D-Xylose/D-glucose isomerases from two strains, a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii are described herein. The enzymes were purified to apparent homogeneity. Both of these D-xylose isomerases are homotetramers with relative subunit molecular masses of 45000 and 53000, respectively, as estimated by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The native molecular masses determined on Superose 12 gel chromatography are 181 kDa for the enzyme from Paenibacillus sp. and 199 kDa for that from A. I ruhlandii. The activity of both enzymes shows a requirement for divalent metal ions; the D-xylose isomerase from Paenibacillus sp. has the highest activity with Mn2+, while the enzyme from A. ruhlandii prefers Mg2+. Both enzymes also accept Co2+ with a somewhat lower efficiency, while Cu2+ inhibits the enzyme reaction. The binding of the metal ions obeys a biphasic characteristic, indicating the presence of two non-identical binding sites per subunit. D-Glucose is converted to D-fructose at a rate that is two- to three-fold slower than for the D-xylose isomerisation. D-Xylitol and D-lyxose are competitive inhibitors of both enzymes. Both enzymes have a pH optimum between 6.5 and 7.0, and they are active up to 60 degrees C. The enzyme from Paenibacillus sp. retained 50% of its activity after 4 days at 55 degrees C, whereas that from A. ruhlandii still retained 50% of its activity after 6 days at 55 degrees C. Polyacrylamide entrapment and immobilisation to both controlled pore glass and cyanogen-bromide-activated Sepharose were achieved for both enzymes with high efficiency.