Food Science and Preservation (한국식품저장유통학회지)

The Korean Society of Food Preservation (한국식품저장유통학회)
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Purification and Characterization of Protease from Bacillus subtilis PANH765

Bacillus subtilis PANH765가 생산하는 Protease의 정제 및 특성

  • 이창호 (경북대학교 식품공학과) ;
  • 우철주 (경북대학교 식품공학과) ;
  • 베동호 (건국대학교 응용생물화학과) ;
  • 김관필 (롯데그룹 중앙연구소)
  • Published : 2003.06.01
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Abstract

Pretense produced by Bacillus subtilis PANH765 was purified from culture supernatant by using ammonium sulfate fractionation DEAE-cellulose ion exchange chromatography, and gel filtration with Sephacryl S 200 HR and Sepharose CL-6B. DEAE-cellulose ion exchange column chromatography, separated the pretense into one fraction. This fraction was further purified using Sephacryl S 200 HR and Sepharose CL-6B gel titration. The molecular mass of pretense was estimated to be 35.0 kDa by the SDS-PAGE and gel filtration using Sepharose CL-6B. The results indicated that the purified pretense are monomeric proteins. Specific activity and purification folds of pretense were 657 U/mg and 4.35, respectively. The optimum temperature, optimum pit stable at a temperature range and pH ranges for the purified protease were 65$^{\circ}C$, 7.05, 50 ∼ 75$^{\circ}C$ and 6.0 ∼ 7.5, respectively. The pretense activity was decreased by the presence of PMSF and DFP, which the protease activity was increased by the presence of Na$\^$+/, K$\^$+/, Mg$\^$2＋/ and NH$_4$$\^$+/ ions.

Bacillus subtilis PANH765 균주가 생산하는 pretense를 황산암모늄에 의한 염석, DEAE-cellulose ion exchange chromatography, Sephacryl S 200 HR 및 Sepharose CL-6B gel filtration을 이용하여 정제하였다. DEAE-cellulose ion exchange chromatography를 행한 결과, 흡착 단백질 부분에서 활성이 높은 분획을 얻었다. 이 분획을 Sephacryl S 200 HR 및 Sepharose CL-6B gel filtration을 행한 결과 protease 활성을 가지는 단일 분획을 얻을 수 있었다. 정제한 protease의 분자량을 SDS-PAGE와 Sepharose CL-6B gel filtration으로 측정한 결과, 분자량은 35.0 kDa으로 추정되었으며, 각 효소의 비활성도는 6.27 U/mg이었으며, 회수율은 각각. 28.0%, 정제도는 4.35배로 나타났다. 최적 반응 온도는 $65^{\circ}C$, 최적 반응 pH는 7.05로 나타났으며, 온도 안정성은50 ∼ 75$^{\circ}C$, pH 안정성은 6.0 ∼ 7.5로 나타났다. 금속 이온의 영향은 $Na^{+}$, $K^{+}$, $Mg^{2＋}$ 및 NH$_4$$^{+}$ 이온이 효소 활성을 촉진하였으며, 그 중에서 $Mg^{2＋}$가 119.5%로 가장 높게 나타났다. 저해제인 경우 PMSF및 DFP에 의해 저해되었고 특히 DFP 보다는 PMSF 첨가시 뚜렷한 저해를 나타내었다.

Keywords

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