Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Overexpression of a Paenibacillus ${\beta}-Glucanase$ in Pichia pastoris: Purification and Characterization of the Recombinant Enzyme

  • Yang, Peilong (Department of Microbiology Engineering, Feed Research Institute, Chinese Academy of Agricultural Science) ;
  • Shi, Pengjun (Department of Microbiology Engineering, Feed Research Institute, Chinese Academy of Agricultural Science) ;
  • Wang, Yaru (Department of Microbiology Engineering, Feed Research Institute, Chinese Academy of Agricultural Science) ;
  • Bai, Yingguo (Department of Microbiology Engineering, Feed Research Institute, Chinese Academy of Agricultural Science) ;
  • Meng, Kun (Department of Microbiology Engineering, Feed Research Institute, Chinese Academy of Agricultural Science) ;
  • Luo, Huiying (Department of Microbiology Engineering, Feed Research Institute, Chinese Academy of Agricultural Science) ;
  • Yuan, Tiezheng (Department of Microbiology Engineering, Feed Research Institute, Chinese Academy of Agricultural Science) ;
  • Yao, Bin (Department of Microbiology Engineering, Feed Research Institute, Chinese Academy of Agricultural Science)
  • Published : 2007.01.31
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Abstract

Isolation, expression, and characterization of a novel $endo-{\beta}-1,3(4)-D-glucanase$ with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a ${\beta}-glucanase$ protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other ${\beta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${\beta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{\beta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{\circ}C$. The $K_m,\;V_{max},\;and\;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{\mu}mol/min{\cdot}mg,\;and\;3,131s^{-1}$, respectively. For barley ${\beta}-glucan$ the values are 3.73mg/ml, $8,939{\mu}mol/min{\cdot}mg,\;and\;4,026s^{-1}$, respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

Keywords

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