In situ proton NMR of glycosidase catalyzed hydrolysis and reverse hydrolysis

Abstract

In situ proton NMR is used to investigate sweet almond ( Prunus dulcis) glycosidase catalyzed hydrolysis and reverse hydrolysis. Time courses of six disaccharide hydrolyses indicate that almond protein extract contains fairly high amounts of β-glucosidase and β-galactosidase, while α-glucosidase activity is negligibly low. For the first time the Michaelis Menten constant ( K m) and maximal reaction rate ( V max) of a biotransformation are determined by in situ proton NMR. The high stability index of the regression line in Lineweaver Burk plot shows the general applicability of this analytical method in kinetic investigations. Glycosidase catalyzed reverse hydrolyses are performed using glucose and galactose as donors as well as several acceptors with one, two, or several hydroxyl groups. Structures of the generated glycosides are determined directly from the reaction mixture using sel-TOCSY and NOESY measurements. The initial reaction rates and final product yields of the reverse hydrolyses are determined by in situ proton NMR. Thermodynamic equilibria of all investigated reverse hydrolyses cause final yields below 7% of predominantly formed primary glycosides.