Purification and characterization of a thermostable xylanase from Bacillus amyloliquefaciens

A Bacillus amyloliquefaciens strain isolated from soil produced xylanolytic enzymes in the extracellular medium when grown on xylan and nitrate. No cellulase activity was detected. Xylanase was purified from the culture supernatant in three steps which comprised anion-exchange adsorption, ammonium sulfate precipitation, and hydrophobic interaction chromatography. The pure enzyme appeared as two close protein bands on SDS-PAGE with molecular weights of 18.4 and 19.6 kD, respectively. The molecular weight obtained by sedimentation equilibrium under native conditions was about 18.5 kD. The isoelectric point was 10.1. The enzyme contains a relatively high content of aspartate, glycine, and threonine. Tryptophan seems to be essential for xylanase activity. The presence of carbohydrate was noted in the pure enzyme. Circular dichroism studies indicated that the protein contains almost equal proportions of α-helix, β-sheet, and β-turns. The optimum pH of activity was 6.8–7.0. The enzyme exhibited good stability even at pH 9.0. Excellent stability was observed at 50°C even though optimal activity was at 80°C. The activity was completely inhibited by Hg 2+ ions and was reduced drastically in the presence of Cu 2+ and Fe 3+ ions. Mn 2+ ions, EDTA, β-mercaptoethanol, and dithiothreitol up to 5 m m stimulated the enzyme activity.