D-xylose isomerases from a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii: isolation, characterization and immobilisation to solid supports

D-Xylose/D-glucose isomerases from two strains, a newly isolated strain, Pa enibacillus sp., and from Alcaligenes ruhlandii are described herein. The e nzymes were purified to apparent homogeneity. Both of these D-xylose isomer ases 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 19 9 kDa for that from A. I ruhlandii. The activity of both enzymes shows a re quirement for divalent metal ions; the D-xylose isomerase from Paenibacillu s sp. has the highest activity with Mn2+, while the enzyme from A. ruhlandi i prefers Mg2+. Both enzymes also accept Co2+ with a somewhat lower efficie ncy, while Cu2+ inhibits the enzyme reaction. The binding of the metal ions obeys a biphasic characteristic, indicating the presence of two non-identi cal binding sites per subunit. D-Glucose is converted to D-fructose at a ra te 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 enzy mes have a pH optimum between 6.5 and 7.0, and they are active up to 60 deg rees C. The enzyme from Paenibacillus sp. retained 50% of its activity afte r 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 an d immobilisation to both controlled pore glass and cyanogen-bromide-activat ed Sepharose were achieved for both enzymes with high efficiency.