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

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 ((H2O)-H-2). The intensity of the D-glucono-1,5-lactone band maximum at 1212cm(-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 epsilon of D-glucono-1,5-lactone was determined to be 1.28mM(-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. (C) 2004 Elsevier Inc. All rights reserved.