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Molecular cloning and heterologous expression of an acid stable xylanase gene from Alternaria sp. HB186

  • Liangwei Mao
  • Po Meng
  • Cheng Zhou
  • Lixin Ma
  • Guimin ZhangEmail author
  • Yanhe Ma
Review

Abstract

A new xylanase gene, named xyn186, was cloned by the genome-walking PCR method from the Alternaria sp. HB186. The sequence of xyn186 contains a 748 bp open reading frame separated by one intron with the size of 52 bp. The cDNA was obtained by DpnI-mediated intron deletion. The cDNA was cloned into pHBM905A and transformed into Pichia pastoris GS115 to screen xylanase-secreting transformants on RBB-xylan plates. The molecular mass of the enzyme was estimated to be 23 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 50°C, respectively. The K m and V max valued for birchwood xylan are 1.404 mg ml−1 and 0.2748 mmol min−1 mg−1, respectively. The inhibitory effects of various metal ions were investigated, Cu2+ and Hg2+ ions inhibited most of the enzyme activity. The gene copy number of xyn186 in the genome of P. pastoris was estimated as two by the Real-time PCR. To date, xyn186 gene is the first xylanase gene cloned from the genus Alternaria.

Keywords

Alternaria sp. HB186 Heterologous expression Pichia pastoris Acid stable xylanase Gene copy number Real-time PCR 

Notes

Acknowledgments

This study was supported by the National Nature Science Foundation (30600014), Hubei Province Nature Science Foundation Key Project, Knowledge Innovative Program of the Chinese Academy of Sciences (KSCX2-EW-G-8) and the Ministry of Sciences and Technology of China (973 programs 2012CB721000).

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Liangwei Mao
    • 1
  • Po Meng
    • 1
  • Cheng Zhou
    • 2
  • Lixin Ma
    • 1
  • Guimin Zhang
    • 1
    Email author
  • Yanhe Ma
    • 2
  1. 1.College of Life SciencesHubei UniversityWuhanChina
  2. 2.State Key Laboratory of Microbial ResourcesInstitute of Microbiology, Chinese Academy of SciencesBeijingChina

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