Construction And Characterization Of A Thermostable Xylanase Mutant With 6 × His-tag

Xylan is a major constituent of hemicellulose complex, which, together with cellulose and lignin, constitutes the majority of plant cell walls. Xylanases(endo-1,4-xylanase, EC 3.2.1.8) are glycosidases that initiates hydrolysis of xylan. During the last decades, xylanases have been successfully introduced to a wide range of industrial applications and processes, including the textile, food, feed, paper and pulp industries. These enzymes have also been reported to offer promising opportunities for the biodegradation and bioconversion of various agro-industrial wastes and by-products. Based on amino acid sequence similarities and the structure characteristics of core catalytic zone, xylanases have been divided into glycosyl hydrolase(GH) families 10 and 11. The GH11 family of xylanases contains low molecular mass enzymes, similar structure and high specificity towards xylan substrates.Thermophilic GH11 xylanase 1YNA from Thermomyces lanuginosus DSM 10635 was isolated and expressed in Escherichia coli. BL21(DE3). In order to increase the stability of this enzyme, a disulfide bridge Q1C–Q24C was introduced into the N-terminal region of the enzyme. The apparent temperature optimum of recombination enzyme DSB at pH 6.5 shifted up wards by about 10 °C to 75 °C.In this study, to simplify the manipulation of purification of xylanase, terminator of the gene was removed by PCR method and the gene was inserted into plasmid pET-22b(+). Expressing recombination xylanase DSB with 6 histidines inserted to the C-terminal, the gene, renamed dsb-his, was expressed in Escherichia coli. BL21(DE3).The SDS-PAGE result showed the enzyme could be purified simply by Ni Sepharose afinity chromatography, while the size and the characterization of enzyme keeps unchanged compared with the enzyme without 6 histidines. The purification of protein had been notably simplified.Although His-tagging engineering has been extensively implemented for protein purification, the effect of His-tagging on enzyme properties has not been systematically studied. Considering the 3-dimensional structure of xylanase in GH 11, it shows the 6 histidines, which were inserted into the C-terminal of xylanase, were exposure on the surface. Meanwhile, the 6 histidines are small and in shape of nonregular coil. In this work, we showed that the biochemical properties of the His-tagged xylanase are very similar to those of the wild-type enzyme, providing a method of highly-efficient purification of xylanase, and a path to that of other little proteins.