Monitoring carbohydrate enzymatic reactions by quantitative in vitro microdialysis

On-line in vitro microdialysis (MD) sampling followed by HPLC separation and UV absorbance detection (HPLC-UV) was used to monitor carbohydrate enzyme systems. Fundamental parameters (i.e., K(m) and V(max)) of hydrolysis reactions of 4-nitrophenyl-beta-D-glucopyranoside, 4-nitrophenyl-beta-d-galactopyranoside, and 4-nitrophenyl-beta-D-xylopyranoside were determined for a model enzyme, almond beta-glucosidase. Accurate quantitation was achieved via internal standard methodology and compared to spectrophotometric data and literature K(m) values, which were found to be 2.6+/-0.5 mM (MD), 2.7+/-0.4 mM (spec), and 2.5 mM (lit), for the substrate 4-nitrophenyl-beta-d-glucopyranoside. A previously unpublished K(m) value for the substrate salicin was also determined by this method. An application is shown for monitoring the glycoside salicin and its hydrolysis product saligenin in a commercially available willow bark product that is used for making tea. This versatile method has far-reaching applications to monitoring a variety of carbohydrates in enzymatic processes without complex sample preparation procedures and without volume loss.