Co-Immobilization and Compartmentalization of Cholesterol Oxidase, Glucose Oxidase and Horseradish Peroxidase for Improved Thermal and H2o2 Stability

Abstract

Glucose oxidase (GO X ) and cholesterol oxidase (CO X ) are enzymes with numerous practical applications, especially in biosensors for detecting glucose and cholesterol, respectively. One of the methods of determining glucose and cholesterol concentrations is colorimetry, where a cascade reaction of glucose and cholesterol oxidation by, respectively, GO X and CO X , is employed to produce H 2 O 2 followed by reaction of the chromogenic substrate 2,2’-azino-bis(3-ehylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) catalyzed by horseradish peroxidase (HRP) to give H 2 O as by-product. We proposed the application of electrospun fibers made of poly(D,L-lactide- co -glycolide) and commercial membrane UFX5 for co-immobilization of glucose oxidase or cholesterol oxidase with horseradish peroxidase combined with simultaneous co-immobilization by adsorption or by compartmentalization of enzyme separation using polyelectrolytes layers. The biosystems resulted in higher conversion efficiency of ABTS using immobilized enzymes at 45 °C and 65 °C compared to use of free enzymes. Moreover, after 5 catalytic cycles, biosystems based on UFX5 membrane and HRP or GO X or CO X immobilized by compartmentalization reached efficiencies of 89% and 34%, respectively, with less than 10% elution of initial amount of immobilized enzymes. In contrast, use of biosystems composed of electrospun fibers and co-immobilized enzymes resulted in enzyme elution of over 90% and lack of ABTS bioconversion.

Keywords: cholesterol oxidase, glucose oxidase, horseradish peroxidase, compartmentalization, co-immobilization