Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge

► An N-terminal disulfide bridge stabilized a thermophilic xylanase by about 10°C. ► The upper limit for the performance of the mutant enzyme at pH 8–9 was about 75°C. ► The presence of substrate decreased slightly the thermostability at alkaline pH. ► The latter effect did not counteract the benefit conferred by the disulfide bridge. In order to increase the stability of thermophilic Thermomyces lanuginosus GH11 xylanase, TLX, a disulfide bridge Q1C–Q24C was introduced into the N-terminal region of the enzyme. The apparent temperature optimum shifted upwards at pH 6.5 by about 10°C to 75°C. The resistance to thermal inactivation also increased by about 10°C. The melting temperature measured by CD spectroscopy increased from 66 to 74°C. Therefore the N-terminal disulfide bridge increased both kinetic and thermodynamic stability almost equally. At pH 8 and 70°C, the disulfide bridge increased the enzyme half-life 20-fold in the presence of substrate. In contrast to the situation in acidic–neutral pH, the substrate decreased the thermostability of xylanases in alkaline pH. The upper limit for the performance of the disulfide bridge mutant at pH 9 was 75°C. This study showed that N-terminal disulfide bridges can stabilize even thermostable family GH11 xylanases.