KSBB Journal

  • 제23권5호
  • /
  • Pages.425-430
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  • 2008
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  • 1225-7117(pISSN)
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  • 2288-8268(eISSN)
한국생물공학회 (The Korean Society for Biotechnology and Bioengineering)
권호 표지

MALDI-TOF MS를 이용한 효모에서의 황화수소 생성 단백질의 동정

The MALDI-TOF MS determination of yeast proteins producing $H_2S$

  • 조현남 (금오공과대학교 자연과학부 응용화학) ;
  • 판루안 (금오공과대학교 자연과학부 응용화학) ;
  • 유동찬 (금오공과대학교 자연과학부 응용화학) ;
  • 양선아 (계명대학교 전통미생물자원개발 및 산업화연구센터) ;
  • 이인선 (계명대학교 전통미생물자원개발 및 산업화연구센터) ;
  • 김재형 (동일시마즈(주)) ;
  • 백효현 (동일시마즈(주)) ;
  • 지광환 (금오공과대학교 자연과학부 응용화학)
  • Cho, Hyun-Nam (Dept. of Applied Chemistry, Kumoh National Institute of Technology) ;
  • Fan, Lu-An (Dept. of Applied Chemistry, Kumoh National Institute of Technology) ;
  • Yoo, Dong-Chan (Dept. of Applied Chemistry, Kumoh National Institute of Technology) ;
  • Yang, Seun-Ah (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • Lee, In-Seon (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • Kim, Jae-Hyung (Bio Institute, Dong-il SHIMADZU Corp.) ;
  • Baek, Hyo-Hyun (Bio Institute, Dong-il SHIMADZU Corp.) ;
  • Jhee, Kwang-Hwan (Dept. of Applied Chemistry, Kumoh National Institute of Technology)
  • Published : 2008.10.31
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⟨ 이전 논문 다음 논문 ⟩

Abstract

생체에서의 황화수소는 의약 분야와 발효 산업에 있어서 중요한 역할을 하고 있다. 본 연구에서는 Saccharomyces cerevisiae을 이용하여 기질인 L-cysteine과 $\beta$-mercaptoethanol로부터 $\beta$-replacement 반응에 인한 황화수소를 발생시킬 수 있는 효소를 간편하고 신속하게 동정하는 방법의 확립을 목적으로 하였다. 효소에 의해 발생된 황화수소와 Pb-acetate의 반응으로 생성된 Pb-S를 gel상에서 간편하게 확인한 후, 확인된 단백질들을 이온교환컬럼를 수행한 후 gel에서 추출하는 방법으로 MALDI-TOFMS의 시료를 간단히 얻을 수 있었다. PMF 방법과 MS/MS ion search 분석을 통해 간편하게 효모에서 황화수소를 형성할 가능성이 있는 세가지 단백질의 동정에 성공하였다. 이 세 가지 단백질은 CYS3, CYS4, MET17 유전자의 단백질로서 cystathionine $\gamma$-lyase, CBS, OASS 임이 밝혀졌다. 그리고 이 세 단백질들은 L-cysteine과 $\beta$-mercaptoethanol의 존재하에서 황화수소를 실제로 생성함을 확인하였다. 본 연구에서 표적의 물질을 생산하는 단백질들을 젤상에서의 활성측정과 MALDI-TOF MS를 이용하여 간단히 그리고 정확하게 동정하는 방법을 확립하였다.

Hydrogen sulfide ($H_2S$) is a by-product of metabolism of amino acids including sulfur and alcoholic fermentation, it is generally thought of in terms of a poisonous gas. Though $H_2S$ can have a negative impact on the perceived quality of fermented drinks due to an undesirable aroma, it plays prominent roles as a neuromodulator in the mammalian brain as well as a smooth muscle relaxant. Nowadays studies on the proteins which produce $H_2S$ are carried out in various fields such as structure, function, and metabolism. Here we propose to develop a simple and rapid $H_2S$ forming assay method, which will lead to speed up preparing the $H_2S$ forming proteins for identification by MALDI-TOF MS analysis. We detected three kinds of proteins which produce $H_2S$ in the crude extract of Saccharomyces cerevisiae. Those proteins were cystathionie $\beta$-synthase, O-acetylserine sulfhydrylase, and cystathionine $\gamma$-lyase.

Keywords

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