Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Expression of the Bacillus stearothermophilus NO2 CGTase gene in Saccharomyces cerevisiae

Saccharomyces cerevisiae 내에서 Bacillus stearothermophilus NO2 CGTnse 유전자의 발현

  • 유동주 (동의대학교 미생물학과) ;
  • 박현이 (동의대학교 미생물학과) ;
  • 전숭종 (일본산업기술종합연구소) ;
  • 권현주 (미쯔비시 화학생명과학연구소) ;
  • 남수완 (동의대학교 미생물학과) ;
  • 김병우 (동의대학교 미생물학과)
  • Published : 2002.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

For the expression of CGTase gene(cgtS) kom Bacillus stearothemophilus NO2 in Saccharomyces cerevisiae, cgtS gene was subcloned into the Eschepichia coll-yeast shuttle vector, pVT103-U. The constructed plasmid, pVT-CGTS was introduced to 5. cemi-siae 2805 cell, and then the cgtS gene under the control of adhl promoter was successfully expressed in the yeast transformant and 87% of the total activity was detected into the fermentation medium. Therefore, the signal peptide of B. stearothemephilus NO2 CeTase showed high secretion efficiency in 5. cerevisiae. Optimal conditions of the recombinant yeast cell f3r expression of CGTase was achieved, when 5. cerevisiae 2805/pv7-CGTS was cultivated on YP medium at 2% dextrose, pH 5.5,$30^{\circ}C$ and the expression level of CGTase was 0.624units/mL for 48 h culture.

Bacillus stearothermophilus의 CCTase 유전자(cgtS) 대장균과 효모의 shuttle vector로서 항구적 promoter인 adh l promoter를 함유한 pVT103-U(6.9Kb)에 도입하여 재조합 plasmid pVT-CCTS (9.0Kb)을 구축하고 효모 숙주 S. cerevisiae 2805에서 발현시켰다. 재조합 균주의 항구적 발현계인 2805/pv7-CGTS의 최적 발현조건은 YP배지에 dextrose 2%, pH 5.5, 30"C에서 최적 발효조건이었으며, CCTase의 최대 발현량은 48시간 배양시 0.624unit/mL을 나타내었다. B. stearothermophilus의 signal peptide가 재조합 효모에서도 높은 분비효율을 나타내어서 발현된 효소의 87%가 세포 외로 분비 생산되었다.산되었다.

Keywords

References

  1. Proc. Natl. Acad. Sci. v.69 Genetic transformation of Escherichia coli by R factor DNA Cohen,S.N.;A.C.Y.Chang;L.Hsu https://doi.org/10.1073/pnas.69.8.2110
  2. Methods in Cell Biology v.12 Cryer,D.R.;R.Eccleshall;J.Marrnur;D.M.Prescott https://doi.org/10.1016/S0091-679X(08)60950-4
  3. Bio. Technol. v.9 Fermentation on a yeast producing A. niger glucose oxidase: scale-up, Purification and characterization of the recombinant enzyme De Baetselier,A.;A.Vasavada;P.Dohet;V.Ha-Thi;M.De Beukelaer;T.Erpicum;L. De Clerck;J.Hanotier;S.Rosenberg https://doi.org/10.1038/nbt0691-559
  4. J. Microbiol. Biotechnol. v.3 Selection and characterization of catabolite repression resistant mutant of Bacillus firmus var. alkalophillus producing cyclodextrin glucanotransferase Do, E. J.;H. D. Shin;C. Kim;Y. H. Lee
  5. J. Ferment. Bioeng. v.70 Purification and properties of cyclodextrin glucanotransferase from Bacillus sp. AL-6 Fujita, Y.;H. Tsubouchi; Y. Inagi;K. Tomita;A. Ozaki;K. Nakanishi https://doi.org/10.1016/0922-338X(90)90174-U
  6. Appl. Environ. Microbiol. v.58 Cyclization characteristics of cyclodextrin glucanotransferase are conferred by the $NH_2$-terminal region of the enzyme Fujiwara,S.;H.Kakihara;B.W.Kim;A.Leujeune;M.Kanemoto;K.Sakaguchi;T.Imanaka
  7. J. Jpn. Soc. Starch Sci. v.3 Application of Cyclodextrin Hara, K.;H. Hashimoto
  8. J. Bacteriol. v.153 Transformation of intact yeast cells treated with alkali cations Ito, H.;Y. Fukuda;K. Murata;A. Kimura
  9. J. Biol. Chem. v.265 Characterization of a nonglycosylated single chain urinary plasminogen activator secreted from yeast Melnick, L. M.;B. G. Turner, P.;Puma, B.;Price-Tillotson;K. A. Salvato;D. R. Dumais;D. T.;Moir;R. J. Broeze;G. C. Avgerinos
  10. Anla. Chem. v.55 Use of dinitrosalicylic acid reagent for determination of reducing sugar Miller,G.L.
  11. Biotech. Lett. v.15 Secretion and localization of invertase and inulinase in recombinant Saccharomyces cerevisiae Nam, S. W.;K. Yoda;M. Yamasaki https://doi.org/10.1007/BF00129936
  12. Denpun Kagaku v.39 Theroanaerobacter sp. CGTase: Its properties and Application Norman,B.E.;S.T.Jogensen
  13. Molecular cloning: a laboratory manual(2nd ed.) Sambrook, J.;E. F. Fritsch;T. Maniatis
  14. Cyclodextrin Technology Szejtli,J.
  15. Gene v.52 A family of yeast expression vectors containing the Phage fl intergenic region Vernet, T.;D. Dignard;D. Y. Themas https://doi.org/10.1016/0378-1119(87)90049-7
  16. Biotehcnol. Bioeng. v.42 Improved protein synthesis sand secretion through medium enrichment in a stable recombinant yeast strain Wang, Z.;N. A. Da Silva https://doi.org/10.1002/bit.260420113