Frontiers of Agriculture in China

, Volume 5, Issue 2, pp 196–200 | Cite as

Cloning of endo-β-glucanase I gene and expression in Pichia pastoris

  • Yu Bai
  • Runfang Guo
  • Hongwei Yu
  • Long Jiao
  • Shuli Ding
  • Yingmin Jia
Research Article

Abstract

Total RNA of Thermoascus aurantiacus was isolated from its mycelium and acted as template for RT-PCR. The full-length cDNA encoding an endo-β-glucanase I was cloned via RACE-PCR method and the cDNA contained an ORF of 1005 bp encoding 305 amino acids. A recombinant plasmid, pPIC9k-egI, was constructed by inserting the ORF sequence of endo-β-glucanase I gene (egI) into the yeast expression vector pPIC9k and transformed to Pichia pastoris GS115. The results showed that the recombinant endo-β-glucanase I was excreted into the fermentation medium. The highest activity of endo-β-glucanase I and the protein content were up to 45.42 U/mL and 788.26 μg/mL at incubation time of 144 h. The optimal temperature and pH for the recombinant endo-β-glucanase I were found to be 70°C and 3.5, respectively.

Keywords

Thermoascus aurantiacus endo-1 4-β-glucanase RACE Pichia pastoris gene expression 

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Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  • Yu Bai
    • 1
  • Runfang Guo
    • 1
  • Hongwei Yu
    • 1
  • Long Jiao
    • 1
  • Shuli Ding
    • 1
  • Yingmin Jia
    • 1
  1. 1.College of Food Science and TechnologyAgricultural University of HebeiBaodingChina

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