Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Purification and Characterization of Bacillus subtilis JS-17 Collagenase.

Bacillus subtilis JS-17이 생산하는 Collagenase의 정제 및 특성

  • Lim Kyoung-Suk (Division of Biological Sciences, Pusan National University) ;
  • Son Shung-Hui (Division of Biological Sciences, Pusan National University) ;
  • Kang Ho Young (Division of Biological Sciences, Pusan National University) ;
  • Jun Hong-Ki (Division of Biological Sciences, Pusan National University)
  • Published : 2005.08.01
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Abstract

Collagenases are generally defined as enzymes that are capable of degrading the polypeptide backbone of native collagen under conditions that do not denature the protein. An extracellular collagenase-producing bacterial strain was isolated from kimchi and identified to be Bacillus subtilis JS-17 through morphological, cultural, biochemical characteristics and 16S rDNA sequence analysis. Optimum culture condition of Bacillus subtilis JS-17 for the production of collagenase was $1.5\%$ fructose, $1\%$ yeast extract, $0.5\%\;K_2HPO_4,\;0.4\%\;KH_2PO_4,\;0.01\%\;MgSO_4\cdot7H_2O,\;0.01\%\; MnSO_4\cdot4H_2O,\;,0.1\%$ citrate and $0.1\%\;CaCl_2$. The production of collagenase was optimal at $30^{\circ}C$ for 72 hr. A collagenase was isolated from the culture filtrate of Bacillus subtilis JS-17. The enzyme was purified using Amberlite IRA-900 column chromatography, Sephacryl S-300 HR column chromatography and DEAE-Sephadex A-50 column chromatography The purified collagenase has an specific activity 192.1 units/mg. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PACE. The purified collagenase has $100\%$ activity up to $55^{\circ}C$.

Collagnase는 천연 collagen의 triple-stranded helix를 분해할 수 있는 protease로서 조직의 수복과 재생 과정에서 collagen의 재형성과 세포의 이동에 아주 중요한 역할을 하고, 숙주 감염시에는 collagen 기질을 빠르게 분해함으로써 감염을 돕는다. 본 연구에서는 일반가정에서 식용하는 김치로부터 collagenase를 생산하는 균주를 분리하여 Bacillus subtilis로 동정하였으며 이를 Bacillus subtilis JS-17이라 명명하였다. Bacillus subtilis JS-17이 생산하는 collagenase의 최적 생산 조건은 $1.5\%$ fructose, $1\%$ yeast extract, $0.5\%\;K_2HPO_4,\;0.4\%\;KH_2PO_4,\;0.01\%\;MgSO_4{\cdot}4H_2O,\;0.1\%\;citrate,\;0.1\%\;CaCl_2(pH\;7.0)$의 배지에서 $30^{\circ}C$, 200 rpm으로 72시간 동안 배양하는 것이다. 최적 조건에서 Bacillus subtilis JS-17이 생산하는 collagenase를 Amberlite IRA-900 column chromatography, Sephacryl S-300 HR column chromatography, DEAE-Sephadex A-30 column chromatography를 거쳐 분리 정제하고, 얻어진 정제 효소의 특성에 대하여 검토하였다. 정제된 collagenase의 비활성은 growth medium에서 192.1 units/mg였고, $1.1\%$의 수율로 얻어졌으며 분자량은 28 kDa이었다. 정제된 collagenase는 $55^{\circ}C$까지는 $100\%$의 활성을 유지하였고 $65^{\circ}C$에서도 $60\%$ 정도의 활성을 유지하였다. 또한 pH $6.0\~9.8$에서 $60\%$ 이상의 활성을 유지하였다. 정제된 collagenase는 metalloprotease inhibitor인 EDTA와 O-phenanthroline에 의해 효소 활성이 감소하였을 뿐만 아니라 Ammoninum persulfate, L-cysteine, N-ethylmaleimide, SDS, $NaN_3$, NaF, $KMnO_4$, PMSF에 대해서도 활성이 감소하였다. 정제된 collagenase를 여러 가지 기질에 대해 효소 활성을 비교한 결과 collagen (type I)에 대해 기질 특이성을 가지고 있었다.

Keywords

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