Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Gene Cloning, Expression, and Characterization of a Novel ${\beta}$-Mannanase from Bacillus circulans CGMCC 1416

  • Li, Yanan (Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yang, Peilong (Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Meng, Kun (Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Wang, Yaru (Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Luo, Huiying (Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Wu, Ningfeng (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Fan, Yuliu (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yao, Bin (Feed Research Institute, Chinese Academy of Agricultural Sciences)
  • Published : 2008.01.31
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Abstract

A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoding 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with $endo-{\beta}-1,4-D-mannanase$ from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by $Ni^{2+}$ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa ${\beta}$-mannanase having a specific activity of 481.55U/mg. The optimal activity of the purified protein, MANB48, was at $58^{\circ}C$ and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.

Keywords

References

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