Abstract
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-Xn or pNP-Xn, n=2-4), and studied for their uses as substrates in a kinetic study. The Kcat and Km of Bacillus pumilus xylanase (EC 3.2.1.8) activities for pNP-X2 were 0.24 s<-1> and 0.5 mM, respectively. The relative xylanase activities with the other substrates to that with pNP-X2, pNP-X, 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 Km and Kcat of the xylanase for oNP-X2 were 0.38 mM and 2.29 s-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.
