Enzymatic Properties of Purified D-Xylose Isomerase from a Thermophilic Alkalophile, Bacillus TX-3

Abstract

A thermophilic alkalophile (Bacillus strain TX-3) which produces D-xylose isomerase (EC 5.3.1.5) in an alkaline medium over 55°C was isolated from soil samples.
The D-xylose isomerase was extracted from cells of this strain and purified 21-fold by DEAE-Toyopearl and hydroxyapatite chromatographies and gel filtration. The molecular weight was 140, 000 or 135, 000 by gel filtration or an ultracentrifugal method, respectively; the enzyme consists of three subunits with a molecular weight of 45, 000. The Km values for D-xylose and D-glucose were 0.1 M and 0.29 M, while V_max values were 28.6 and 1.6 μmol/min/mg protein, respectively. The optimum pH for activity was 7.5-9 and the optimum temperature was 80°C.
This enzyme was activated by the addition of Mn²⁺, Co²⁺, or Mg²⁺, but considerable activity was observed in the absence of these metal ions. If the enzyme was treated with EDTA, however, it showed no activity in the absence of metal ions, and its activity was restored by the addition of Mn²⁺, Co²⁺, or Mg²⁺. This enzyme was quite tolerant to SDS, and the residual activity was 50% after treatment with 5% SDS solution at 60°C for 30 min. Metal ions such as Mn²⁺ or Co²⁺ enhanced the inactivation of the enzyme with SDS.