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A Simple Enzymatic Synthesis of 4-nitrophenyl beta-1,4-D-xylobioside, a Chromogenic Substrate for Assay and Differentiation of Endoxylanases

A Simple Enzymatic Synthesis of 4-nitrophenyl beta-1,4-D-xylobioside, a Chromogenic Substrate for Assay and Differentiation of Endoxylanases

Vladimír Puchart, Peter Biely

J Biotechnol. 2007 Feb 20;128(3):576-86.

PMID: 17223215

Abstract:

A simple procedure has been elaborated for preparation of 4-nitrophenyl beta-d-xylopyranosyl-1,4-beta-d-xylopyranoside (NPX(2)), a chromogenic substrate of some endo-beta-1,4-xylanases. The procedure is based on a self-transfer reaction from 4-nitrophenyl beta-d-xylopyranoside catalyzed by an Aureobasidium pullulans and Aspergillus niger beta-xylosidases. Both enzymes catalyzed only the formation of 4-nitrophenyl glycosides of beta-1,4-xylobiose with a small admixture of 4-nitrophenyl glycoside of beta-1,3-xylobiose. The highest yields of the NPX(2) (19.4%) was obtained at pH 5.5. The removal of the beta-1,3-isomer from NPX(2) is not necessary for quantification of endo-beta-1,4-xylanase activity since it is not attacked by endo-beta-1,4-xylanases. In contrast to GH family 5 xylanase from Erwinia chrysanthemi, which did not attack NPX(2), all family 10 and 11 xylanases cleaved the chromogenic substrate exclusively between xylobiose and the aromatic aglycone. Significant differences in the K(m) values of GH10 and GH11 xylanases suggested that activities of these enzymes could be selectively quantified in the mixtures using various concentrations of NPX(2). Moreover, NPX(2) could serve as an ideal substrate to follow the interaction of endo-beta-1,4-xylanases with various xylanase inhibitors.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP2001969	4-Nitrophenyl β-D-xylopyranoside		2001-96-9	Price

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