PURIFICATION AND CHARACTERIZATION OF ALKALINE XYLANASES FROM BACILLUS-POLYMYXA

By applying different classical and fast protein liquid chromatographic techniques, three xylanases (beta-1,4-D-xylan xylanhydrolase) were purified to homogeneity from the extracellular enzymatic complex of Bacillus polymyxa. The three enzymes (X34C, X34E, and X22) were small proteins of 34, 34, and 22 kDa and basic pIs 9.3, >9.3, and 9.0, respectively. X34C and X34E are closely related and seem to be isoforms of the same enzyme. However, they differ in some characteristics. The three enzymes had different pH and temperature optima. One of them, X34E, showed a high thermal stability. The V(max) values determined for X34C, X34E, and X22 enzymes on oat spelts xylan were 14.9, 85.5, and 64.0 U mg-1, respectively, and 16.1, 62.0, and 150.6 U mg-1 on birchwood xylan. When oat spelts xylan was the substrate used, K(max) values of 3.4, 2.4, and 1.9 mg ml-1 were obtained for X34C. X34E, and X22 enzymes, respectively, and 0.65, 6.3, and 0.32 mg ml-1 were the respective K(m) values determined with birchwood xylan as the substrate. The enzymes were nondebranching endo-beta-xylanases. Xylose was one of the products of xylan hydrolysis by xylanases X34C and X34E, but this monosaccharide was not released by X22 enzyme. However, neither of the enzymes was able to degrade xylobiose.