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., Km and Vmax) of hydrolysis reactions of 4-nitrophenyl-β-d-glucopyranoside, 4-nitrophenyl-β-d-galactopyranoside, and 4-nitrophenyl-β-d-xylopyranoside were determined for a model enzyme, almond β-glucosidase. Accurate quantitation was achieved via internal standard methodology and compared to spectrophotometric data and literature Km 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-β-d-glucopyranoside. A previously unpublished Km value for the substrate salicin was also determined by this method.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.