(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal → NAD Glucose

温度

90 °C

Next, in order to evaluate thermal stability, 1 ml of a 20 mM phosphate buffer solution (pH 7.4) was added to 5 mg of the NAD-GDH-immobilizing mesoporous silica, and then the resulting mixture solution was heated at 90° C. for 30, 60, 90, or 120 minutes. After the heating, the mixture solution was centrifuged to obtain NAD-GDH-diaphorase-immobilizing silica. The obtained silica was washed twice with pure water. To this heated NAD-GDH-immobilizing silica, a 20 mM NAD solution prepared by using a 0.1 M tris-HCl buffer solution (pH 8.0) was added. After the stirring at room temperature for 5 minutes, a 1.5 M glucose solution was added to the silica solution. The resulting mixture solution was further stirred for 10 minutes. After the reaction, the solution was centrifuged at 20000 g for 10 minutes at 4° C. The activity of NAD-GDH for catalyzing a reduction reaction of NAD to NADH was measured by using the absorption maximum of NADH in the supernatant at 340 nm. For comparison, a solution of NAD-GDH that was not immobilized on mesoporous silica was prepared and similarly heated, and the activity of the NAD-GDH was measured. The relative activity of NAD-GDH was determined by using the absorbance of a solution obtained by adding 1.5 M of glucose to unheated NAD-GDH as a reference. The results confirmed the NAD-GDH immobilized on mesoporous silica retained a high relative activity even if the NAD-GDH was heated at 90° C. for 120 minutes. On the other hand, free NAD-GDH that was not immobilized on mesoporous silica was completely inactivated by heating at 90° C. for 30 minutes. The results suggest that NAD-GDH is prevented from thermal denaturation by being immobilized on pore walls of mesoporous silica.