Cloning and expression of a xylanase xynB from Aspergillus niger IA-001 in Pichia pastoris

The high-level expression of the xylanase GH11 gene from Aspergillus niger IA-001 called xynB was successfully completed in Pichia pastoris. The xynB gene encoding a mature xylanase of 225 amino acid was subcloned into the pPICZ alpha A vector and was transformed into P. pastoris X-33 under the control of the alcohol oxidase I (AOX1) promoter. The xynB gene was ligated with a sequence encodingmodified a-factor signal peptide (pPICZ alpha mA) and the recombinant xylanase activity, which was measured 1280 U ml(-1), was 1.5-fold higher than when it was inserted into pPICZ alpha A and was 19.39-fold greater than the native xylanase in the original strain. In a 10 L fermenter, the recombinant xylanase activity measured 10,035 U ml(-1) after 114 h. The SDS-PAGE analysis revealed that the purified xynB protein migrated as a single band with an apparent molecular weight of 24 kDa. The specific activity, using beechwood xylan as a substrate, was 1916 U mg(-1). The xylanase activity was optimal at pH 5.0 and at 50 degrees C. In addition, the xynB was active over a pH range of 2.2 to 10.0. The apparent K-m and V-max values were 4.429 mg ml(-1) and 1429 U mg(-1), respectively.