Characterization of mannanase S1 from Klebsiella oxytoca KUB-CW2-3 and its application in copra mannan hydrolysis

The mannanase Si from Klebsiella orytoca KUB-CW2-3 was purified by a single anion exchange chromatography to electrophoretic homogeneity. Si is a protein with a molecular mass of approximately 165 kDa and a pI value of 3.5. The optimum pH and temperature of mannanase activity were 4.0 and 40 degrees C, respectively. The enzyme exhibited good stability over the broad pH range of 3-6. The mannanase SI exhibited specific activity for different mannan substrates including galactomannan from locust bean gum (LBG), copra meal, and glucomannan from konjac, while neither xylanase nor cellulase activity were detectable. The Michaelis-Menten constants (K-m), maximum velocity (V-max), and the catalytic constant (k(cat)) values of Si against LBG and konjac mannan were 1.038-1.056 mg/ml, 6.149-6.183 mu U ml(-1) min(-1), and 0.047 s(-1), respectively. In addition, mannanase activity was activated by Co2+ (129%) but completely inhibited by EDTA and Zn2+. The N-terminal amino acid sequence (GRVGEAGPHGPHGPH) of mannanase S1 is different from the N-terminal region of other bacterial mannanases belonging to the glycoside hydrolase family GH5. The degradation products of mannanase S1 from LBG hydrolysis were galactose and unknown oligosaccharides with a different molecular structure to mannobiose, triose, and tetraose indicating the cleavages of alpha-1,6-galactosidic and beta-1,4-mannosidic linkages. Its hydrolysis of 100 mM CoCl2-treated copra mannan enhanced the growth of Lactobacillus reuteri KUB-AC5 but inhibited that of Salmonella serovar Enteritidis S003, indicating potential prebiotic properties by the action of mannanase from K oxytoca.