title: A simple assay for xylanase using o-nitrophenyl-beta-D-xylobioside.
abstract: We measured xylanase activities upon eight chromogenic substrates, o- or p-nitrophenyl-beta-D-xylopyranoside (oNP-X or pNP-X) and o- or p-nitrophenyl-beta-D-xylooligosaccharides (oNP-Xn or pNP-Xn, n = 2-4), and studied for their uses as substrates in a kinetic study. The Kcat and K(m) of Bacillus pumilus xylanase (EC 3.2.1.8) activities for pNP-X2 were 0.24s-1 and 0.5 mM, respectively. The relative xylanase activities with the other substrates to that with pNP-X2, pNP-X3, pNP-X4, oNP-X, oNP-X2, oNP-X3, and oNP-X4 were < 0.001, 9.4, 9.7, < 0.001, 19, 190, and 200, respectively. HPLC analysis of the digestion products of oNP-X2 or pNP-X2 showed that the xylanase hydrolyzed each of the substrates only at the ether bond between nitrophenol and xylobiose. All ether bonds of pNP-X3, oNP-X3, pNP-X4, or oNP-X4 were hydrolyzed by the xylanase and further hydrolyses proceeded in the digestion products, e.g., oNP-X2 and oNP-X3 from oNP-X4. Therefore, oNP-X2 was screened as a useful substrate in a kinetic study of the xylanase. The K(m) and Kcat of the xylanase for oNP-X2 were 0.38 mM 2.29s-1, respectively. Thermodynamic studies showed that the higher reaction rate obtained with oNP-X2 than that with pNP-X2 was due to a significant decrease in the activation energy change, despite a decrease in the activation entropy change.
authors.affiliation: Department of Applied Microbial Technology, Kumamoto Institute of Technology, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Taguchi
authors.forename: T
authors.initials: T
authors.lastname: Hamasaki
authors.forename: T
authors.initials: T
authors.lastname: Akamatsu
authors.forename: H
authors.initials: H
authors.lastname: Okada
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000378:metabolism*; D007700:Kinetics; D013379:Substrate Specificity; D013816:Thermodynamics; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000032:analysis*
chemical_list: C100564:2-nitrophenylxylobioside; D004187:Disaccharides; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase