Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy (original) (raw)

2004, Analytical Biochemistry

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 D-glucose at carbon 1 into D D-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide (2 H 2 O). The intensity of the D D-glucono-1,5-lactone band maximum at 1212 cm À1 due to CAO stretching vibration was measured as a function of time to study the kinetics of D D-glucose oxidation. The extinction coefficient e of D D-glucono-1,5-lactone was determined to be 1.28 mM À1 cm À1. 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 À1 protein, 10.07 ± 1.75 mM, 1095.07 ± 151.19 s À1 , and 108.74 s À1 mM À1 , 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 À1 protein, 6.47 ± 0.85 mM, 1187.77 ± 108.16 s À1 , and 183.58 s À1 mM À1 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.