Cloning and expression of a xylanase xynB from Aspergillus niger IA‐001 in Pichia pastoris. (21st June 2013)

• Record Type: Journal Article
• Authors: Fang, Wei; Gao, He; Cao, Yunhe; Shan, Anshan
• Is Part Of: Journal of basic microbiology. Volume 54:issue 1(2014:Jan.)
• Page Start: S190
• Page End: S199
• Publication Date: 2013-06-21
• Subjects: Microbiology -- Periodicals; 579
• DOI: 10.1002/jobm.201300078
• Languages: English
• ISSNs: 0233-111X

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="jobm201300078-sec-0001" sec-type="section"> <p>The high‐level expression of the xylanase GH11 gene from <italic>Aspergillus niger</italic> IA‐001 called <italic>xynB</italic> was successfully completed in <italic>Pichia pastoris</italic>. The <italic>xynB</italic> gene encoding a mature xylanase of 225 amino acid was subcloned into the pPICZαA vector and was transformed into <italic>P. pastoris</italic> X‐33 under the control of the alcohol oxidase I (AOX1) promoter. The <italic>xynB</italic> gene was ligated with a sequence encoding modified α‐factor signal peptide (pPICZαmA) and the recombinant xylanase activity, which was measured 1280 U ml<sup>−1</sup>, 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<sup>−1</sup> 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<sup>−1</sup>. 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 <italic>K</italic><sub><italic>m</italic></sub> and <italic>V</italic><sub><italic>max</italic></sub> values were 4.429 mg ml<sup>−1</sup> and 1429 U mg<sup>−1</sup>, respectively.</p> </sec> </abstract>