Cloning and expression of a broad pH stable GH11 xylanase from Aspergillus niger C71

Objective: To clone a full-length cDNA sequence of xynB, encoding endo-1,4-beta-xylanase of Aspergillus niger C71 and express in Escherichia coli BL21(DE3). Methods: The xynB was cloned using rapid amplification of cDNA ends (RACE) methods. The sequenced DNA was compared with the available sequences from GenBank using the BLASTX program, and the phylogenetic tree of the xylanases was then constructed using MEGA version 5.0. The amino acid sequences of XYNB were submitted to the ESyPred3D Web Server 1.0 for Homology modeling. The XYNB was purified by MonoQ an ion exchange chromatography and analyzed by SDS-PAGE. Results: The results showed that xynB is 678 bp in length, and encodes a 18 amino acid signal peptide as well as a 22 amino acid mature peptide with a calculated molecular weight of 24.127 kDa. Phylogenetic analysis of xynB, three-dimensional structure and overlap analysis of XYNB demonstrated that XYNB has a beta-jelly-roll architecture, which is more conversation in the catalytic domain of GH11 xylanases, belonging to the family 11 of glycosyl hydrolases. A maximum enzyme activity of 62,242.33 U.ml(-1) was obtained from XYNB. The XYNB with MW of 41 kDa demonstrated a broad pH stability from pH 3.0 to pH 12. 0.5 mM of Fe2+ could greatly enhance activity of XYNB to 567.02%. The mainly hydrolysis products of beechwood xylan were xylobiose. The hydrolysis products of XYNB effecting on beechwood xylan were analysed by HPLC. Conclusion: The xynB had been successfully expressed in Escherichia coli BL21, with high enzyme activity and a broad pH stability, and it would be a very good application prospect as the feed enzyme.