Abstract:
Glucose oxidase (GOD) was produced from Aspergillus fumigatus by submerged fermentation system. To monitor the kinetics of GOD, peroxidase is used as coupling enzyme. This research was carried out to isolate and purify peroxidase from low cost, P. vulgaris (green beans) in order to use it for GOD assay which could be a robust and cost effective approach. Peroxidase and GOD activities in crude extract were measured spectrophotometrically. The supernatant was assayed for peroxidase activity, 35.5 U/mL with protein content of 0.82 mg/mL. The crude peroxidase was partially purified to the level of gel filtration chromatography (SG-200). With 80 % ammonium sulfate saturation, it was found suitable to precipitate peroxidase with enzyme activity of 26.9 U/mL and protein content of 0.44 mg/mL. After gel filtration, the specific activity was 143.6 U/mg with a purification fold of 3.3 for green beans peroxidase (gPOD). Peroxidase extracted from green beans was used as a coupled enzyme for fungal glucose oxidase. Glucose oxidase which was purified through ammonium sulphate precipitation (90 %), dialyisis, and gel filtration chromatography (Sephadex G-200) showed a specific activity of 67.5 U/mg proteins with 7.7 degree of purification and 1.2 % yield. Glucose oxidase was characterized using partially purified peroxidase, gPOD as coupled enzyme based on the pH effect on enzyme activity, temperature and effect of substrate concentration. Maximum enzyme activity (50.43 U/mL) was obtained at pH 5.5 and was considered optimum pH of GOD; glucose oxidase showed highest activity (34.72 U/mL) at 45 °C, hence the optimum temperature. Moreover, the substrate (glucose) concentration effect was carried out and the Michaelis-Menten constant, Km and maximum velocity, Vmax obtained from Line-Weaver-Burk plot were 5.66 mM and 16.67 U/mL, respectively. It is more evident that peroxidase is the most heat stable enzyme; therefore, it is concluded that it may be potentially useful for coupling with GOD and other industrial purposes.
