Characterization of alkaline thermoactive cellulase-free xylanases from alkalophilic Bacillus (NCL 87-6-10)

Two alkaline xylanases designated as "A" and "C", respectively, were isolated from the culture filtrates of the alkalophilic Bacillus grown on a wheat bran-yeast extract medium. The two xylanases occurred in the culture filtrate in a ratio of 10:90. These xylanases were purified to homogeneity on a CM-Sephadex matrix followed by further separation of Xylanase "A" on a phenyl sepharose column and preparative electrophoresis. The two xylanases differed considerably in their physico-chemical properties, kinetics and in their mode of action. Xylanase "C" had a molecular weight of 25,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was a cationic protein with a pI of 8.9. In contrast xylanase "A" had a molecular weight of 45,000 with a pI of 5.3. The two xylanases showed distinct differences in their hydrolysis pattern. Xylanase "A" produced comparatively larger amounts of small molecular weight oligosaccharides and xylose namely xylotriose (X(3)), xylobiose (X(2)) and xylose even in the initial stages of hydrolysis (2 and 5 h) while xylanase "C" produced negligible amounts of X(2) and no xylose for the same period of incubation. At 24 h only traces of xylose was produced by xylanase "C" while substantial amounts of the monomer was produced by xylanase A in 24 h. Xylanase "A" had a broad pH optimum ranging from pH 6.0-10.0 at 40-60 degrees C while xylanase "C" had an optimum pH of 8.0 at 40-60 degrees C. Xylanases "A" and "C" differed in their K(m) and V(max) values. Xylanase "A" had a K(m) of 1.67 mg/ml and a V(max) of 3.85 x 10(2) micromol/ml/min, whereas xylanase "C" had a K(m) of 10 mg/ml and a V(max) of 1.43 x 10(4) micromol/ml/min.