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Disulphide bridges were introduced in different combinations into the N-terminal region and the single a-helix of mesophilicTrichoderma reeseixylanase II (TRX II). We used earlier disulphide-bridge data and designed new disulphide bridges for the combination mutants. The most stable mutant contained two disulphide bridges (between positions 2 and 28 and between positions 110 and 154, respectively) and the mutations N11D, N38E, and Q162H. With a half-life of ~56 h at 65°C, the thermostability of this sevenfold mutant was ~5,000 times higher than that of TRX II, and the half-life was 25 min even at 75°C. The thermostability of this mutant was ~30 times higher than that of the corresponding mutant missing the bridge between positions 2 and 28. The extensive stabilization at two protein regions did not alter the kinetic properties of the sevenfold mutant from that of the wild-type TRX II. The combination of disulphide bridges enhanced significantly the pH-dependent stability in a wide pH range. [ABSTRACT FROM AUTHOR]

Xylanase was produced by growing Thermomyces lanuginosus DSM 10635 in different carbohydrate containing media. The xylanase was purified to electrophoretic homogeneity and some of its enzymatic properties were studied. The molecular mass of this xylanase estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 25.5 kDa, and the pI was approximately 3.7. Applying mass spectrometric analysis, the average molecular mass of the xylanase was determined to be 21295.17 Da, which is practically the same as the theoretical molecular mass of the T. lanuginosus DSM 5826 xylanase (crystal structure 1yna). Accordingly, the thermostability and enzymatic properties of DSM 10635 xylanase were similar to DSM 5826 xylanase. The inactivation of DSM 10635 xylanase during the elevation of temperature proceeded slowly at pH 6.5 and 8.0, whereas at pH 5.0, the inactivation profile was steep, indicating that changes in the electrostatic interactions decrease the stability at lower pH. The thermostabilizing effect of the substrate on DSM 10635 xylanase was studied and it was observed to be significant only under acidic conditions. The half-life in the presence of 1% birchwood xylan (w/v) was 7-fold, 4-fold and 1.5-fold higher than without the substrate at pH 4.0, 5.0 and 6.5, respectively. No increase in the stability by the presence of substrate was observed at pH 9.0. [Copyright &y& Elsevier]

Highlights: [•] N-terminal disulphide bridge in an extremophilic GH11 xylanase increased enzyme activity even at 110°C. [•] The half-life increased 10-fold at 100°C by the disulphide bridge. [•] The biomass-dissolving ionic liquid, [emim]OAc, affected enzyme activity in a greater extent than thermostability. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]