Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression and Secretion of Serratia marcescens 58 KD Chitinase in Escherichia coli

대장균에서 Serratia marcescens 58KD 키티나아제의 발현과 분비

  • 장규일 (경상대학교 자연과학대학 미생물학과) ;
  • 강송옥 (경상대학교 자연과학대학 미생물학과) ;
  • 신용철 (경상대학교 식물분자생물학 및 유전자조작연구센터)
  • Published : 1992.10.01
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Abstract

We subcloned a 58 KD chitinase gene of Serratia marcescens into Escherichia coli and investigated the expression and secretion of the chitinase. Chitinase was produced in E. coli by using its own promoter but the levels of enzyme were very low, less than 5 mU/m$\ell$. However, by the combined action of the chitinase and lac promoters, the chitinase activity increased up to about 80 mU/m$\ell$. The most of the chitinase produced in E. coli was localized in periplasm and the small amounts were observed in cytosol and culture medium. Intracellular chitinase activities increased in proportion to the growth of E. coli up to the early stationary phase but rapidly decreased thereafter, which was assumed to be degradation of the chitinase by E. coli proteolytic enzymes.

Serratia marcescens ATCC 27117에서 부터 클로닝한 58KD 키티나아제 유전자를 subcloning 하여 2.6Kb DNA 삽입단편을 가진 플라스미드 pCHI26을 제조하고 대장균에서 발현과 분비를 살펴보았다. 키티니아제 유전자는 대장균에서 자신의 prmoter를 이용하여 매우 낮은 수준(<5mU/m$\ell$)으로 발현되었으며 lac promoter를 이용하는 경우 키티니아제 발현이 증가되어 약 80mU/m$\ell$가 되었다. 발현된 키티니아제는 거의 전적으로 대장균의 periplasm에 위치(약 87.8)하고 있었다. 배양시간에 따라서 세포내 키티니아제 활성을 측정해본 결과 초기정지기까지는 균체 성장과 비례해서 세포내 효소활성이 증가되었으나 정지기부터 세포내 효소활성이 급격히 줄어드는 양상을 보였다. 그러나 이 기간 동안 세포의 효소활성의 변화는 거의 없었다. 이러한 결과로 보아 periplasm에 위치한 키티나아제가 대장균의 단백분해효소에 의해서 분해되는 것으로 추정되었다.

