A Simple Assay for Xylanase Using o-Nitrophenyl-β-D-xylobioside

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We measured xylanase activities upon eight chromogenic substrates, o- or p-nitrophenyl-β-D-xylopyranoside (oNP-X or pNP-X) and o- or p-nitrophenyl-β-D-xylooligosaccharides (oNP-X_n or pNP-X_n, n=2-4), and studied for their uses as substrates in a kinetic study. The K_<cat> and K_m of Bacillus pumilus xylanase (EC 3.2.1.8) activities for pNP-X_2 were 0.24 s<-1> and 0.5 mM, respectively. The relative xylanase activities with the other substrates to that with pNP-X_2, pNP-X, pNP-X_3, pNP-X_4, oNP-X, oNP-X_2, oNP-X_3, and oNP-X_4 were <0.001, 9.4, 9.7, <0.001, 19, 190, and 200, respectively. HPLC analysis of the digestion products of oNP-X_2 or pNP-X_2 showed that the xylanase hydrolyzed each of the substrates only at the ether bond between nitrophenol and xylobiose. All ether bonds of pNP-X_3, oNP-X_3, pNP-X_4,or oNP-X_4 were hydrolyzed by the xylanase and further hydrolyses proceeded in the digestion products, e.g., oNP-X_2 and oNP-X_3 from oNP-X_4. Therefore, oNP-X_2 was screened as a useful substrate in a kinetic study of the xylanase. The K_m and K_<cat> of the xylanase for oNP-X_2 were 0.38 mM and 2.29 s^<-1>, respectively. Thermodynamic studies showed that the higher reaction rate obtained with oNP-X_2 than that with pNP-X_2 was due to a significant decrease in the activation energy change, despite a decrease in the activation entropy change.
社団法人日本農芸化学会の論文
1996-06-23