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KMID : 0545120030130010077
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Journal of Microbiology and Biotechnology
2003 Volume.13 No. 1 p.77 ~ p.84
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Purification of Chitinase from an Antagonistic Bacterium Bacillus sp. 7079 and Pro-Inflammatory Cytokine Gene Expression by PCTC
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HAN, OK KYUNG
LEE, EUN TAG/LEE, YOUNG SUN/KIM, SANG-DAL
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Abstract
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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 molecular weight of purified chitinase (PC-1) was approximately 66.5 kDa on SDS-PAGE. PC-1 exhibited optimum pH and temperature of pH 7.5 and 45℃, respectively. More than 80% of PC-1 was stable at pH 5.0 to 9.0, and more 90% at 40℃. Fe^2+ and Ca^2- inhibited the chitinase activity about 20%, and EDTA and p-CMB 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-1α and ⅠL-1β mRNA gene was investigated using reverse transcriptase polymerase chain reaction (RT-PCR). ⅠL-1α and ⅠL-1β mRNA were induced by the treatment of RCTC 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-1α and ⅠL-1β mRNA were more strongly expressed by the treatment of PCTC than chitin treatment alone in RAW 264.7 cells.
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