Keywords

References

  1. Can. J. Microbiol. v.15 The chitinase of Serratia mareescens. Monreal,J.;E.T.Reese
  2. J Bacteriol v.170 Cloning of the genes of the chitin utilization regulon of Serratia liquefaciens Joshi,S.;M.Kozlowski;G.Selvaraj;V.N.lyer;R.W.Davies
  3. Chitin Chitosan and related enzymes. Cloning of Vibrio harvaeyi a chitinase genes in Excherichia coli Soto-Gil,R.W.;J.W.Zyskind;J.P.Zakikas(ed.)
  4. Appl. Environ. Microbiol v.52 Chitinase determinants of Vibrio vulnificus: gene cloning and applications of a chitinase probe Wortman,A.T.;C.C.Somerville;R.R.Colwell
  5. J. Bacteriol v.263 Cloning and expression of a Streptomyces plicatus chitinase(chitinase-63) in Escherichi coli Robbins,P.W.;C.Albright;B.Benfield
  6. J. Bacteriol v.172 Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degrdation Watanabe,T.;K.Suzuki;W.Oyanagi;K.Ohnishi;H.Tanaka
  7. J. General Microbiol v.145 Purification and some properties of two forms of chitinase from mycelial cells of Mucor rouxii Pedraza-Reyes,M.;E.Lopez-Romero
  8. J. Biol. Chem. v.257 Endochitinase a mannan-associated enzyme from Saccharomyces cerevisiae Correa,J.U.;N.Elango;I.Polacheek;E.Cabib
  9. Cellular and Molecular Biology of Plant Stress Induction of hydrolases as a defense reaction against pathogens Boller,T.;J.L.Key;T.Kosuge(ed.)
  10. Plant Physiol. v.93 Isolation and characterization of the genes encoding basic and acidic chitinase in Arabidopsis thaliana Samac,D.A.;C.M.Hironaka;P.E.Yallaly;D.M.Shan
  11. Planta v.182 The effect of ethylene on the cell-type specific and intracellular localization of β-1.3-glucanase and chitinase in tobacco leaves Keefe,D.;U.Hinz;F.Meins,Jr.
  12. Plant Physiol. v.89 Identification of several pathogenesis-related proteins in tomato leaves inoculated with Cladosporium fulvum(syn. Fulvia fulva) as 1.3-β-glucanases and chitinases Joosten,M.H.A.J.;P.J.G.M.De Wit
  13. J. Agric. Chem. Soc. Jpn. v.16 Enzymatic studies on exuvial fluid of Bombyx mori II. Chitinase Hamamura,Y.;Y.Kanehara
  14. Insect Biochem. v.13 Insect endochitinases: glycoproteins from moulting fluid, integument and pupal hemolymph Koga,D.;J.Jilka;K.J.Kramer
  15. Biochim Biophys. Acta v.44 Chitinase system from Carcinus maenas Lunt,M.R.;P.W.Kent
  16. Chitin in Nature and Technology Origin of chitinase activity in cod. Gadus morhua Danulat,E.;R.Muzzarelli;C.Jeuniaux;G.W.Gooday(ed.)
  17. Arch. Biochem. Biophys. v.272 Rat liver chitobiase: purification properties and role in the lysosomal degradation of asn-linked glycoproteins Aronson,N.N.Jr.;M.Backes,;M.J.Kuranda
  18. The EMBO Journal v.5 Isolation and charaterization of genes encoding two chitinase enzymes from Serratia marcescens Jones,J.D.G.;K.L.Grady;T.V.Suslow.;J.R.Bedbrook
  19. Appl. Environ. Microbiol v.51 Cloning of Serratia marcescens gene encoding chitinase Fuchs,R.L.;S.A.Mcpherson;D.J.Drahos
  20. Korean Biochem. J. v.24 Identification of five chitinase isozymes from Serratia marcescens Hwang,J.R.;S.W.Gal;K.A.Lee;Y.C.Shin;M.J.Cho;S.Y.Lee
  21. Kor. J. Appl. Microbiol. Biotechnol. v.20 Molecular clonining of Serratia marcescens chitinase gene into Escherichia coli Chang,K.I.;K.S.Kim;M.J.Cho;S.Y.Lee;Y.C.Shin
  22. Phytopathol v.78 The role of chitinase of Serratia marcescens in biocontrol of Sclerotium rolfsii Ordentlich,A.;Y.Elad;I.Chet
  23. J. General Microbiol. v.134 Plant and bacterial chitinases differ in antifungal activity Roberts,W.;C.P.Selitrennikoff
  24. Nucleic acids Res. v.17 Nucleotide sequence of the chitinas B gene of Serratia marcescens QMB 1466 Mark,H.H.;P.Dunsnueir
  25. Soil Sci. Soc. Am. Proc. v.26 Microbial processes associated with the use of chitin for biological control. Mitchell,R.;M.Alexander
  26. Phytopathol v.79 Control of plant diseases by chitinase expressed from cloned DNA in Escherichia coli Shapira,R.;A.Ordentlich;I.Chet;A.Oppenheim
  27. Plant Physiol. v.91 Bacterial chitinase is modified and secreted in transgenic tobacco Lund,P.;R.Y.Lee;P.Dunsmuir
  28. Molecular Cloning A laboratory Manual v.2 Sambrook,J.;E.F.Fritsch;T.Maniatis
  29. Proc. Natl. Acad. Sci. USA v.69 Nonchromosomal antibiotic resistance in bateria: genetic transformation of Excherichia coli by R Factro DNA Cohen,S.N.;A.C.Y.Chang;L.Hsu
  30. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Laemmli,U.K.
  31. Anal. Biochem. v.178 Detection of chitinase activity after polyacrylamide gel electrophoresis Trudel,J.;A.Asselin
  32. Mol. Gen. Genet v.186 Cloning and expression of a Bacillus coagulans amylase gene in Excherichia coli Cornelis,P.;C.Digneff;K.Willemot
  33. J. Biol. Chem. v.195 Notes on sugar determination Somogyi,M.
  34. J. Bacteriol v.174 Strueture of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to the other porkaryotic chitinases ans class III plant chitinases Watanabe,T.;W.Oyanage;K.Suzuki;K.Ohnishi;H.Tanaka
  35. J. Biol. Chem. v.265 Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin Watanabe,T.;K.Suzuki;W.Oyanagi;K.Ohnishi;H.Tanaka
  36. J. Bacteriol v.172 Chitnase system of bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation Watanabe,T.;W.Lyanagi;K.Suzuki;H.Tanaka