Expression, Mutation And Characterization Of Thermomyces Lanuginosus Xylanase 1YNA

Xylan can be hydrolyzed by xylanase to oligoxylose and xylose. Nowadays xylanases are widely used in the field of feed, food, paper, and so on. Most of the commercial xylanases are produced by large-scale fermentation of the engineering strains. Until now, many xylanase genes from different organisms have been expressed in some expression hosts, like Escherichia coli, Bacillus subtilis, yeast and even fungi. Xylanase secreted by Thermomyces lanuginosus DSM10635 belongs to G11 family, whose isoelectric point is 3.7. The optimal temperature and pH of this enzyme is 70℃and 6.5 separately. This xylanase keeps high relative activity at pH 5.5-9.0.Xylanase gene was gained from total DNA of Thermomyces lanuginosus DSM10635 by using polymerase chain reaction (PCR) technology with degenerate primers. There is an intron found in this xylanase gene after BLASTN at NCBI webpage. The coding DNA sequences were got with the help of overlap extension PCR technology. The target fragment was correctly ligased into expression plasmid pET-22b(+) by double digestion at Endonuclease sites of NcoⅠand XhoⅠ, to form recombination expression plasmid. This plasmid was routinely transformed into expression host BL21(DE3) by the method of CaCl2. Active xylanase was successfully synthesized after induction. Characterization of some properties of this enzyme shows that the optimal temperature of 1YNA is 65℃, which is lower than that of the original xylanase which secreted from DSM10635. Some other results also show that the thermostability of 1YNA become worse, when compare with the origin xylanase.Some mutations were introduced into xylanase 1YNA to make several mutants, which were expressed in term of the same process. Analysis of some special properties shows that these mutants do have some changes, which are more or less different from that of 1YNA. The mutant with newly-constructed disulfide bridges, like DSB1, has an optimal temperature of 75℃, which is 10℃higher than that of 1YNA. The thermostability of DSB1 is also enhanced. It retains 40% relative residual activity after incubation at 80℃for 30min. Xylanases 1YNA and DSB2 achieved a specific activity of 5912 IU/mg and 6756 IU/mg, after the procedures of ammonium sulfate precipitation, hydrophobic interaction chromatography, ion exchange interaction chromatography and ultrafiltration. The purified xylanases are bases for further research.