Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Purification and Characterization of Glyoxalase I from Pleurotus ostreatus

Pleurotus ostreatus에서 분리된 Glyoxalase I의 특성

  • Kim, Seong-Tae (Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University) ;
  • Yang, Kap-Seok (Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University) ;
  • Seok, Yeong-Jae (Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University) ;
  • Huh, Won-Ki (Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University) ;
  • Kang, Sa-Ouk (Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University)
  • 김성태 (서울대학교 자연과학대학 미생물학과, 분자미생물학연구센터) ;
  • 양갑석 (서울대학교 자연과학대학 미생물학과, 분자미생물학연구센터) ;
  • 석영재 (서울대학교 자연과학대학 미생물학과, 분자미생물학연구센터) ;
  • 허원기 (서울대학교 자연과학대학 미생물학과, 분자미생물학연구센터) ;
  • 강사욱 (서울대학교 자연과학대학 미생물학과, 분자미생물학연구센터)
  • Published : 1994.01.01
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Abstract

Glyoxalase I was purified 2,294-fold from Pleurotus ostreatus by S-hexylglutathione affinity chromatography, Sephadex G-150 gel filtration chromatography and DEAE-sepharose A-50 CL-6B ion exchange chromatography with an overall yield of 21.7%. The molecular mass determined by gel filtration was found to be approx. 34 kDa. SDS-PAGE revealed that the enzyme consists of two identical subunits with a molecular mass of approx. 17 kDa. The K sub(m) values of this enzyme for methylglyoxal and phenylglyoxal were 0.39 mM and 0.22 mM, respectively. And this enzyme had a strong affinity for L-xylosone and hydroxypyruvaldehyde. The enzyme showed its optimal activity at pH 6.5-7.5 and at $40^{\circ}C$. $^1H$-NMR spectroscopic analysis of enzymic reaction showed that this enzyme catalyzes intramolecular proton transfer.

Pleurotus ostreatus로부터 glyoxalase I(S-lactoyl-glutathione methylglyoxal lyase, EC 4.4.1.5)이 S-hexylglutathione affinity chromatography, Sephadex G-150 gel permeation chromatography, DEA-sepharose A-50 CL-6B ion exchange chromatography를 통해 순수 분리되었다. 이 결과, 전체 활성도의 21.7% fmf 수확하였으며, 분리 배수는 2,294 배 이었다. Gel filtration chromatography로 측정한 효소의 분자량은 34 kDa이며, SDS-PAGE 결과 본 효소는 분자량 17 kDa인 동일한 소단위체 두 개로 구성된 이합체라고 생각된다. Methylglyoxal과 phenylglyoxal에 대한 $K_m$ 값은 각각 0.39 mM 과 0.22 mM 이며 L-xylosone과 hydroxypyruvaldehyde에 대해서도 강한 친화력을 보여주었고, pH 6.5~7.5, $35~45^{\circ}C$에서 활성도가 가장 높았다. 이 효소의 반응 과정을 핵자기공 명분광법으로 분석한 결과, 분자내의 양성자 전달과정이 뚜렷이 관찰되었다.

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