A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-glucuronidase

Anal Biochem. 2000 Nov 15;286(2):289-94. doi: 10.1006/abio.2000.4810.

Abstract

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 al., 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus niger / enzymology
  • Aspergillus niger / genetics
  • Chemistry Techniques, Analytical / methods
  • Chromogenic Compounds*
  • Genes, Fungal
  • Glycoside Hydrolases / analysis*
  • Glycosides*
  • Kinetics
  • Nitrobenzenes*
  • Recombination, Genetic
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Xylosidases* / genetics

Substances

  • 4-nitrophenyl-2-(4-O-methylglucopyranuronosyl)xylopyranoside
  • Chromogenic Compounds
  • Glycosides
  • Nitrobenzenes
  • Glycoside Hydrolases
  • Xylosidases
  • alpha-glucuronidase
  • exo-1,4-beta-D-xylosidase