Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

Abstract

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ( 2H 2O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm −1 due to C O stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient ε of d-glucono-1,5-lactone was determined to be 1.28 mM −1 cm −1. The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants ( V max, K m, k cat, and k cat/ K m) determined by Lineweaver–Burk plot were 433.78 ± 59.87 U mg −1 protein, 10.07 ± 1.75 mM, 1095.07 ± 151.19 s −1, and 108.74 s −1 mM −1, respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36 ± 42.83 U mg −1 protein, 6.47 ± 0.85 mM, 1187.77 ± 108.16 s −1, and 183.58 s −1 mM −1 for V max, K m, k cat, and k cat/ K m, respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.