The Reform and Study Ofsite-directed Mutagenesis of Acid Xylanase XynⅢ from Aspergillus Niger
|School||Henan Agricultural University|
|Keywords||Xylanase pH stability over-1ap PCR site-directed mutagenesis|
Although xylanases have widely been used in the paper industry, the feed industry the brewing, the energy industry as well as in the foodstuff industry. For example they can be u`sed in biological bleaching of paper pulping, improving the environment and the nutritive value of the forage . The application of xylanases in industry have the different requirements in pH. In most condition,we require them reaction in the low pH condition. for example,in the feed industry, as the feed additive xylanases should effect in the low pH condition.But,the most acid resistance of the much xylanases are very poor.,it is essential to improve the properties of xylanases acid resistance for broader applications.In this experiment, we dislodge the extra alignment by PCR, and link the pET-20b. The results show that the extra alignment impact the character of xylanases.In our study, we change the amino acid which after the amino acid Y to amino acid S, because the Amino acid diad YS was Negative correlation to the XynⅢenzyme about pH in former study.We got 9 Mutated xylanase gene, by PCR and overlap PCR, and link them to pMD19-T Vector respectively, then digest with NdeⅠand XhoⅠ, and connected with the digested pet-20b. and now we have connected No.1, No.2 and No.4 to expression vector pet-20b, then transformed the recombinant plasmids with the mutant gene to BL-21 and got expressed, then we got the proteins with the activity of xylanase.After the research of the character of the recombinases, we find that The optimum pH of the reformed xylanase was about 4.0, both the No.1 No.4 of the mutant xylanase were lower than the reformed xylanase, and the change of No.2 was not obvious.