Bacillus megaterium KSM B-404으로부터 생산되는 Maltopentaose생성 Amylase의 정제 및 특성

Purification and Characterization of a Maltopentaose-producing Amylase from Bacillus megaterium KSM B-404.

  • 발행 : 2002.12.01

초록

토양으로부터 maltopentaose생산성 amylase를 분비하는 세균 KSM B-404를 분리하여 그의 형태적, 생리적인 특성을 고려한 결과 Bacillus megaterium으로 동정되었다. 효소는 ($NH_4$)$_2$$SO_4$ 침전 분획, DEAE-Toyopearl 및 Superdex 75 HR 10/30 크로마토그래피로 129배 정제되었으며 21.4%의 활성이 회수되었다. 정제된 효소를 SDS-PACE로 분석한 결과 분자량은 약 68 kDa이었고, 최적 반응 온도는 $50^{\circ}C$이며 $Ca^{2+}$ 이온의 존재 시 열 안정성이 증가하였다. 한편 최적 반응 pH는 6.0~7.0부근이며 알칼리 조건에서도 안정하였다. 또한 효소의 활성은 $Cu^{2+}$ , $Hg^{2+}$ 그리고 특히 Fe/eup 3+/이온 등의 금속이온에 의해 강하게 저해 받았고 acetic anhydride, EDTA , hydroxylamine-HCI, $\rho$ - chloromercuribenzoate 등의 저해제에 의해서 활성이 저해되었으나 concanavalin A에 의한 저해 효과는 나타나지 않았다. 전분의 가수분해 산물을 HPLC로 분석한 결과 maltopentaose가 주산물로 나타났으며 반응 24시간 후 총 가수분해 산물의 약 52%를 차지하였다.

An amylase that hydrolyzes starch into maltopentaose as a main product was found in the culture supernatant of a strain of Bacillus megaterium KSM B-404 isolated from local soil. The enzyme was purified 129-fold by ammonium sulfate precipitation, DEAE-Toyopearl and Superdex 75 HR 10/30 column using a FPLC system. The molecular weight of the amylase was determined as about 68 kDa by using SDS-PAGE. Optimum pH and temperature of amylase were found to be $50^{\circ}C$ and pH 6.0~7.0, respectively. The enzyme was stable up to $60^{\circ}C$ by addition of $Ca^{2+}$ and its pH stability was in the range of 6.0~10.0. The activity of enzyme was inhibited by $Cu^{2+}$ $Hg^{2+}$ , and $Fe^{3+}$ and maintained by $Ca^{2+}$ and $Mg^{2+}$ . EDTA and pCMB also showed inhibitory effect to the enzyme. TLC and HPLC analysis of the products of the enzyme reaction showed the presence of maltopentaose(52%), maltotriose (25%), maltose (11%), glucose, and maltotetraose in the starch hydrolysates.

