PURIFICATION AND PROPERTIES OF EXTRACELLULAR ENDOXYLANASES FROM ALKALOPHILIC THERMOPHILIC BACILLUS SP

An alkalophilic thermophilic Bacillus sp.(NCIM 59) isolated from soil produced two types of cellulase-free xylanase at pH 10 and 50-degrees-C. The two enzymes (xylanase I and II) were purified to homogeneity by ethanol precipitation followed by Bio Gel P-10 gel filtration and preparative polyacrylamide gel electrophoresis. The molecular weights of xylanase I and II were estimated to be 35 000 and 15 800, respectively, by sodium dodecyl sulfate gel electrophoresis' The enzymes inhibited immunological cross-reactivity and were glycoproteins. They had similar temperature (50-60-degrees-C) and pH (6) optima. Both xylanases were stable at 50-degrees-C at pH 7 for 4 days. However, xylanase I was comparatively more stable than xylanase II at 60-degrees-C. The isoelectric points of xylanase I and II were 4 and 8, respectively. The apparent K(m) values, using xylan as substrate, were 1.58 and 3.5 mg/mL, and V(max) values were 0.0172 and 0.742-mu-mol . min-1 . mg-1, respectively. Both xylanases were inhibited by N-bromosuccinimide, suggesting the involvement of tryptophan in the active site. The hydrolysis patterns demonstrated that the xylanases were endoenzymes. Xylanase I and II yielded mainly xylobiose, xylotriose, and higher xylooligosaccharides, with traces of xylose from x