| Xylanase (EC3.2.1.8) can hydrolyze xylan into xylo-oligosaccharides and D-xylose. Endo-β-D-1, 4-xylanase is one of the major hemicellulose hydrolases catalyzes the hydrolysis of internal β-1, 4 bonds of xylan. Xylanases are wildly used in paper and pulp industry, feed industry, foodstuff industry and energy industry. Xylanase XYNB from Streptomyces olivaceoviridis Al has been used as feed additive. XYNB has good properties such as high specific activity and resistance to pepsin and trypsin whereas its thermostability is not good enough.This study aimed at the thermostability improvement. Methods of site-directed mutagenesis and DNA shuffling were used for the purpose. Different mutants were expressed in Pichia pastoris and purified, and then the enzymatic properties of these mutants were analyzed to test and verify the effects of mutations. Furthermore, the catalytic residues of XYNB and the factors influencing the optimal pH have also been analyzed.The mutants Tl1Y, N13D, S40E, XS and TB were obtained by site-directed mutagenesis. The thermostability of these mutants increased with different degrees. The thermostability of N13D and S40E improved 24.76% and 14.46% respectively, at 70℃ for 5 min, compared with XYNB;The thermostability of T11Y mutant is 1.8 times higher than that of XYNB;The half-life of XS (with a disulfide bridge mutation) was increased to 9 min instead of 3 min of XYNB, at 70 ℃ and pH 6.0;TB (with the N-terminal replacement mutation) further increase the half-life at 70 ℃ to 20 min.We optimized and combined the mutations mentioned above to obtain a new mutant TS (with replacement of N-terminal and introduction of a disulfide bridge). The synergism of N-terminal and disulfide bridge made the thermostability of TS increased 12.4-fold compared with that of XYNB at 70 ℃, pH 6.0 for 20 min, and the half-life of TS increased to 150 min instead of 3 min of XYNB. The mutant TS with high stability is potentially useful in industrial applications.A homology modeling of xylanase XYNB was made by Swiss-Model and BLAST. Then the E87F, E177F and N46D mutations was introduced into XYNB respectively by site-directed mutagenesis. The mutants E87F and E177F lost almost all of their activities, the E87 and the E177 were deduced to be the catalytic residues. The mutant N46D expressed in Pichia pastoris was purified and its enzymatic properties were determined. The result revealed that the optimal pH of N46D reduced from 5.2 to 4.2 and the pH stability of N46D changed to acid pH. The optimal temperature and thermostability of N46D also increased. A very good correlation is shown between N46 and the optimal pH. N46 is an important amino acid to catalysis.Moreover, a mutant pool of XYNB was obtained by DNA shuffling method. A thermostable mutant sh-94# was screened from 700 recombinants, which thermostability was as twice as XYNB at 70℃. A system to screen the mutants of xylanase with better thermostability was founded, which will be helpful to further improvement of xylanase.The thermostability of xylanase XYNB was improved and the important catalytic amino acids were found by protein engineering methods in this study. The mutants obtained in this study can be good materials for further research into the relationship between structure and function of xylanase.
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