Purification and Characterization of Acetyl Xylan Esterase II from Escherichia coli Cells Harboring Recombinant Plasmid pKMG7

재조합 균주 Escherichia coli가 생산하는 Bacillus stearothermophilus Acetyl Xylan Esterase II의 정제 및 특성

  • 김희선 (고려대학교 자연자원대학 유전공학과) ;
  • 서정한 (고려대학교 자연자원대학 유전공학과) ;
  • 최용진 (고려대학교 자연자원대학 유전공학과)
  • Published : 1995.08.01

Abstract

Acetylxylan esterase II was produced by Escherichia coli HB101 harboring the recombinant plasmid pKMG7 which contained the estII gene of Bacillus stearothermophilus. Optimal medium for the production of the acetylxylan esterase by E. coli HB101/pKMG7 was determined to contain 0.5% galactose, 1% yeast extract and 1% NaCl. The enzyme produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The temperature and pH optimum of the esterase were 45$\circ$C and pH 6, respectively. The essential amino acids for the esterase activity were found to be methionine, serine, and cysteine. Molecular weight of the esterase was determined to be 28 kDa by SDS-polyacrylamide gel electrophoresis, and 120 kDa by gel filtration. This suggests that the functional enzyme is a homomeric tetramer. The esterase had an isoelectric point of pH 3.4. The N-terminal amino acid sequence of the enzyme was Ala-Leu-Phe-Glu-Ser-Arg-Phe-Phe-Ser-Glu-Val-Leu-Gly-Leu.

Keywords

Purification;characterization;acetylxylan esterase II;recombinant plasmid;Bacillus stearothermophilus

References

  1. Kor. J. Appl. Microbial. Biotech. v.20 Molecular cloning and expression of Bacillus stearothermophilus $\beta$-D-xylosidase gene in Escherichia coli Oh,S.W.;S.S.Park;Y.I.Park;Y.J.Choi
  2. Protein structure: A practical appoach See,Y.P.;G.Jackowski;T.E.Creighton(ed.)
  3. Biochem. et Biophys. Acta. v.1174 Cloning andsequence analysis of an esterase gene from Pseudomonas sp KWI-56 Yuji Shimada;T.Nagao;A.Sugihara;Yoshoi,T.
  4. J. Biol. Chem. v.240 The release of enzymes from Escherichia coli by osmotic shock and during the formation of shperplast Neu,H.C.;Heppel,L.A.
  5. Anal. Chem. v.35 Determination of Molecular Weights of Proteins by Gel filtration on Sephadex Whitaker,J.R.
  6. Mol. Gen. Genet. v.186 Cloning and Expression of a Bacillus coagulans Amylase Gene in Escherichia coli Cornelis,P.;C.Digneffe;K.Willemot
  7. Kor. J. Appl. Microbiol. Biotechnol. v.22 Purification and Characterization the acetyl xylan esterase from Escherichia coli Cells harboring the recombinant plasmid pKMG6 Kim,I.S.;C.W.Lee;Y.J.Choi
  8. Kor. J. Appl. Microbiol. Biotechnol. v.17 Production of xylanase by Bacillus stearothermophilus Song,H.S.;Y.J.Choi
  9. Meth. Enzymol. v.160 Measurement of acetyl xylan esterase in Streptomyces Johnson,K.G.;J.D.Fontana;C.R.MacKenzie
  10. Kor. J. Appl. Microbiol. Biotechnol. v.22 Molecular cloning and Expression of the acetyl xylan esterase gene(estⅡ) of Bacillus stearothermophilus in Escherichia coli Kim,H.S.;S.J.Eom;S.G.Cho;Y.J.Choi
  11. Kor. J. Appl. Microbial. Biotech. v.20 Molecular cloning and expression of an endo-xylanase from Bacillus stearothermophilus into Escherichia coli Cho,S.G.;S.S.Park;Y.I.Park;Y.J.Choi
  12. J. Biol. Chem. v.193 Protein measurement with Folin phenol reagent Lowry,O.H.;N.J.Rasebrough;A.L.Farr;R.J.Randall
  13. Kor. J. Appl. Microbiol. Biotechnol. v.22 Molecular cloning and Exspression of the $\alpha$-L-arabinofuraosidase gene of Bacillus stearothermophilus in Escherichia coli Eom,S.J.;H.S.Kim;S.G.Cho;Y.J.Cho
  14. Enzyme Microb. Technol. v.15 Esterases of xylan-degrading microorganisms: Production, properties, and significance Lyudmil Pavlov Christov;Bernard Alexander Prior
  15. Nature(London) v.227 Cleavage of structure proteins during the assembly do the head of bacteiophage T4 Laemmli,U.K.
  16. Kor. J. Appl. Microbiol. Biotechnol. v.21 Molecular cloning and Expression of the acetyl xylan esterase gene of Bacillus stearothermophilus in Escherichia coli Kim,I.S.;S.G.Cho;Y.J.Choi
  17. Agric. Biol. Chem. v.47 Purification of four esterases from Aspergillus niger Iwai,M.;S.Okumura;E.L.de Leal;Y.Tsujisaka
  18. Protein Method Daniel, M. Bollag;Stuart, J. Edelstein