Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification of \alpha-Cyclodextrin Glucanotransferase Excreted from Themophilic Geobacillus thermosac-chalytycus and Characterization of Transglycosylation Reaction of Glucosides.

호열성 Geobacillus thermosacchalytycus가 생산하는 \alpha-Cyclodextrin Glucanotransferase의 분리정제와 당전이 반응 특성

  • 이미숙 (경북대학교 자연과학대학 유전공학과) ;
  • 신현동 (이엔지바이오㈜) ;
  • 김태권 (경북대학교 자연과학대학 유전공학과) ;
  • 이용현 (경북대학교 자연과학대학 유전공학과, 이엔지바이오㈜)
  • Published : 2004.03.01
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Abstract

$\alpha$-Cyclodextrin glucanotransferase excreted from a newly isolated Geobacillus thermosacchalytycus was purified through the ultrafiltraion, hydrophobic Sepharose CD-4B affinity chromatography, and gel filtration on Sephadex G-75, respectively. The molecular weight of the purified CGTase was 69 kDa and its N-terminal amino acid sequence was determined to be Asn-Leu-Asn-Lys-Val-Asn-Phe-Val-Ser-Asp-Val-Val-Val-Gln-Ile. The optimum pH and temperature were pH 6.0 and$ 60^{\circ}C$, respectively, and stably at the pH range of 6.0-8.0 and $60^{\circ}C$ in the presence of $Ca^{++}$. The excreted CGTase from the thermophilic G. thermosacchalytycus was $\alpha$-type showing a high coupling activity for the transglycosylation on various glucosides. The coupling reaction was carried out according to the random ternary complex mechanism.m.

토양으로부터 CGTase를 생산하는 새로운 호열성 세균을 분리하였으며, 형태 및 생리적 특성과 16S ribosomal DNA의 염기서열을 분석한 결과 Geobacillus thermosacchalytycus 로 동정하였다. 호열성 G. thermosacchalytycus가 분비하는 CGTase를 한외여과, 소수성 Phenyl Sephadex CL-4B 클로마토그래피 , 그리고 gel filtration의 순서로 분리 정제하여 정제도 28.2배, 정제수율 12.2%, 그리고 specific activity가 4952.5 units/mg인 CGTase를 얻을 수 있었다. 정제된 CGTase의 분자량은 69,000 dalton이었고, terminal amino sequence는 Asn-Leu-Asn-Lys-Val-Asn-Phe-Thr-Ser-Asp-Val-Val-Tyr-Gln-Ile이었다. 최적 pH 및 온도는 각각 pH 6와$ 50^{\circ}C$였고, pH 6-8과 $60^{\circ}C$에서 장기간 보존할 수 있는 중성 pH의 내열성 효소임을 알 수 있었다. 내열성 CCTase의 cyclization과 coupling 반응의 특정을 검토한 결과 G. thermosacchalytycus가 생산하는 내열성 효소는 $\alpha$-type CGTase이었고, cyclization 반응보다는 coupling반응에 적합한 당전이성이 높은 효소임을 알 수 있었다. 당전이 반응에서 기질 특이성을 검토한 결과 당공여체로는 가용성 전분 그리고 당수용체로는 배당체인 stevioside에 대해 높은 기질 특이성을 보였다. 당전이 반응은 당수용체와 당공여체가 효소와 무작위로 결합하여 진행되는 random ternary complex mechanism으로 반응이 수행되었다.

