Gene Cloning, Expression And Characterization Of Xylanases From Bispora Betulina And Nesterenkonia Xinjiangensis

Because of the complexity of xylan, the complete hydrolysis of xylan requires cooperation of a great variety of enzymes. Among them, xylanase plays an important role in the xylanolytic enzyme system by hydrolyzing theβ-1,4-glycoside linkages in xylan backbone. Due to the important industrial application great attention was focused on xylanase system. Nowadays, screening enzyme-producing strains from special environments and then obtaining enzymes with superior properties have been important research method.In this study, a fungus, Bispora betulina and an alkaliphilic bacterium, Nesterenkonia xinjiangensis were used for the strains. To determine whether they could produce xylanase, mediums containing oat spelt xylan, birchwood xylan, wheat bran or corncob were used to induce them. Results showed that all the carbon sources could induce the two strains to produce xylanase.Based on sequence analysis of xylanase, a GH11 xylanase gene, designated as Xyn11BB, was cloned from B. betulina. Meanwhile, the other GH11 xylanase gene, designated as xyn11NX, was cloned from N. xinjiangensis. By homology searches using BLAST, the sequences of nucleotides and coded amino acids of Xyn11BB or xyn11NX share the highest identities which are less than 70%among the known sequences of GH11 xylanases, respectively. The results demonstrated that both of them are novel.Xyn11BB and xyn11NX were expressed in Pichia pastoris and Escherichia coli, respectively. The corresponding activities were 121.15 and 65.5 U/mL, which verified the function of Xyn11BB and xyn11NX.The recombinant proteins were purified and further characterized. Xyn11BB from Pichia pastoris shows the optimal pH of 4.5, optimal temperature at 50℃, and is stable over pH 4.0-10.0; Km is 113.06 mg/mL; Vmaxis 303.03μmol-min-1mg-1; the specific activity is 470 U/mg when using oat spelt xylan as substrate; rXyn11BB has good resistance to collagenase, trypsin and a-chymotrypsin. Xyn11NX from Escherichia coli shows the optimal pH of 7.0, optimal temperature at 55℃; It is thermostable, retains nearly 60%of the initial activity after incubation at 70℃for 1 h and more than 40%of the activity at 90℃for 15 min; Km is 16.08 mg/mL; Vmaxis 45.66μmol·min-1mg-1; The specific activity is 2158 U/mg when using oat spelt xylan as substrate. In addition, the enzyme has no cellulase activity either. Above all, the characteristics make it promising for various applications, especially in pulp and paper industry. Both of two recombinant xylanases have good prospects in industrial application and scientific research due to the enzymical characterizations.