Purification and Characterization of D-Xylose Isomerase from Bifidobacterium adolescentis

Abstract

D-Xylose isomerase was purified to homogeneity from cell-free extracts of Bifidobacterium adolescentis by ammonium sulfate fractionation and chromatographies on DEAE-cellulose and Butyl-Toyopearl. The molecular weight of the purified enzyme was estimated to be 168, 000 by gel filtration on TSKgel G-3000SW, and 53, 000 on SDS-polyacrylamide gel electrophoresis. The optimum pH was around 7 and the enzyme was stable at pH 7-8. The enzyme required bivalent cations, Mg²⁺, Co²⁺, or Mn²⁺ for the activity, particularly Mn²⁺ to be best. The enzyme had a pI of 4.3, and the K_m for D-xylose was 4mM. The N-terminal amino acid sequence of the enzyme was not similar to those of D-xylose isomerases from other sources such as Clostridium thermosulfurogenes, Escherichia coli, or Bacillus subtilis.