Cloning and Expression of an $\alpha$-Amylase Gene from Bacillus circulans in B. subtilis and B. megaterium

Bacillus circulans $\alpha$-amylase 유전자의 Basillus subtilis와 Bacillus megaterium에서의 클로닝 및 발현

  • 이동석 (인제대학교 의생명공학대학 임상병리학과) ;
  • 김지연 (인제대학교 생물건강산업유성지원 센터) ;
  • 김한복 (호서대학교 자연과학부 생명과학전공)
  • Published : 2000.09.01


A Baczllus circdans KCTC3004 $\alpha$-amylase gene contained in a recombinant plasmid pAL850 was transferred into a new shuttle vector plasmid pALSIlI by ligating linearlzed DNAs of pUC19 and pUB110. B. subtilis RM125 and B. megatenurn ATCC14945 transfonned with pALS111 produced the $\alpha$-amylase substantially Most of the enzyme was produced during the exponential growth period. The maxiinurn activities of the $\alpha$-amylase produced by the Bucillus transformants were compared with that of the B. circulans gene donor strain. The B. subtilis RM125(pALS111) enzyme showed the actlvicy 95 times higher than that of the gene donor cells, followed by the B, nzegaterium ATCC14945(pALSlll) enzyme with activity 34 limes higher than that of the gene donor cells. While E coli secreted about 10% of the produced enzyme, B. subtilis excreted the enzyme inlo the medium wholly and B. megaterirun about 98% ofthe total product. The plasmid pALSI11 was quite stable inB. nzegaterium (92%), inoderately stable in B. subtilis (76%), but was unstable in E. coli (38%). The SDS-PAGE and zymogram of this enzyme produced in E. coli(pALS111), B. subtilis( pALS111) or B. megateril~m (pALS111) indicated a molecular weight of 55,000. The enzymes overproduced in three different host cells hydrolyzed starch to produce mainly maltoaiose and mallooligosaccharides.


$\alpha$-amylase;Bacillus circulans;B. megaterium;B. subtilis;shuttle vector;stability


  1. Meth. Enzymol. v.68 Plasmid of Escherichia coli as cloning vectors Bolivar,F.;K.Backman
  2. Mol. Gen. Genet. v.168 High frequency transformation of Bicillus subtilis protoplasts by plasmid DNA Chang,S.;S.N.Cohen
  3. Crit. Rev. Biochem. Mol. Biol. v.24 Microbial amylolytic enzymes Vihinen,M.;P.Mantsala
  4. Proc. Natl. Acad. Sci. U.S.A. v.74 Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis Ehrlich,S.D.
  5. Anal. Chem. v.31 Use of dinitrosalicylic acid reagent for determnation of reducing sugar Miller,G.L.
  6. J. Bacteriol v.142 Plasmid-mediated transformation in Bacillus megaterium Brown,B.J.;B.C.Carlton
  7. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacterophage T4 Laemmli,U.K.
  8. Restriction maps of plasmids pUB110 and pBD9, Gene v.14 Jalanko,A.;I.Palva;H.Soderlund
  9. DNA cloning v.Ⅰ Techniques for transformation of Escherchia coli Hanahan,D.;D.M.Glover(ed.)
  10. Bacillus. Bacteriol. Rev. v.41 Extracellular enzyme synthesis in the genus Priest,F.G.
  11. Kor. J. Microbiol. v.36 Clomng and expression of an acidophilic α-amylase gene from Bacillus crrculans in Escherchia coli Lee,J.S.;J.Y.Kim;H.B.Kim;D.S.Lee
  12. J. Appl. Chem. Biotech. v.25 Kinetics of utilization of highly polymerized carbon source(starch)in a chemostat cluture of Klebsnella aerogenes pullulanase and α-amylase activities Hemandez,E.;S.J.Pilt
  13. In Advances in Carbohydrate Chemistry v.23 Starch degrading and synthesizing enzymes Greenwood,C.T.;E.A.Milne