Cloning and Expression of Thermophilic Xylanase and Its Application in Xylooligosaccharides Production

Objective:A thermostable xylanase was obtained and characterized,the results showed that it could be used for preparation of xylo-oligosaccharides(XOSs). Methods:The xylanase(CsXynB)gene from Caldicellulosiruptor saccharolyticus DSM 8903 was analyzed by bioinformatics methods. Then amplified CsXynB gene was cloned into pET-20b. The recombinant plasmid was transformed into Escherichia coli BL21(DE3). The recombinant CsXynB was purified by heat treatment and the Ni-NTA chromatography. The purified CsXynB was applied to determinate the enzymatic properties and was used in the production of XOSs. Results:CsXynB was classified into the glycoside hydrolase family 10(GH10)by the bioinformatics analyses. CsXynB exhibited optimal activity at pH6.5 and 80℃. Further,CsXynB was stable up to 65℃ and pH values of 5.5 to 7.5. When beechwood xylan was used as substrate,the K_m,V_max,and K_cat values were 1.8 mg/mL,46.0 U/mg,and 60.7 s^-1,respectively. Production of XOSs from beechwood using CsXynB was investigated. Upon incubation with substrate recombinant CsXynB at pH6.5 and 65℃ for 1 h,beechwood xylan was hydrolyzed to XOSs with chain length of 2 and 3.Conclusion:CsXynB was a new and thermostable enzyme which could be used to produce xylooligosaccharides.