PRODUCTION AND PURIFICATION OF EXTRACELLULAR D-XYLOSE ISOMERASE FROM AN ALKALIPHILIC, THERMOPHILIC BACILLUS SP

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracel lular xylose isomerase at pH 10 and 50 degrees C by using xylose or wh eat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and i ntracellular marker enzymes such as xylanase and beta-galactosidase re vealed that xylose isomerase was truly secreted as an extracellular en zyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, an d ion-exchange chromatography. The molecular weight of xylose isomeras e was estimated to be 160,000 by gel filtration and 50,000 by sodium d odecyl sulfate-polyacrylamide gel electrophoresis, indicating the pres ence of three subunits. The enzyme is most active at pH 8.0 and with i ncubation at 85 degrees C for 20 min. Divalent metal ions Mg2+, Co2+ a nd Mn2+ were required for maximum activity of the enzyme. The K-m valu es for D-xylose and D-glucose at 80 degrees C and pH 7.5 were 6.66 and 142 mM, respectively, while K-cat values were 2.3 x 10(2) s(-1) and 0 .5 x 10(2) s(-1), respectively.