키워드

참고문헌

  1. Anal. Biochem. v.72 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing for the principle of protein-dye binding Bradford,M.M. https://doi.org/10.1016/0003-2697(76)90527-3
  2. Kor. J. Food Sci. Technol. v.26 Characterization of Streptomyces sp. KSM-35 and purification of its maltotertaose forming amylase Cha,J.;Y.B.Kim;B.C.Seo;K.H.Park
  3. Appl. Environ. Microbiol. v.59 Regulation and cloning of the gene encoding amylase activity of the ruminal bacterium Streptococcus bovis Cotta,M.A.;T.R.Whitehead
  4. Appl. Environ. Microbiol. v.63 Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing α-amylase from Streptococcus bovis Eiichi,S.;T.Uchimura;T.Kudo;K.Komagata
  5. Microbial Enzymes and Biotechnology(2nd ed) Recent advances in microbial amylase Fogarty,W.M.;C.T.Kelly;W.M.Fogarty(ed.);C.T.Kelly(ed.)
  6. Appl. Environ. Microbiol. v.36 A novel maltohexaose-forming amylase from Bacillus caldovelox: Patterns and mechanisms of action Fogarty,W.M.;F.Bealin-Kelly;C.T.Kelly;E.M.Doyle
  7. Biotechnol. Lett. v.16 Extracellular maltotetraose-forming amylase of Pseudomonas sp. IMP 353. Fogarty,W.M.;C.T.Kelly;A.C.Bourke;E.M.Doyle https://doi.org/10.1007/BF01023328
  8. J. Bacteriol. v.166 Structural genes encoding the thermophilic α-amylases of Bacillus stearothermophilus and Bacillus licheniformis Gray,G.L.;S.E.Mainzer;M.W.Rey;M.H.Lamsa;K.L.Kindle;C.I.Carmona;C.Pequart https://doi.org/10.1128/jb.166.2.635-643.1986
  9. J. Biol. Chem. v.267 Catalytic properties of the cloned amylase from Bacillus licheniformis Kim,I.C.;J.H.Cha;J.R.Kim;S.Y.Jang;B.C.Seo;T.K.Cheong;D.S.Lee;Y.D.Choi;K.H.Park
  10. Bioindustry News v.6 The developing state and the view of microbial industry: teh sate and the view of microbial enzymes production Kim,C.S.;H.W.Seo
  11. M.S.thesis, Korea Univ. Characterization of Maltopentaose-producing Amylase from Bacillus megaterium KSM B-404 Kim,B.J.
  12. Kor. J. Biotechnol. Bioeng. v.16 Isolation of Bacillus sp. AIR-5 producing maltopentaose forming amylase and optimization of maltopentaose production Kim,Y.M.;E.S.Seo;D.Kim;D.W.Kim;J.H.Lee;A.Kimura
  13. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Laemmli,U.K. https://doi.org/10.1038/227680a0
  14. J. Biol. Chem. v.262 Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluroide membranes Matsudaira,P.
  15. Anal. Chem. v.31 Use of dinitrosalicylic acid reagent for determination of reducing sugar Miller,G.L. https://doi.org/10.1021/ac60147a030
  16. J. Agric. Food Chem v.46 Cloning of novel maltooligosaccha-ride-producing amylases as anti-staling agents for bread Min,B.C.;S.H.Yoon;J.W.Kim;Y.W.Lee;Y.B.Kim;K.H.Park https://doi.org/10.1021/jf970755y
  17. Gene v.15 Nucleotide sequence of the promoter and NH₂-terminal signal peptide region of the α-amylase gene from Bacillus amyloliquefaciens Palva,I.;R.F.Petersson;N.Kalkkinen;P.Lehtovara;M.Sarvas;H.Soderlund;K.Takkinen;L.Kaarianinen https://doi.org/10.1016/0378-1119(81)90103-7
  18. Food Sci. and Technol. v.25 The development of new carbohydrate materials Park,K.H.
  19. Clin. Chem. v.22 A new enzymatic kinetic method for determination of α-amylase Pierre,K.J.;K.K.Tung;H.Nadj
  20. Arch. Biochem. Biophys. v.145 Isolation, purification and characterization of a maltotetraose-producing amylase from Pseudomonas stutzeri Robyt,J.F.;R.J.Ackerman https://doi.org/10.1016/0003-9861(71)90015-4
  21. Arch. Biochem. Biophys. v.155 A thermophilic extracellular α-amylase from Bacillus licheniformis Saito,N. https://doi.org/10.1016/0003-9861(73)90117-3
  22. Biosci. Biotech. Biochem. v.56 Cloning and nucleotide sequence of malto-pentaose-forming amylase gene from Pseudomonas sp. KO-8940. Shida,O.;T.Takano;H.Takagi;K.Kadowaki;S.Kobayashi https://doi.org/10.1271/bbb.56.76
  23. The Prokaryotes(2nd ed.), Vol. 2 The genus Bacillus-nonmedical Slepecky,R.A.;H.E.Hemphill;A.Balows(ed.);H.G.Truper(ed.);M.Dworkin(ed.);W.Harder(ed.);K.Schleifer(ed.)
  24. Bergey's Manual of Systematic Bacteriology v.2 Endospore forming gram positive rods and cocci Sneath,P.H.A.(ed.);N.S.Mair(ed.);M.E.Sharpe(ed.);J.G.Holt(ed.)
  25. Agric. Biol. Chem. v.46 Production of maltohexaose by amylase from Bacillus circulans G-6 Takasaki,Y. https://doi.org/10.1271/bbb1961.46.1539
  26. Agric. Biol. Chem. v.49 An amylase producing maltotriose from Bacillus subtilis Takasaki,Y. https://doi.org/10.1271/bbb1961.49.1091
  27. Agric. Biol. Chem. v.53 Novel maltose-producing amylase from Bacillus megaterium G-2 Takasaki,Y. https://doi.org/10.1271/bbb1961.53.341
  28. Agric. Biol. Chem. v.55 Maltotetraose-producing amylase from Bacillus sp. MG-4 Takasaki,Y.;H.Shinohara;M.Tsuruhisa;S.Hayashi;K.Imada https://doi.org/10.1271/bbb1961.55.1715
  29. Handbook of Amylases and Related Enzymes Yamamoto,T.
  30. Agric. Biol. Chem. v.49 Characterization of maltopentaose-producing bacterium and its cultural conditions Yoshigi,N.;T.Chikano;M.Kamimura https://doi.org/10.1271/bbb1961.49.2379