Engineering the activity of thermophilic xylose isomerase by site-directed mutation at subunit interfaces

Some of the conserved residues at subunit interfaces of thermophilic xylose isomerases (XIs) were selected by means of both multiple sequences alignment and subunit interactions analysis of XIs, and then were mutated for improving the activity of Thermus thermophilus xylose isomerase (TtXI). By site-directed mutagenesis, single (D375G, K355A, V144A) and double (D375G/V385A) mutations were introduced into TtXI containing a N91D mutation site, namely, TtXI-N91D. It was shown that the specific activities of mutants D375G, K355A and V144A were remarkably increased over a temperature range of 40–90 °C at pH 7.0. The activities of mutants D375G/V385A, D375G, V144A and K355A were 1.14-, 1.62-, 2.49- and 3.02-fold greater than that of TtXI-N91D at 75 °C, respectively. Over the pH range of 5.0–9.0, the activities of mutants D375G, K355A and V144A were greater than that of TtXI-N91D at 60 °C. The thermostability of all mutants, except K355A, was lower than that of TtXI-N91D. The results suggest that the activity of TtXI could be engineered by site-directed mutagenesis on the conserved residues at subunit interfaces. This method could be employed for improving the activity of other thermophilic XIs.