Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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High-Level Expression of an Aspergillus niger Endo-$\beta$-1,4-Glucanase in Pichia pastoris Through Gene Codon Optimization and Synthesis

  • Zhao, Shumiao (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University) ;
  • Huang, Jun (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University) ;
  • Zhang, Changyi (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University) ;
  • Deng, Ling (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University) ;
  • Hu, Nan (College of Life Science and Pharmacy, Nanjing University of Technology) ;
  • Liang, Yunxiang (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University)
  • Received : 2009.08.25
  • Accepted : 2009.10.21
  • Published : 2010.03.31
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Abstract

To improve the expression efficiency of recombinant endo-$\beta$-1,4-glucanase in P. pastoris, the endo-$\beta$-1,4-glucanase (egI) gene from Aspergillus niger was synthesized using optimized codons. Fourteen pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egI gene was generated by two-step PCR-based DNA synthesis. In the synthesized endo-$\beta$-1,4-glucanase gene syn-egI, 193 nucleotides were changed, and the G+C content was decreased from 54% to 44.2%. The syn-egI gene was inserted into pPIC9K and transformed into P. pastoris GS115 by electroporation. The enzyme activity of recombinant P. pastoris stain 2-7# reached 20.3 U/ml with 1% barley $\beta$-glucan and 3.3 U/ml with 1% carboxymethylcellulose (CMC) as substrates in shake flasks versus 1,270.3 U/ml and 220.7 U/ml for the same substrates in 50-1 fermentors. The molecular mass of the recombinant protein was approximately 40 kDa as determined by SDS-PAGE analysis, the optimal temperature for recombinant enzyme activity was $70^{\circ}C$, and the optimal pH was 5.0 when CMC was used as the substrate.

Keywords

References

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