Isolation and characterization of an extremely thermostable D-xylose isomerase from Thermus aquaticus HB 8

Abstract

Summary: The extremely thermophilic organism Thermus aquaticus possesses high activities of enzymes catalysing the degradation of xylans and metabolizing D-xylose via the pentose phosphate pathway. The D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5), an important enzyme of this process, is efficiently induced by its substrate D-xylose, and, to a lesser extent, by related pentoses and some derivatives of D-xylose. The D-xylose isomerase from T. aquaticus has been purified by anion-exchange chromatography, chromatography on D-xylose agarose and gel filtration. A single band migrating according to an M_r of 50000 was obtained by SDS-PAGE. An M_r of 196000 for the native enzyme, determined by gel filtration and ultracentrifugation in a glycerol gradient, suggested that the D-xylose isomerase is a homomeric tetramer. Arrhenius plots of the enzyme activity of the D-xylose isomerase were linear up to a temperature of 85 °C. At 70 °C the enzyme was inactivated in the absence of divalent cations, with a half-life of 4 d, while in the presence of Mn²⁺ or Co²⁺ it remained fully active for at least 1 month. The enzyme had an isoelectric point at 4.4 and showed a broad optimum in the pH range from 5.5 to 8.5. No significant differences in the pH and temperature behaviour could be observed when D-xylose was compared with D-glucose as substrate. Different methods of immobilization of the enzyme to solid supports as well as inclusion into nylon beads were studied. Attachment of the enzyme to epoxy-activated agarose and its co-aggregation with bovine serum albumin gave immobilized preparations with the same stability as free enzyme supplemented with Mn²⁺ or Co²⁺.