Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Cloning and Identification of Essential Residues for Thermostable β-glucosidase (BgIB) from Thermotoga maritima

Thermotoga maritima로부터 고온성 β-glucosidase (BgIB)의 클로닝과 필수아미노산 잔기의 확인

  • Hong, Su-Young (Division of Applied Life Science, Gyeongsang National University) ;
  • Cho, Kye-Man (Division of Applied Life Science, Gyeongsang National University) ;
  • Kim, Yong-Hee (Division of Applied Life Science, Gyeongsang National University) ;
  • Hong, Sun-Joo (Division of Applied Life Science, Gyeongsang National University) ;
  • Cho, Soo-Jeong (Department of Microbiological Engineering, Chinju National University) ;
  • Cho, Yong-Un (Department of Microbiological Engineering, Chinju National University) ;
  • Kim, Hoon (Department of Agricultural Chemistry, Sunchon National University) ;
  • Yun, Han-Dae (Research Institute of Agriculture & Life Science, Gyeongsang National University)
  • 홍수영 (경상대학교 응용생명과학부) ;
  • 조계만 (경상대학교 응용생명과학부) ;
  • 김용희 (경상대학교 응용생명과학부) ;
  • 홍선주 (경상대학교 응용생명과학부) ;
  • 조수정 (진주산업대학교 미생물공학과) ;
  • 조용운 (진주산업대학교 미생물공학과) ;
  • 김훈 (순천대학교 생물환경화학과) ;
  • 윤한대 (경상대학교 농업생명과학연구원)
  • Published : 2006.12.01
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Abstract

A hyperthermophilic bacterium Thernotoga maritima produced thermostable ${\beta}-glucosidase$. The gene encoding ${\beta}-glucosidase$ from T. maritima MSB8 was cloned and expressed in Escherichia coli. The en-zyme (BgIB) hydrolyzed ${\beta}-glucosidase$ linkages between glucose and alkyl, aryl of saccharide groups such as salicin, arbutin, and $_pNPG$. The insert DNA contained ORF with 2,166 bp encodes a 721 amino acids (calculated molecular mass of 80,964 and pl of 4.93). The amino a.id sequence of BglB showed the similarity to family 3 glycosyl hydrolases. The molecular weight of the enzyme was estimated to be approximately 81kDa by MUG-nondenaturing PAGE (4-methylumbelliferyl 13-D-glucoside-nondenaturing polyacrylamide gel electophoresis) and SDS-PACE. The ${\beta}-glucosidase$ exhibited maximal activity at pH 7.0 and $80^{\circ}C$. By exchanging two possible residues (Glu-232 and Asp-242) to Ala by site-directed mutagenesis method, it was found that these were essential for enzymatic activity.

초고온성 세균인 Thermotoga maritima로부터 ${\beta}-glucosidase$ 유전자를 클로닝한 후 대장균 숙주에서 발현시켰다. 이 효소는 salicin, arbutin, $_pNPG$과 같은 탄소원의 ${\beta}$-글루코시드 결합을 가수분해하였다. 721개의 아미노산을 암호화하는 2,166 bp의 DNA 염기서열로된 유전자이였다. 다른 ${\beta}-glucosidase$ 효소들과 단백질 유사성을 비교한 결과 glycosyl hydrolase family 3에 속하였으며 MUG-nondenaturing PAGE와 SDS-PAGE에 의해 확인된 단백질의 크기는 약 81 kDa이었다. 효소활성은 pH 7.0, $80^{\circ}C$에서 가장 높은 활성을 나타냈으며 이 효소의 아미노산 서열에 있는 두 개의 아미노산 잔기 (232번 글루탐산과 242번 아스파르트산 잔기)를 알라닌으로 치환시켜 활성이 없어지는 것으로 보아 이 두 잔기가 효소활성에 중요한 역할을 하는 것으로 추정된다.

Keywords

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