毕赤酵母
木聚糖酶
黑曲霉
重组DNA
化学
生物化学
发酵
表达式向量
分子生物学
酶
生物
基因
作者
Wei Fang,He Gao,Yunhe Cao,Anshan Shan
标识
DOI:10.1002/jobm.201300078
摘要
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.
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