Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal 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 degrees C to 75 degrees C. The resistance to thermal inactivation also increased by about 10 degrees C. The melting temperature measured by CD spectroscopy increased from 66 to 74 degrees C. Therefore the N-terminal disulfide bridge increased both kinetic and thermodynamic stability almost equally. At pH 8 and 70 degrees 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 degrees C. This study showed that N-terminal disulfide bridges can stabilize even thermostable family GH11 xylanases. (C) 2012 Elsevier Ltd. All rights reserved.