한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제45권1호
  • /
  • Pages.63-70
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  • 2017
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  • 1598-642X(pISSN)
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  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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독도 심해토 메타게놈 유래 신규 내열성 에스테라아제의 생화학적 특성규명

Biochemical Characterization of a Novel Thermostable Esterase from the Metagenome of Dokdo Islets Marine Sediment

  • 이창묵 (농촌진흥청 국립농업과학원) ;
  • 서소현 (이수 앱시스글로벌 연구개발 연구소) ;
  • 김수연 (농촌진흥청 국립농업과학원) ;
  • 송재은 (농촌진흥청 국립농업과학원) ;
  • 심준수 (농촌진흥청 국립농업과학원) ;
  • 한범수 (농촌진흥청 국립농업과학원) ;
  • 김동헌 (농촌진흥청 국립농업과학원) ;
  • 윤상홍 (농촌진흥청 국립농업과학원)
  • Lee, Chang-Muk (Metabolic Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Seo, Sohyeon (Global R&D Center, ISU ABXIS) ;
  • Kim, Su-Yeon (Metabolic Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Song, Jaeeun (Metabolic Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Sim, Joon-Soo (Metabolic Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Hahn, Bum-Soo (Metabolic Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Kim, Dong-Hern (Metabolic Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Yoon, Sang-Hong (Metabolic Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration)
  • 투고 : 2016.09.20
  • 심사 : 2016.11.04
  • 발행 : 2017.03.28
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초록

독도 해저 2,000 미터 퇴적토를 이용한 메타게놈 유전자 은행의 60,672 클론을 기름 성분 tributyrin이 첨가된 배지에서 스크리닝 하였다. 활성을 가진 클론에서 EstES1 유전자를 선발하였다. EstES1은 553개 아미노산으로 구성된 분자량 59.4 kDa 단백질로, 가장 높은 유사성은 Haliangium ochraceum의 carboxylesterase와 44%이었다. EstES1 서열 내부에는 carboxylesterase의 전형적인 penta-peptide motif, catalytic triad 및 N-terminal 부위 37개의 leader sequence가 존재했다. 서열기반 계통분석 결과, EstES1은 신규한 esterase 임을 보여주었다. EstES1 효소의 leader 서열을 제거한 재조합 수용성 RLES1 효소는 탄소 2-12까지 포함된 long acyl ethyl ester 기질을 모두 이용할 수 있지만, p-Nitrophenyl butyrate (C4)에 가장 높은 활성과 turn-over 값을 보였다. 최적 활성은 $45^{\circ}C$, pH 9.0이다(specific activity 255.4 U/mg). 또한 강알칼리 상태인 pH 10.5까지 80% 이상의 활성이 유지되었다. EstES1의 활성은 $60^{\circ}C$에서 내열성을 보여, 1시간 동안 활성을 100% 유지할 수 있다. 효소 활성은 여러 종류의 유기용매 하에서도 안정하게 유지되었다. 따라서, EstES1은 배양이 불가능한 난배양 미생물로부터 유래된 신종 효소 유전자로서, 고온의 지방산 가수분해, 알칼리 상태나유기용매가 존재하는 여러 공정분야에 활용될 수 있다.

A functional screen of 60,672 fosmid metagenomic clones amplified from marine sediment obtained from the Dokdo islets in Korea identified the gene EstES1, whose product, EstES1, displayed lipolytic properties on tributyrin-supplemented media. EstES1 is a 576 amino acid protein with a predicted molecular weight of 59.4 kDa including 37 N-terminal leader amino acids. EstES1 exhibited the highest sequence similarity (44%) to a carboxylesterase found in Haliangium ochraceum DSM14365. Phylogenetic analysis indicated that EstES1 belongs to a currently uncharacterized family of lipases. Within the conserved domain, EstES1 retains the catalytic triad that consists of the consensus penta-peptide motif, GESAG. EstES1 demonstrated a broad substrate specificity toward the long acyl group of ethyl esters (C2-C12), and its optimal activity was recorded toward p-Nitrophenyl butyrate (C4) at pH 9.0 and $40^{\circ}C$ (specific activity of 255.4 U/mg). The enzyme remained stable in the ranges of $60-65^{\circ}C$ and pH 9.0-10.5 and in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, EstES1 has potential for use in industrial applications involving high temperature, organic solvents, and/or alkaline conditions.

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피인용 문헌

  1. A strategy for securing unique microbial resources - focusing on Dokdo islands-derived microbial resources vol.64, pp.1, 2018, https://doi.org/10.1163/22244662-20181024