Cloning and expression of a xylanase xynB from Aspergillus nigerIA‐001 in Pichia pastoris

Abstract

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 pPICZαA 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 encoding modified α‐factor signal peptide (pPICZαmA) and the recombinant xylanase activity, which was measured 1280 U ml⁻¹, was 1.5‐fold higher than when it was inserted into pPICZαA 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 °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.