A chromogenic substrate for a β-xylosidase-coupled assay of α-glucuronidase

4-Nitrophenyl 2-(4-O-methyl-alpha -D-glucopyranuronosyl)-beta -D-xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha -D-glucopyranosyluronate)-beta -D-xylopyranoside (Hirsch et at, Carbohydr. Res. 310, 145-149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic alpha -glucuronidase activity, A new precise alpha -glucuronidase assay was developed by coupling the alpha -glucuronidase-catalyzed formation of 4-nitrophenyl beta -D-xylopyranoside with its efficient hydrolysis by beta -xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta -xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of beta -xylosidase, The activity values of beta -xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the alpha -glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions, (C) 2000 Academic Press.