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