A simple enzymatic synthesis of 4-nitrophenyl beta-1,4-D-xylobioside, a chromogenic substrate for assay and differentiation of endoxylanases

作者：Puchart Vladimir; Biely Peter^*

来源：Journal of Biotechnology, 2007, 128(3): 576-586.

DOI：10.1016/j.jbiotec.2006.12.011

• beta-xylosidase
• enzymatic synthesis
• 4-nitrophenyl beta-1,4-D-xylobioside
• chromogenic substrate
• endo-beta-1,4-xylanase

A simple procedure has been elaborated for preparation of 4-nitrophenyl beta-D-xylopyranosyl-1,4-beta-D-xylopyranoside (NPX2), a chromogenic substrate of some endo-beta-1,4-xylanases. The procedure is based on a self-transfer reaction from 4-nitrophenyl P-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 NPX2 (19.4%) was obtained at pH 5.5. The removal of the beta-1,3-isomer from NPX2 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 Enwinia chrysanthenti, which did not attack NPX,, all family 10 and 11 xylanases cleaved the chromogenic substrate exclusively between xylobiose and the aromatic aglycone. Significant differences in the Kn, values of GH10 and GH11 xylanases suggested that activities of these enzymes could be selectively quantified in the mixtures using various concentrations of NPX2 Moreover, NPX2 could serve as an ideal substrate to follow the interaction of endo-beta-1,4-xylanases with various xylanase inhibitors.

• 出版日期2007-2-20