Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Cell Surface Display of Cycloinulooligosaccharide Fructanotransferase Gene in Saccharomyces cerevisiae

Saccharomyces cerevisiae에서 Cycloinulooligosaccharide Fructanotransferase 유전자의 표층 발현

  • Kim, Hyun-Jin (Department of Biomaterial Control, Dong-Eui University) ;
  • Lee, Jae-Hyung (Department of Biomaterial Control, Dong-Eui University) ;
  • Kim, Hyun-Chul (Bioneer Corp.) ;
  • Kim, Yeon-Hee (Department of Biotechnology, Osaka University) ;
  • Kwon, Hyun-Ju (Department of Biomaterial Control, Dong-Eui University, Department of Life Science & Biotechnology, Dong-Eui University) ;
  • Nam, Soo-Wan (Department of Biomaterial Control, Dong-Eui University)
  • 김현진 (동의대학교 대학원 바이오물질제어학과) ;
  • 이재형 (동의대학교 대학원 바이오물질제어학과) ;
  • 김현철 ((주)바이오니아) ;
  • 김연희 (오사카대학 응용생물공학과) ;
  • 권현주 (동의대학교 대학원 바이오물질제어학과, 동의대학교 생명응용학과) ;
  • 남수완 (동의대학교 대학원 바이오물질제어학과)
  • Published : 2007.02.28
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Abstract

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTECFTN (9.0 kb) was introduced to S. cerevisiae EBY100 cell and then east transformants were selected on the synthetic defined medium lacking uracil and on the inulin containing medium. The surface display of CFTase was confirmed by immunofluorescence microscopy and its enzymatic ability to form cycloinulooligosaccharides(cyclofructans, CFs) from inulin. The total activity of the CFTase was reached about 5.52 unit/1 by cultivation of yeast transformant on YPDG medium. The optimized conditions determined were as follows; pH, 8.0; temperature, $50^{\circ}C$ ; substrate concentration, 5%; inulin source, Jerusalem artichoke. By the reaction with inulin, CFs consisting of cycloinulohexaose (CF6), cycloinuloheptaose (CF7), and cycloinulooctaose (CF8) were produced and CF6 was the major product.

Paenibacillus macerans 유래의 cycloinulooligosaccharide fructanotransferase (CFTase) 유전자(cft, 2832 bp, 103.8 kDa)를 효모 표층발현 vector인 pCTcon (GAL1 promoter)에 subcloning 하였다. 구축된 pCTECFTN (9.0 kb)를 숙주세포인 s. cerevisiae EBY100에 형질전환한 후, uracil이 결핍된 배지와 inulin 함유배지에서 선별하였다. cft 유전자는 GAL1 promoter에 의해 효모 형질전환체에서 성공적으로 발현되었다. inulin으로부터 cyclofructans (CFs)로 생산하는 효소적 능력으로부터 표층발현 유무를 확인하였다. YPDG배지에서 48시간 배양 후 분획한 균체는 5.52 unit/ l 의 활성을 보였다. CF 생산을 위한 효소의 최적 반응 조건으로 pH 8.0, 반응온도 $50^{\circ}C$, 기질농도 5%, 기질은 Jerusalem artichoke 등의 inulin과 표층 발현 CFTase 효소반응 결과, cycloinulohexaose (CF6), cycloinuloheptaose (CF7), 그리고 cycloinulooctaose (CF8)이 생성되었고, 이 중에서 CF6가 주 생성물이었다.

Keywords

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