Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50 degrees C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and beta-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme 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, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85 degrees C for 20 min. Divalent metal ions Mg, Co, and Mn were required for maximum activity of the enzyme. The K(m) values 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 s and 0.5 x 10 s, respectively.