A Chromogenic Substrate for a β-Xylosidase-Coupled Assay of α-Glucuronidase

✍ Scribed by Peter Biely; Ján Hirsch; Daniel C. la Grange; Willem H. van Zyl; Bernard A. Prior

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 86 KB
• Volume: 286
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4810

✦ Synopsis

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