Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Separation and Purification of Protease from Bacillus subtlils CCKS-111 in Korean Traditional Soy Sauce

한국재래간장으로 부터 분리한 Bacillus subtilis CCKS-111이 생성하는 Protease의 분리 및 정제

  • Kim, Sung (Dep. of Food Science & Technology, Yeungnam University) ;
  • Lim, Seong-Il (Korea Food Research Institute) ;
  • Lee, Hee-Duck (Dep. of Food Science & Technology, Yeungnam University) ;
  • Lee, Seon-Ho (Dep. of Food Science & Technology, Yeungnam University) ;
  • Son, Jun-Ho (Dep. of Food Science & Technology, Yeungnam University) ;
  • Choi, Hee-Jin (Dep. of Food Science & Technology, Yeungnam University) ;
  • Kim, Yeung-Hweal (Dep. of Clinical Pathology, Daegu Junior College) ;
  • Choi, Cheong (Dep. of Food Science & Technology, Yeungnam University)
  • 김성 (영남대학교 식품가공학과) ;
  • 임성일 (한국식품개발연구원 생물공학연구부) ;
  • 이희덕 (영남대학교 식품가공학과) ;
  • 이선호 (영남대학교 식품가공학과) ;
  • 손준호 (영남대학교 식품가공학과) ;
  • 최희진 (영남대학교 식품가공학과) ;
  • 김영활 (대구보건전문대학 임상병리과) ;
  • 최청 (영남대학교 식품가공학과)
  • Published : 1997.06.30
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Abstract

A protease was purified from Bacillus subtilis CCKS-111 by ammonium sulfate treatment, DEAE-cellulose ion-exchange chromatography, Sephadex G-100 gel filtration and high performance liquid chromatography (HPLC). The specific activity of the purified enzyme was 24.3 unit/mg protein and the purification fold of enzyme was 50.6. Molecular weight of the purified enzyme estimated about 28,000 by HPLC gel filtration. The amino acid residues of this enzyme were 251.3 except threonine, serine and glycine. This result was similar to Bacillus subtilis subtilisin DY. From the first N-terminal amino acid to the 32th amino acid, the amino acid sequence was estimated after RP-HPLC elution. N-terminal and the 32th amino acids were alanine and aspartic acid. Alanine, serine, glycine and arginine were four major acids in the enzyme.

한국재래간장으로 부터 분리한 Bacilius subtilis CCKS-111이 생성하는 proteae를 분리 정제하였다. 황산암모늄 염석, DEAE cellulose ion-exchange chromatography, Sephades G-100 및 HPLC를 이용한 겔여과법에 의해 비활성도 24.3 unit/mg, 정제배수 50.6배로 효소를 정제하였으며 high performance liquid chromatography (HPLC)에 의하여 단일 단백질임을 확인하였다 정제효소의 분자량은 HPLC에 의하여 분자량 28,000 정도의 monomer로 추정되었고, 본 효소의 아미노산 잔기수는 251.3으로, 분해되기 쉬운 threonine, serine, glycine을 제외한 아미노산 잔기수는 Bacillus subtilis subtilisin DY 잔기수(274)와 유사한 것으로 나타났다. 아미노산 조성은 alanine, serine, glycine 및 arginine의 함량이 많았다. Reverse phase (RP)-HPLC로 분리한 주 peak로 N-말단에서 32번 까지 아미노산 배열을 확인한 결과 Bacillus subtilis subtilisin DY와 동일하였다.

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