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FSB1 – 2004 Food Science and Biotechnology in Developing Countries Properties of glucose oxidase immobilised in liposomes Berenice Ordóñez, Angélica Delgadillo, Elizabeth Contreras, Judith Jaimez, José Manuel Rodriguez-Nogales* Chemistry Research Centre. University of Hidalgo State..Carretera Pachuca- Tulancingo, Km 4.5. CP 42070. Pachuca. Hidalgo. México. * rjosem@uaeh.reduaeh.mx ABSTRACT Glucose oxidase was encapsulated in liposomes by the dehydration-rehydration method. Some characteristics of the liposomal and free glucose oxidase were compared. The enzyme encapsulated shows an apparent inhibition by glucose with a apparent Km value higher than that of the free enzyme. The Vmax of liposomal enzyme decreased by a factor of 0.35. The optimum temperature of the both enzyme forms remained similar but the liposomal enzyme showed maximal activity at a more acid pH. KEY WORDS Antimicrobial enzymes; DRV; glucose oxidase; liposome. INTRODUCTION Antimicrobial enzymes are ubiquitous in nature, playing a significant role in the defence mechanisms of living organisms against infection by bacteria and fungi. 1 Emerging preservation techniques, such as glucose oxidase and lactoperoxidase system, have received particular attention. 2 The antimicrobial activity of the glucose oxidase is due to the cytoxicity of the H2O2 formed. 2 One of the major problems with the use of glucose oxidase is achieving its temporal protection against microbial infection. The major factors responsible for this behaviour are the uncontrolled and high production rate of hydrogen peroxide and the high reactivity of hydrogen peroxide. 3 To obtain slow release of hydrogen peroxide at least one of the components of the hydrogen peroxide generating system must be
FSB1 – 2004 Food Science and Biotechnology in Developing Countries immobilized. This can be the enzyme (glucose oxidase) and/or the substrate (glucose). The immobilization of glucose oxidase in liposome may be useful for the sustained release of hydrogen peroxide. In this context, the present paper reports on several attempts to gain knowledge of the immobilization of glucose oxidase in liposomes. The effects of microencapsulation on the catalytic efficiency of the enzyme compared to those of its native counterpart were investigated. MATERIALS AND METHODS Liposomes were prepared by the dehydratation-rehydratation vesicle method (DRV) described by Kirby and Gregoriadis. 4 Enzymatic activity of free and liposomal glucose oxidase were determined using D-glucose as substrate. The quantity of H2O2 liberated, as a result of oxidation of glucose, was determined spectrophometrically using peroxidase in combination with o-dianisidine. 5 The apparent kinetic constants of Michaelis for the free and liposomal glucose oxidase were determined by measuring the enzymatic reaction rates at different substrate concentration ranging from 0.06 to 1.39 mol dm -3 at 25°C and at pH 6.0. Michaelis constants were calculated by analysing the data according to the Leonora program, an application specially designed for the analysis of enzyme kinetic. 6 The effect of pH and temperature of both free and microencapsulated glucose oxidase activity was studied using a response surface design. 7 . The design of the statistical experiments and the evaluation were performed using the computer program Statgraphics® Plus for Windows 4.0 (Statistical Graphics Corp., Rockville, MD 20852-4999 USA). All experiments were carried out in triplicate under identical conditions and at least three replicated samples were used in analytical determination. Mean values and standard deviations are present in figures and tables.
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PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

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