A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-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 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.
Biely
P
P
Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, 84238, Slovakia. chempbsa@savba.sk
Hirsch
J
J
la Grange
D C
DC
van Zyl
W H
WH
Prior
B A
BA
D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002863:Chromogenic Compounds; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000032:analysis*; D006027:Glycosides; D007700:Kinetics; D009578:Nitrobenzenes; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014995:Xylosidases / Q000235:genetics
C421252:4-nitrophenyl-2-(4-O-methylglucopyranuronosyl)xylopyranoside; D002863:Chromogenic Compounds; D006027:Glycosides; D009578:Nitrobenzenes; D006026:Glycoside Hydrolases; D014995:Xylosidases; C096734:alpha-glucuronidase; C026579:exo-1,4-beta-D-xylosidase