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Saccharomyces cerevisiae에서 Cycloinulooligosaccharide Fructanotransferase 유전자의 표층 발현

Cell Surface Display of Cycloinulooligosaccharide Fructanotransferase Gene in Saccharomyces cerevisiae

  • 김현진 (동의대학교 대학원 바이오물질제어학과) ;
  • 이재형 (동의대학교 대학원 바이오물질제어학과) ;
  • 김현철 ((주)바이오니아) ;
  • 김연희 (오사카대학 응용생물공학과) ;
  • 권현주 (동의대학교 대학원 바이오물질제어학과, 동의대학교 생명응용학과) ;
  • 남수완 (동의대학교 대학원 바이오물질제어학과)
  • 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)
  • 발행 : 2007.02.28

초록

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.

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.

키워드

참고문헌

  1. Bader, E. T. and K. D. Wittrup. 1997. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553-557 https://doi.org/10.1038/nbt0697-553
  2. Duagherty, P. S., M. J. Olsen, B. L. Linverson and G. Georgiou. 1999. Development of an optimized expression system for the screening of antibody libraries displayed on the Esherichia coli surface. Protein Eng. 12, 613-621 https://doi.org/10.1093/protein/12.7.613
  3. Georgiou, G., H. L. Poetschke, C. Stathopoulos and J. A. Francisco. 1993. Practical applications of engineering Gram-negative bacterial cell surfaces. Trends Biotechnol. 11, 6-10 https://doi.org/10.1016/0167-7799(93)90068-K
  4. Ito, H., Y. Fukuda, K. Murata and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168
  5. Jeon, S. J., D. J. You, H. J. Kwon, S. Kanaya, N. Kunihiro, K. H. Kim, Y. H. Kim and B. W. Kim. 2002. Cloning and characterization of cycloinulooligosaccharide fructanotransferase (CFTase) from Bacillus polymyxa MGL21. J. Microbiol. Biotechnol. 12, 921-928
  6. Jin, Z., J. Wang, B. Jiang and X. Xu. 2005. Production of inulooligosaccharides by endoinulinases from Aspergillus ficuum. Food Res. Int. 38, 301-308 https://doi.org/10.1016/j.foodres.2004.04.011
  7. Kawamura, M., T. Uchiyama, T. Kuramoto, Y. Tamura and K. Mizutani. 1989. Formation of a cycloinulooligosaccharide from inulin by an extracellular enzyme of Bacillus circulans OKUMZ31B. Carbohydr. Res. 192, 83-90 https://doi.org/10.1016/0008-6215(89)85167-5
  8. Kawamura, M. and T. Uchiyama. 1993. Reaction catalyzed by cycloinulooligosaccharide fructanotransferase. Biosci. Biotechnol. Biochem. 57, 343-350 https://doi.org/10.1271/bbb.57.343
  9. Kawamura, M., H. Nakai, T. Uchiyama, Y. Takai and M. Sawada.1997. Synthesis of methyl 6-O-${\beta}$-inulotriosyl-${\alpha}$ -D-glucopyranoside by intermolecular transglycosylation reaction of cycloinulo-oligosaccharide fructanotransferase, Carbohydr. Res. 297, 187-190 https://doi.org/10.1016/S0008-6215(96)00264-9
  10. Kim, D. H., Y. J. Choi, S. K. Song and J. W. Yun. 1997 . Production of oligo- saccharides using endo-inulinase from Pseudomonas sp. Biotechnol. Lett. 19, 369-371 https://doi.org/10.1023/A:1018311219788
  11. Kim, H. C., J. H. Kim, S. J. Jeon, W. B. Choi and S. W. Nam. 2005. Expression of Paenibacillus macerans Cycloinulooligosaccharide Fructanotransferase in Saccharomyces cerevisiae. J. Life Sci. 15, 317-322 https://doi.org/10.5352/JLS.2005.15.3.317
  12. Kim, H. Y. and Y. J. Choi. 2001. Molecular characterization of cycloinulo- oligosaccharide fructanotransferase from Bacillus macerans. Appl. Environ. Microbiol. 67, 995-1000 https://doi.org/10.1128/AEM.67.2.995-1000.2001
