at different temperatures between o o peroxidase. The assays were carried out at 28oC 25 C and 60 C while maintaining constant the and the reaction contained the followings in a final glucose oxidase concentration, glucose volume of 10ml: glucose (0.01M, pH=7.0); concentration, pH and ionic strength at 0.2M.The Horseradish peroxidase (4mg/ml, pH=7.0), glucose pseudo-first-order rate constants at each oxidase (0.025mg/ml, pH=7.0); o-dianisidine temperature were then estimated from the slopes of (0.156g/mg, pH=7.0). The reaction was initiated by the plots of log(absorbance) versus time. the addition of the enzyme to the glucose solution and the progress of the reaction was followed by RESULTS AND DISCUSSION: measuring at 1 minute interval, the increase in Effect of reactant concentration: The reaction absorbance at 430nm with a Camspel M105 velocities, kobs, were measured at different initial spectrophotometer. The average initial rates, kobs, concentrations of glucose oxidase but at constant over three independent measurements were glucose concentration, constant ionic strength, and determined by linear regression from the slope of temperature at 28oC and pH at 7.0. The results the concentration versus time plots. The rate show that the rate increased as the concentration of constants were averages of at least three the enzyme increased. In another set of measurements. experiments, the reaction velocities were determined at different initial concentrations of Effect of reactants concentration: The glucose while maintaining constant glucose oxidase dependence of the reaction rate on glucose concentration and temperature at 28oC. The concentration was measured by keeping constant reaction velocities increased as the glucose the concentration of glucose oxidase, pH at 7.0, concentration increased. The plot of reaction o temperature at 28 C and the ionic strength at 0.2M. velocities (kobs) against the glucose concentration in The kinetic procedure as described above was then fig. 1 shows two regions of concentration followed and the pseudo-first-order rate constants, dependence of reaction velocity. In the low kobs, were estimated from the slopes of the concentration region, kobs increased almost linearly log(absorbance) versus time plots. In another set of with concentration and a plot of logkobs versus experiments, kinetic procedure as described above log[glucose] is linear with a slope of 1,(Owalude, was followed keeping constant the concentration of 2004) showing that the reaction rate is first order the glucose, the pH at 7.0, ionic strength at 0.2M within this range. However in the high glucose o and the temperature at 28 C while varying the concentration region, the rate of increase in kobs glucose oxidase concentration varied between 3.12 decreased progressively with concentration and it -9 -9 x 10 M and 15.6 x 10 M. First-order rate eventually levelled off to a constant value that is constants were estimated from the plots of independent of further increase in glucose log(absorbance) versus time plots. concentration. The reaction rate tends towards zeroth order in glucose in this high concentration Effect of pH: The kinetic measurements were also range, an indication that the reaction obeyed carried out at different pH values from 5.0 to 8.0 saturation kinetics (Bright and Porter, 1975). using the NaOH and NaHPO4 solutions mixed in * Corresponding author: Owalude, S O Kinetic and thermodynamic studies of glucose oxidase 97 0.06 0.05 0.04 -1 0.03 min obs k 0.02 0.01 0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 [GLUCOSE] M O Fig 1: Plot of kobs against glucose concentration at 28 C, pH =7.0, -9 [Glucose oxidase] = 6.24 x 10 M, [NaClO4] = 0.2M Effect of pH: The reaction velocities, kobs , were large there is always a decrease in catalytic activity determined at different pH values but at constant of glucose oxidase (Weibel and Bright, 1971). This glucose concentration, glucose oxidase has been attributed to the protonation of the active concentration, ionic strength and temperature at site histidine in the FAD molecule, i.e. the 28oC. The results show that the reaction rate protonated histidine results in a reduction of the increased with increase in pH of the medium but as kinetic barrier for the electron transfer to the the pH becomes large the rates started to decrease oxygen. Thus at low pH glucose oxidase provides a and showed a maximum at pH = 6.5 (fig 2.). A rigid and polar environment of low polarizability kinetic study at different pH values has established which reduces the intrinsic barrier of the charge that as the pH of the reaction medium becomes transfer to oxygen (Gibson and Bright, 1967).
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