Korean Chemical Engineering Research

The Korean Institute of Chemical Engineers (한국화학공학회)
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Optimization of Anion-exchange Chromatography for the Separation of Agarase from Culture Broth of Pseudoalteromonas sp.

Pseudoalteromonas sp. 배양액으로부터 아가레이즈 분리를 위한 음이온교환 크로마토그래피 최적화

  • Kim, Yu-Na (Department of Chemical & Biological Engineering and ERI, Gyeongsang National University) ;
  • Lee, Jae-Ran (Department of Chemical & Biological Engineering and ERI, Gyeongsang National University) ;
  • Kim, Mu-Chan (Department of Marine Environmental Engineering, Gyeongsang National University) ;
  • Kim, Sung-Bae (Department of Chemical & Biological Engineering and ERI, Gyeongsang National University) ;
  • Chang, Yong-Keun (Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology) ;
  • Hong, Soon-Kwang (Division of Bioscience and Bioinformatics, Myong Ji University) ;
  • Kim, Chang-Joon (Department of Chemical & Biological Engineering and ERI, Gyeongsang National University)
  • 김유나 (경상대학교 생명화학공학과 및 공학연구원) ;
  • 이재란 (경상대학교 생명화학공학과 및 공학연구원) ;
  • 김무찬 (경상대학교 해양환경공학과) ;
  • 김성배 (경상대학교 생명화학공학과 및 공학연구원) ;
  • 장용근 (한국과학기술원 생명화학공학과) ;
  • 홍순광 (명지대학교 생명과학정보학부) ;
  • 김창준 (경상대학교 생명화학공학과 및 공학연구원)
  • Published : 2011.12.01
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Abstract

Degradation products of agarose are biologically active and thus used as an ingredient in pharmaceuticals or functional cosmetics. Furthermore, it has been strongly considered as a substrate for bio-ethanol fermentation. Recently, we isolated new agarase-producing microorganism, Pseudoalteromonas sp. from south sea of Korea. In this study, we aimed to separate and purify the agarase from culture broth of this strain. Separation of agarase was performed by ion- exchange chromatography on DEAE-Sepharose resin. Equilibrium pH and volume ratio of resin to the amount of protein were optimized for the efficient adsorption of protein. 410 ${\mu}g$ of protein was completely adsorbed to 3 mL of resin at pH 7.5. The total amount of eluted protein increased as NaCl concentration increased to 400 mM at isocratic elution. Agarase was separated by linear gradient elution of NaCl (0~1,000 mM). Three major protein peaks were observed and the presence or absence of agarase in these eluted proteins was measured by Lugol's staining technique. Only six eluted protein fractions showed strong agarase activity.

아가로오스 분해산물은 생리활성이 우수하여 의약품 및 기능성 화장품 원료로 사용될 뿐만 아니라 바이오에탄올 생산을 위한 효모 발효용 기질로 검토되고 있어 상당한 주목을 받고 있다. 이에 따라 고성능 아가레이즈 탐색에 대한 연구가 활발히 진행되고 있다. 최근 본 연구팀에서는 남해안에서 신규 아가레이즈를 생산하는 미생물인 Pseudoalteromonas sp.를 분리하였다. 본 연구에서는 음이온교환수지를 이용하여 미생물 배양액으로부터 아가레이즈를 분리 정제하기 위한 크로마토그래피 조건을 최적화 하였다. 황산암모늄을 첨가하여 배양 상등액으로부터 침전된 단백질을 회수하고 이를 투석하여 조효소액을 얻었다. 음이온 교환수지인 DEAE-Sepharose 레진이 충진된 칼럼에 조효소액을 로딩하여 아가레이즈를 분리하였다. 410 ${\mu}g$의 단백질 로딩 시 흡착에 적합한 평형 pH는 7.5~8.0 이었고, 적정한 resin의 부피는 3 mL였다. 등용매 용출에서 용출액 중의 NaCl 농도가 증가함에 따라 용출되는 단백질 양이 증가하여 400 mM의 NaCl에서 최대에 달하였다. 최종적으로, NaCl 농도를 1,000 mM까지 선형적으로 증가시키며 아가레이즈를 분리하였다. Lugol 용액을 이용한 염색법으로 용리액 중의 아가레이즈의 존재를 확인하였다.

Keywords

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