Cloning, purification, and characterization of xylose isomerase from Thermotoga naphthophila RKU-10

A 1.3kb xyl-A gene encoding xylose isomerase from a hyperthermophilic eubacterium Thermotoga naphthophila RKU-10 (TnapXI) was cloned and over-expressed in Escherichia coli to produce the enzyme in mesophilic conditions that work at high temperature. The enzyme was concentrated by lyophilization and purified by heat treatment, fractional precipitation, and UNOsphere Q anion-exchange column chromatography to homogeneity level. The apparent molecular mass was estimated by SDS-PAGE to be 49.5kDa. The active enzyme showed a clear zone on Native-PAGE when stained with 2, 3, 5-triphenyltetrazolium chloride. The optimum temperature and pH for D-glucose to D-fructose isomerization were 98 degrees C and 7.0, respectively. Xylose isomerase retains 85% of its activity at 50 degrees C (t(1/2) 1732min) for 4h and 32.5% at 90 degrees C (t(1/2) 58min) for 2h. It retains 90-95% of its activity at pH 6.5-7.5 for 30min. The enzyme was highly activated (350%) with the addition of 0.5mM Co2+ and to a lesser extent about 180 and 80% with the addition of 5 and 10mM Mn2+ and Mg2+, respectively but it was inhibited (54-90%) in the presence of 0.5-10mM Ca2+ with respect to apo-enzyme. D-glucose isomerization product was also analyzed by Thin Layer Chromatography (R-f 0.65). The enzyme was very stable at neutral pH and sufficiently high temperature and required only a trace amount of Co2+ for its optimal activity and stability. Overall, 52.2% conversion of D-glucose to D-fructose was achieved by TnapXI. Thus, it has a great potential for industrial applications.