Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Molecular Cloning and Expression of a Cellulolytic Xylanase Gene from Bacillus circulans in Escherichia coli

Bacillus circulans 기원의 Cellulolytic Xylanase 유전자의 대장균에서의 클로닝 및 발현

  • 이동석 (인제대학교 의생명공학대학 임상병리학과) ;
  • 김지연 (인제대학교 생물건강산업유성지원 센터) ;
  • 김한복 (호서대학교 자연과학부 생명과학전공)
  • Published : 2000.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

A gene for cellulolytic xylanase of Bacillus circulnns ATCC21365 was cloned on pUC 19 in Eschwichia coli. The recombinant plasniid pXLI80 contained an 1.8 id, inselt composed of0.5 kb and 1.3 kb PslI fragments derived from B, circulans. The 0.5 kh fragment in the upstream region of 1.3 kb one was confirmed lo be indispensable for not only expression but also hyperexpression of the cloned gene. The transformant overproduced the xylanase 135 times greater than that produced by the orlginal B circulnns. The optimum pH and temperature of the cloned enzyme we]-e pH 5.2 and $60^{\circ}C$, respectively. Heal pretl-eatment at TEX>$55^{\circ}C$C for 1 Indid not cause inhibition of the activity of this enzyme. The elm.ynie could hydl-olyre CMC and lichenan as well as xylan to produce xylose(or GI), xylohiose(or G2) and xylolnose(or G3) as inah products. Hence We defined the cloned enzyme as a cellulolytic xylanase. The SDS-PAG electrophoretic mobility and zyiiogram of this enzyme derived from whole cell extracts or c~~lture supematants or E. coli(pXL180) indicated a molecular weight of 45,000 and nonprocessing of the enzyme in the peilplasln of E. coli.

Bacillus circulans ATCC21367의 Cellulolytic Xylanase 유전자를 pUC19을 vector 로 하여 대장균내에서 클로닝 하였다. 재조합 plasmid pXL180은 B. circulans 로부터 유래 된 0.5 kb와 1.3 kb Pst I 절편으로 이루어진 총 1.8 kb 삽입 절편을 포함 하고 있었다. 1.3 kb 절편의 상류영역에 있는 0.5 kb 절편은 클로닝된 유전자의 정상 발현뿐만아니라 과잉 발 현에 대해서도 필수 불가결함을 확인하였다. 형질전환체는 모균 B. circulans에 의해 생산된 것보다 135배 이상 많은 xylanase를 생산하였다. 클로닝된 효소의 최적 pH와 온도는 각각 pH 5.2와 $60^{\circ}C$ 였다. $55^{\circ}C$에서 1시간 동안의 사전 연처리에도 이 효소는 활성의 감소를 일 으키지 않았다. 이 효소는 xylan과 carboxymethyl cellulose (CMC), lichenan에 대하여 기질 특이성을 보였는데, 주요 산물로서 xylose(EH는 G1)와 xylobiose(또는 G2), xylotriose(또는 G3)를 생산하였다. 그러므로 우리는 클로닝된 유전자의 효소를 cellulolytic xylanase로 명명 하였다. 액체 배양된 Eschericxhia coli DH5$\alpha$(pXL180)의 전체 세포 추충물이나 배양 상등 액으로부터 조제된 시료들의 SDSPAGE와 zymogram을 통하여 이 효소의 분자량은 45kDa 이었으며, 대장균 내에서 주목할 만한 processing은 일어나지 않았다.

Keywords

References

  1. Trends Biotechnol. v.3 Biely,P.
  2. Meth, Enzymol. v.68 Plasmid of Escherichia coli as cloning vectors Bolivar,F.;K.Backman
  3. Carbohydr: Res. v.101 The degradation of isolated hemicellulose and lignin-hemi-cellulose complex by cell-free, rumen hemicellulose Brice,R.E.;I.M.Morrison
  4. J. Chem. Soc, v.12 The construction of xylan from Esparto Grass Chanda,S.K.;E.L.Hirst;J.K.N.Jones;E.G.V.Percival
  5. J. Appl. Bacteriol. v.60 Degradation of isolated grass mesophyll, epidermus and fibre cell walls in the rumen and by cellulolytic rumen bacteria in axemic culture Chesson,A.;C.S.Stewart;K.Delgarno;T.D.King
  6. DNA cloning v.Ⅰ Techniques for transformation of Escherichia coli Hanahan,D.
  7. FEMS Microbiol. Lett. v.51 A catalogue of Clostridum thermocellum endoglucanase,β-glucosidase and xylanase genes cloned in Escherichia coli Hazlewood,G.P.;M.P.M.Romamec;K.Davidson;O.Grepinet;P.Begum;J.Millet;O.Raynaud;J.P.Aubert
  8. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Laemmli,U.K.
  9. Appl. Environ. Microbiol. v.53 Purification and characterization of two endoxylanases from Clostridium acetobutylicum ATCC8824 Lee,S.F.;C.W.Forsberg;J.B.Paittray
  10. Molecular cloning, A Laboratory Manual. 2nd ed, Manistis,T.;E.F.Futsch;J.Sambrook
  11. Anal. Chem. v.31 Use of dimtrosalicylic acid reagent for determination of reducing sugar Miller,G.L.
  12. Recombinant DNA Techniques, An Introduction Rodriguez,R.L.;R.C.Tait
  13. Appl. Environ. Microbiol. v.53 Molecular cloning of a xylanase gene from Bacteroides succmogenes and its expression in Escherichia coli Sipat,A.;K.A.Taylor;R.Y.C.Lo;C.W.Forsberg;P.J.Krell
  14. Complete mucleotide sequence of the Eschericha coli plasmid pBR322 v.43 Sutcliffee,J.G.
  15. Wood and agricultural residues Properties of xylanase from Clostridum thermocellum using a trinitrophenyl-xylan assay Tang,J.C.;T.P.McGray;P.Chen;B.Ferguson;G.Carson;W.S.Kelly
  16. Plant Physiol v.63 Structure of cell walls Weinstein,L.;A.Peter
  17. Adv. Carbohydr. Chem. Biochem. v.36 The hemicellulose of grasses and cereals, Wilkie,K.C.B.
  18. Microbiol. Rev. v.52 Multiplicity of β-1,4-xylanase in microorganisms : funtion and applications Wong,K.K.Y.;L.U.L.Tan;J.N.Saddler
  19. Appl. Environ. Microbiol. v.55 Identification of two distinct Bacillus circulans xylanases by molecular cloning of the genes and expression in Escherichia coli Yang,R.C.A.;C.R.MacKenzie;D.Bilous;S.A.Narang
  20. Appl. Microbiol Biotechnol. v.27 Cloning and expression of a xulanase gene from Clostridum acetobutylicum P262 in Escherichia coli Zappe,H.;D.T.Jones;D.R.Woods
  21. Ann. Rev. Microbiol. v.34 Chemical and fuel production by anaerobic bacteria Zeikus,J.G.