  13. Kim, K. Y., M. D. Kim, N. S. Han and J. H. Seo. 2002. Display of Bacillus macerans cyclodextrin glucanotransferase on cell surface of Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 12, 411-416
  14. Kondo, A. and M. Ueda. 2004. Yeast cell-surface display-applications of molecular display. Appl. Microbiol. Biotechnol. 64, 28-40 https://doi.org/10.1007/s00253-003-1492-3
  15. Lipke, P. Nand J. Kurjan. 1992. Sexual agglutination in budding yeasts: Structure, function, and regulation of adhesion glycoproteins. Microbiol. Rev. 56, 180-194
  16. Little, M., P. Fuchs, F. Breitling and S. Dubel. 1993. Bacterial surface presentation of proteins and peptides: and alternative to phage technology. Trends Biotechnol. 11, 3-5 https://doi.org/10.1016/0167-7799(93)90067-J
  17. Ohta, K., S. Hamada and T. Nakamura. 1993. Production of high concentrations of ethanol from inulin by simultaneous saccharification and fermentation using Aspergillus niger and Saccharomyces cerevisiae. Appl. Environ. Microbiol. 59, 729-733
  18. Romanos, M. A., C. A. Scorer and J. J. Clare. 1992. Foreign gene expression in yeast : a review. Yeast 8, 423-488 https://doi.org/10.1002/yea.320080602
  19. Sawada, M., T. Tanaka, Y. Takai, T. Hanafrsa, T. Taniguchi, M. Kawamura and T. Uchiyama. 1991 The srystal structure of cyclo-inulohexaose produce from inulin by cycloinulooligosaccharide fructanotransferase. Carbohydr. Res. 217, 7-17 https://doi.org/10.1016/0008-6215(91)84112-R
  20. Schmid, G. 1989. Cyclodextrin glycosyltransferase production; yield enhancement by overexpression of cloned genes. Trends Biotechnol. 7, 244-248 https://doi.org/10.1016/0167-7799(89)90015-2
  21. Schreuder, M. P., A. T. Mooren, H. Y. Toschka, C. T. Verrips and F. M. Klis. 1996. Immobilizing proteins on the surface of yeast cells. Trends Biotechnol. 14, 115-120 https://doi.org/10.1016/0167-7799(96)10017-2
  22. Schreuder, M. P., C. Deen, W. J. A. Boersma, P. H. Pouwels and F. M. Klis. 1996. Yeast expressing hepatitis B virus surface antigen determinants on its surface: implications for a possible oral vaccine. Vaccine 14, 383-388 https://doi.org/10.1016/0264-410X(95)00206-G
  23. Takai, Y., Y. Okumura, S. Takahashi, M. Sawada, M. Kawamura and T. Uchiyama. 1993. A permethylated cyclic fructo-oligosaccharide host that can bind cation in solution. J. Chem. Soc. Chem. Commun. 1, 53-54
  24. Uchiyama, T., M. Kawamura, T. Uragami and H. Okuno. 1993. Complexing of cycloinulo- oligosaccharides with metal ions. Carbohydr. Res. 241, 245-248 https://doi.org/10.1016/0008-6215(93)80111-Q
  25. Vandamme, E. J. and D. G. Derycke. 1983. Microbial inulinase: fermentation process, properties, and applications. Adv. Appl. Microbiol. 29, 139-176 https://doi.org/10.1016/S0065-2164(08)70356-3
  26. Valentin, E., E. Herrero, J. F. I. Pastor and R. Sentandreu. 1984. Solubilization and analysis of mannoprotein molecules from the cell wall of Saccharomyces cerevisiae. Arch. Microbiol. 130, 1419-1428 https://doi.org/10.1099/00221287-130-6-1419
  27. van der Vaart, J. M., L. H. P. Caro, J. W. Chapman, F. M. Klis and C. T Verrips. 1995. Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J. Bacteriol. 177, 3104-3110 https://doi.org/10.1128/jb.177.11.3104-3110.1995
  28. Washida M., S. Takahachi, M. Ueda and A. Tanaka. 2001. Spacer-mediated display of active lipase on the yeast cell surface. Appl. Microbiol. Biotechnol. 56, 681-686 https://doi.org/10.1007/s002530100718