Bivalent cations and amino‐acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase

Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro‐organisms has revealed a strong bias against specific thermolabile amino‐acid residues (i.e. N and Q) in hyperthermophilic proteins. The N + Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperthermophiles was examined. It was found to correlate inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus licheniformis DSM13 (BLXI), which had an N + Q content comparable to that of homologs from much more thermophilic sources. To determine whether BLXI behaves as a thermostable enzyme, it was expressed in Escherichia coli, and the thermostability and activity properties of the recombinant enzyme were studied. Indeed, it was optimally active at 70–72 °C, which is significantly higher than the optimal growth temperature (37 °C) of B. licheniformis. The kinetic properties of BLXI, determined at 60 °C with glucose and xylose as substrates, were comparable to those of other class II XIs. The stability of BLXI was dependent on the metallic cation present in its two metal‐binding sites. The enzyme thermostability increased in the order apoenzyme