Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification of Chitinase from an Antagonistic Bacterium Bacillus sp.7079 and Pro-Inflammatory Cytokine Gene Expression by PCTC

  • Han, Ok-Kyung (Department of Applied Microbiology, College of natural Resources, Yeungnam University, Efficacy and safety Research Center for traditional Oriental Medicines, Kyungsan University) ;
  • Lee, Eun-Tag (Department of Applied Microbiology, College of natural Resources, Yeungnam University) ;
  • Lee, Young-Sun (Efficacy and safety Research Center for traditional Oriental Medicines, Kyungsan University) ;
  • Kim, Sang-Dal (Department of Applied Microbiology, College of natural Resources, Yeungnam University)
  • Published : 2003.02.01
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Abstract

Chitinase was purified from an antagonistic bacterium Bacillus sp. 7079 by ammonium sulfate precipitation, QAE-Sephadex anion exchange chromatography, Sephadex G-100 gel filtration, and SP-Sephadex cation exchange chromatography. The molecula. weight of purified chitinase (PC-1) was approximately 66.5 kDa on SDS-PACE. PC-1 exhibited optimum pH and temperature of pH 7.5 and $45^{\circ}C$, respectively. More than $80\%$ of PC-1 was stable at pH 5.0 to 9.0, and more than $90\%$ at $40^{\circ}C$. $Fe^2+\;and\;Ca^2+$ inhibited the chitinase activity about $20\%$, and EDTA and p-CMB by about $30\%$, whereas $Ag^+$ inhibited the activity up to $65\%$. The $K_m$ value of PC-1 was 1.215 mg/ml with colloidal chitin as a substrate. We also investigated the effect of PC-1 treated chitin (PCTC) on the pro-inflammatory cytokine gene expression in macrophage RAW 264.7 cells. The expression of IL-$1{\alpha}$ and IL-$1{\beta}$ mRNA gene was investigated using reverse transcriptase polymerase chain reaction (RT-PCR). IL-$1{\alpha}$ and IL-$1{\beta}$ mRNA were induced by the treatment of PCTC and chitin only in RAW 264.7 cells. These expressions were induced as early as 2 h and sustained up to 24 h in RAW 264.7 cells. IL-$1{\alpha}$ and IL-$1{\beta}$ mRNA were more strongly expressed by the treatment of PCTC than chitin treatment alone in RAW 264.7 cells.

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