Keywords

References

  1. Kor. J. Appl. Microbiol. Biotechnol. v.29 Production of 2-O-α-D-glucopyranosyl L-ascorbic acid by cyclodxtrin glucanotransferase from Paenibacillus sp. JB-13 Bae,K-M.;Y.kang;H-K.Jun
  2. J. Agric. Food Chem. v.46 Characterization of a thermostable cyclodextrin glucanotransferase isolated from bacillus stearothermophilus ET1 Chung,H.C.;S.H.Yoon;M.J.Lee;M.J.Kim;K.Kwon;I.W.Lee;J.W.Kim;B.H.Oh;H.S.Lee;V.A.Spiridonova;K.H.Park
  3. Kor. J. Appl. Microbiol. Biotechnol. v.18 Purification and characterization of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. Chung,Y.J.;I.S.Kong;Y.S.Kang;J.H.Yu
  4. J. Microbiol. Biotechnol v.3 Selection and characterization of catabolite repression rresistant mutant of Bacillus firmus var alkalpphilus producting cyclodextrin glucanotransferase Do,E.J.;H.D.Shin;C.Kim;Y.H.Lee
  5. J. Bacteriol. v.174 Analysis of mutantions in cyclodextrin glucanotransferase from Bacillus stearothermophilus which affect cyclization characteristics and thermostability Fujiwara,S.;H.Kakihara;K.Sakaguchi;T.Imanaka
  6. J. Ferment. Bioeng. v.70 Purification and properties of cyclodextrin glucanotansferase from Bacillus sp. AL-6 Fujita,Y.;H.Tsubouchi;Y.Inagi;K.Tomita;A.Ozaki;K.Nakanishi
  7. FEMS Microbiol. Lett. v.164 Cloning and sequencing of a cyclodextrin glucanotransferase gene from Brevibacillus brevis CD 162 and its expression in Escherichia coli Kim,M.H.;C.B.Sohn;T.K.Oh
  8. Agric. Chem. Biotechnol v.40 purification and characterization of cyclodextrin glycosyltansferase from Bacillus brevis CD162 Kim,M.H.;C.B.Sohn;Y.H.Lim;T.K.Oh
  9. J. Biochem. v.79 Studies on cyclodextrin glycosyltransferase?. Enzymatic synthesis of 3-O-α-D-glucopyranosyl-D-xylose using cyclodextring glycosyltransferase Kitahata,S.;S.Okada
  10. J. Ferment. Bioeng. v.74 Cloning and sequencing of the gene encoding cyclodextrin glucanotransferase from Bacillus sp. KC201 Kitamoto,N.;T.Kimura,Y.Kito,K.Ohmiya
  11. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophange T4 Laemmli,U.K.
  12. Eru. J. Biochem v.270 Thermoanaerobacterium themosulfurigenes cyclodextrin glycosyltransferase Leemhuis,H.;B.W.dijkstra;L.dijkhuizen
  13. Enzme Microbiol. Technol v.14 Enzymatic production of α-cyclodextrin with the cyclodextrin glucanotransferase of Klebsiella oxytoca 19-1 Lee,J.H.;K.H.Choi;J.Y.Choi;Y.S.Lee;I.B.Kwon;J.H.Yu
  14. Enzyme. Microb. Technol. v.30 A new cyclodextrin glucanotransferase from an alkaliphilic Bacillus agaradhaerens isolate: purivication and characterization Martins,R.F.;R.H.Kaul
  15. FEMS Letters v.337 The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a ping-pong mechanism Nakamura,A.;K.Haga;K.Yamane
  16. Denpun Kagaku v.39 Thermoanaerobacter sp. CGTTase: Its properties and application Norman,B.E.;F Fritsch;T.Maniatis
  17. Denpun Kagaku v.39 Thermoanaerobacter sp. CGTase: its properties and application Norman,B.E.;Jorgensen,S.T.
  18. In Proc. of International Symposium and 1997 Fall Scientific Meeting of KSAM Intermolecular transglycosylation reaction of cyclodextion glucanotransferase in the heterogeneous enzyme rection system Park,D.C.;T.K.Kim;Y.H.Lee
  19. Biochemistry v.34 Site-directed mutation in tyrosine 195 of cyclodextrin glucanotransferase of Bacillus circulans strain 251 affect activity and product specificity penninga,D.;B.Strokopytov;H.J.Rozeboom;C.L.Lawson;B.W.Dijkstra;J.Bergsma;L.Dijkhuizen
  20. Syst. Appl. Microbiol. v.16 Phylogenetic evidence for the relationship of Saccharococcus thermophilus to Bacillus thermoglucosidaisus, Bacillus Kaustophilus and Bacillus sterothermophilus Rainey,F.A.;E.Stackebrandt
  21. Enzyme Kinetics Segal.I.H.
  22. Biosci. Biotech. Biochem v.57 Enzymatic synthesis of novel trisaccharide, glucosyl lactoside Shibuya,T.;Y.Miwa;M.Nakano;T.Yamauchi;H.Chaen;S.Sakai;M.Kurimoto
  23. Appl. Microbiol. Biotechnol. v.35 Cloning and sequenciong of cyclodextrin glucanortansferase gene from Bacillus ohbensis and its expression in Escherichia coli Sin,K.A.;A.Nakamura;K.Kobayashi;H.Masaki;T.Uozumi
  24. FEMS Microbiol. Lett. v.172 Phylogenetic analysis of transformable strains of thermophilic Bacillus species Studholme,D.J.;R.A.Jackson;D.J.Leak
  25. Agric. Biol. Chem. v.55 Enzymatic formation of 4G-α-glucopyranosyl-rutin Suzuki,Y.;K.Suzuki
  26. J. Biochem. v.133 Biochemical and genetic analysis of a novel a-cyclodextrin glucanotransferase from an alkalophilic Bacillus clarkii 7364 Takada,M.;Y.Nakagawa;M.Tamamoto
  27. Biochim. Biophys. Acta. v.1543 Engineering of cyclodextrin glycosyltransferase reaction and product specificity van der Veen,B.A.;J.C.M.Uitdehaag;B.W.Kijkstar;L.Dijkhuizen
  28. Carbohydr. Res. v.223 Directed enzymatic synthesis of linear and branched glucooligosaccharides using cyclodextrin glucaosyltransferase Vetter,D.;W.Thorn;H.Brunner;W.A.King
  29. Biochemistry v.39 Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into mechanism of transglycosylation activity and cyclodextrin size specificity Uitdehaag,J.C.M.;G.W.M.Alebeek;B.A.Veen;L.Dijkhuizen;B.W.Dijkstra