Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Production of Cyclofructan by Cycloinulooligosaccharide Fructanotransferase Expressed in Saccharomyces cerevisiae.

Saccharomyces cerevisiae에서 발현된 Cycloinulooligosaccharide Fructanotransferase을 이용한 Cyclofructan의 생산

  • 임채권 (동의대학교 미생물학과) ;
  • 김현철 (동의대학교 바이오물질제어학과) ;
  • 김광현 (동의대학교 미생물학과) ;
  • 김병우 (동의대학교 미생물학과) ;
  • 남수완 (동의대학교 생명공학과)
  • Published : 2004.03.01
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Abstract

The cycloinulooligosaccharide fructanotransferase(CFTase) gene (cft) from Paenibacillus polymyxa was subcloned into the E. coli-yeast shuttle vector, pYES2.0 (GALI promoter). The constructed plasmid, pYGCFT (9.9 kb) was introduced into S. cerevisiae SEY2102 cell and then the yeast transformant was selected on the synthetic defined media lacking uracil Based on the cyclofructan(CF) spots on thin-layer chromatogram, the gene under the control of GALI promoter was successfully expressed in the yeast transformant. The recombinant CFTase was not secreted into the medium and was predominantly localized in the periplasmic space. CF was started to be produced after 3h of enzymatic reaction with inulin. The pH and temperature optimum for the CF production from inulin was pH 8.0 and 45$^{\circ}C$, respectively. Enzyme activity was stably maintained up to the pH of 10.0. The examination of the inulin sources revealed that a dahlia tuber and Jerusalem artichoke were the best for the production of CF.

Paenibacillus polymyxa 유래의 cycloinulooligosaccharide fructanotransferase(CFTase) 유전자(cft)를 Saccharomyces cerevisiae SEY2102 에 발현시키기 위해 대장균과 효모의 shuttle vector인 pYES 2.0(GALI) promoter)에 subcloning하였다. 구축된 pYGCFT(9.9kb) plasmid를 S. cerevisiae SEY2102에 형질전환하였고, uracil이 결핍된 SD 배지에서 선별하였다. 선별된 형질전혼체(S. cerevisiae SEY2102/pYGCFT)는 galactose 첨가에 의해 성공적으로 발현되어 cyclofructan(CF)을 생셩함을 TLC로 확인하였다. 그러나 균체외로의 효소 분비는 이루어지지 않았고 cytoplasm 보다 periplasmic space에 많이 존재하였다. 효소반응 3시간부터 CF가 생성됨을 확인하였고, 최적온도와 pH는 각각 45$^{\circ}C$와 pH 8.0로 나타났으며, pH 10.0에서도 효소활성이 안정적으로 유지되었다. Inulin 기질에 따른 반응산물 분석결과, Jerusalem artichoke와 dahlia tuber로부터 CF가 가장 효과적으로 생성되었다.

Keywords

References

  1. Enz. Microbial. Technol. v.13 Cultivation and utilization of jerusalem artichkoke for ethanol, single cell protein, and high fructose syrup production Bajpai,P.K.;P.Bajpai
  2. Proc. Natl. Acad. Sci. v.80 An Ma$\'{a}$l-SUC2 (α-factor-invertase) gene fusion for study of protein localization and gene expression in yeast Emr,S.D.;R.Schekman;M.C.Flessel;J.Thorner
  3. J. Microbiol. Biotechnol v.4 Secretion of a Bacillus endoglucanase in Saccharomyces cerevisiae by its own signal sequence Han,Y.J.;D.O.Kang;S.C.Lee;B.Y.Kim;H.H.Suh;J.M.Kim;T.I.Mheen;J.S.Ahn
  4. J. Chem. Soc v.72 Studies on fructosans Hirst,E.L.;D.J.Mcgilvary;E.G.V.Percival
  5. J. Bacteriol. v.153 Transformation of intact yeast cells treated with alkali cations Ito,H.;Y.Fukuda;K.Murata;A.Kimura
  6. J. Microbiol. Biotechnol v.12 Cloning and characterization of cycloinulooligosaccharide fructanotransferase(CFTase) from Bacillus polymyxa MGL21 Jeon,S.J.;d.J.You;H.J.Kwon;s.Kanaya;N.Kunihiro;K.H.Kim;Y.H.Kim;B.W.Kim
  7. Appl. Environ. Microbiol. v.63 Recombinant thermostable cycloinulo-oligosaccharide fructanotransferase produced by Ssccharomyces cerevisiae Kanai,T.;N.Ueki;T.Kawaguchi;Y.Teranishi;H.Atomi;C.Tomorbaatar;M.Ueda;A.Tanaka
  8. Carbohydr. Res. v.192 Formation of cycloinulo-oligosaccharide from inulin by an extracclular enzyme of Bacillus circulans OKUMZ31B Kawamura,M.;T.Uchiyama;K.Takashi;T.Yukiyoshi;M.Kenji
  9. Carbohydr. Res. v.260 Purification and some properties of cycloinulooligosaccharide fructanotransferase from Bacillus circulans OKUMZ31B Kawamura.M.;T.Uchiyama
  10. Biosci. Biotechnol. Biochem. v.57 Reaction catalyzed by cycloinulooligosaccharide fructanotransferase Kawamura,M.;T.Uchiyama
  11. Appl. Environ. Microbiol v.67 Molecular characterization of cycloinulooligosaccharide fructanotransferase from Bacillus macerans Kim.H.Y.;Y.J.Choi
  12. Annu. rev. Biochem v.54 Assembly of asparaginelinked oligosaccharides Kornfeld,R.;s.Komfeld
  13. Biosic. Biotechnol. Biochem v.58 Purification and catalyztion of cycloinulooligosaccharide fructanotransferase (CFTase) Kushibe,S.;K,Mitsui;M.Yamagishi;K.Yamada;Y.Morimoto
  14. Anal. Chem. v.55 Use of dinitrosalicylic acid teagent for determination of reducing sugar Miller,G.L.
  15. Biotechnol. Lett. v.23 Expression of Bacillus macerans cyclodextrin glucanotransferase gene in Saccharomyces cerevisiae Nam,S.W.;H.Y.Park;J.H.Kim;J.H.Seo;N.S.Han;B.W.Kim
  16. Biotechnol. Lett. v.15 Secretion and localization of invertase and inulinase in recombinant Saccharomyces cerevisiae Nam,S.W.;K.Yoda;M.Yamasaki
  17. Carbohydr. Res. v.217 The crystal structure of cycloinulohexaose produced from inulin by cycloinulooligosaccharide fructanotransferase Sawada,M.;T.Tanaka;Y.Takai;T.Hanafrsa;T.Taniguchi;M.Kawamura;T.Uchiyama
  18. Trends Biotechnol. v.7 Cyclodextrin glycosyltransferase production;yield enhancement by overexpression of cloned genes Schmid,G.
  19. Yeast v.12 The targeting of Bacillus subtillis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N-terminus mature part Sotti,P.A.;M.Praestegaard,R.Chambert;M.R.PetitGlatron
  20. J. Chem. Soc. Chem. Commun. v.1 a permethylated cyclic fructo-oligosaccharide host that can bind cation in solution Takai,Y.;Y.Okumura;S.Takahashi;M.Sawada;M.Kawamura;t.Uchiyama
  21. Carbohydr. Res. v.241 Complexing of cycloinulo-oligosaccharides with metalions Uchiyama,T.;M.Kawamura;T.U.ragami;H.Okuno
  22. Carbohydr. Res. v.241 Complexing of cycloinulo-oligosaccharides with metal ions Uchiyama,T.;M.Kawamura;T.Uragami;H.Okuno