Taguchi H, Hamasaki T, Akamatsu T, Okada H
Department of Applied Microbial Technology, Kumamoto Institute of Technology, Japan.
Biosci Biotechnol Biochem. 1996 Jun;60(6):983-5. doi: 10.1271/bbb.60.983.
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.
我们测定了芽孢杆菌木聚糖酶在8种显色底物（邻硝基苯基-β-D-木吡喃糖苷或对硝基苯基-β-D-木吡喃糖苷（oNP-X或pNP-X）以及邻硝基苯基-β-D-木寡糖或对硝基苯基-β-D-木寡糖（oNP-Xn或pNP-Xn，n = 2 - 4））上的活性，并研究了它们在动力学研究中作为底物的用途。短小芽孢杆菌木聚糖酶（EC 3.2.1.8）对pNP-X2的催化常数（Kcat）和米氏常数（Km）分别为0.24 s-1和0.5 mM。与pNP-X2、pNP-X3、pNP-X4、oNP-X、oNP-X2、oNP-X3和oNP-X4相比，该木聚糖酶对其他底物的相对活性分别<0.001、9.4、9.7、<0.001、19、190和200。对oNP-X2或pNP-X2消化产物的高效液相色谱分析表明，木聚糖酶仅在硝基苯酚和木二糖之间的醚键处水解每种底物。pNP-X3、oNP-X3、pNP-X4或oNP-X4的所有醚键均被木聚糖酶水解，并且在消化产物中进一步发生水解，例如oNP-X4水解生成oNP-X2和oNP-X3。因此，oNP-X2被筛选为木聚糖酶动力学研究中的有用底物。该木聚糖酶对oNP-X2的Km和Kcat分别为0.38 mM和2.29 s-1。热力学研究表明，尽管活化熵变有所降低，但oNP-X2比pNP-X2具有更高的反应速率是由于活化能变化显著降低所致。
