Purification and Characterization of Acetyl Xylan Esterase from Escherichia coli Cells Harboring the Recombinant Plasmid pKMG6

제조합 균주 Escherochia coli가 생산하는 Bacillus stearothermophilus Acetyl Xylan Esterase의 정제 및 특성

  • 김인숙 (고려대학교 자연자원대학 유전공학과) ;
  • 이철우 (고려대학교 자연자원대학 유전공학과) ;
  • 최용진 (고려대학교 자연자원대학 유전공학과)
  • Published : 1994.10.01

Abstract

Acetyl xylan esterase was produced by E. coli HB101 harboring a recombinant plasmid pKMG6 which contained the estI gene of Bacillus stearothermophilus. The maximum production was observed when the E. coli strain was grown at 37$\circC for 12 hours in the medium containing 0.5% acetyl xylan, 1.0% tryptons, 1.0% sodium chloride, and 0.5% yeast extract. The esterase produced was purified to homogeneity using a combination of ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 gel filtration. The native enzyme had an apparent molecular mass of 60 kd and was composed of two identical subunits of 29 kd. The N-terminal amino acid sequence of the polypeptide was Ala-X-Leu-Gln- Ile-Gln-Phe-X-X-Gln. The acetyl esterase displayed a pH optimum of 6.5 and a temperature opti- mum of 45$\circC. The heavy metal ions such as Ag$^{++}$, Hg$^{++}$ and Cu$^{++}$ inhibited nearly completely the activity of the esterase, and no specific metal ion was found to be required for the enzyme activity. The enzyme readily cleaved MAS, $\beta$-D-glucose pentaacetate, $\alpha$-naphthyl acetate, $\rho$-nitrophenyl acetate as well as acetyl xylan, but had no activity on $\rho$-nitrophenyl propionate, $\beta$-nitrophenyl butyrate or $\beta$-nitrophenyl valerate. The Km and Vmax values for MAS were 2.87 mM and 11.55 $\mu$mole/min, respectively. Synergistic behavior was demonstrated with a combination of xylanase and esterase from B. stearothermophilus in hydrolyzing acetyl xylan.

Keywords

Purification;characterization;acetyl xylan esterase;plasmid pKMG6;Bacillus strarothermophilus

References

  1. Agric. Environ. v.6 Deacetylation and enhancement of digestibility Bacon,J.S.D.;A.Chesson;A.H.Gordon
  2. Adv. Carbohydr. Chem. Biochem. v.32 Hemicellulases: their occurrence, purification, properties, and mode of action Dekker,R.F.H.;G.N.Richards
  3. Enzyme Microb. Technol. v.9 α-Glucuronidase in two microbial xylanolytic systems Puls,J.;O.Schmidt;C.Granzow
  4. Kor. J. Appl. Microbiol. Bioeng. v.20 no.2 Molecular cloning and expression of Bacillus Stearothermophilus β-D-xylosidase gene in E.coli Oh,S.W.;S.S.Park;Y.I.Park;Y.J.Choi
  5. Appl. Environ. Microbiol. v.56 Purification and properties of an acetyl xylan esterase from Fibrobacter succinogenes S85 McDermid,K.P.;C.W.Forsberg;C.R.Mackenzie
  6. Appl. Biochem. Biotechnol. v.20;21 The role of ester groups in resistance of plant cell wall polysaccharides to enzymatic hydrolysis Grohmann,K.;D.J.Mitchell;M.E.Himmel;B.E.Dale;H.A.Schroeder
  7. Appl. Microbiol. Biotechnol. v.23 Hydrolysis of steamed birchwood hemicellulose by enzymes produced by Trichoderma reesei and Asperillus awamori Poutanen,K.;J.Puls;M.Linko
  8. Meth. Enzymol. v.160 Measurement of acetyl xylan esterase in Streptomyces Johnson,K.G.;J.D.Fontana;C.R.MacKenzie
  9. FEBS Lett. v.186 Acetylxylan esterases in fungal cellulolytic systems Biely,P.;J.Puls;H.Schneider
  10. Kor. J. Appl. Microbiol. Bioeng. v.20 Molecular cloning and expression of an endo-xylanase gene from Bacillus stearothermophilus into Escherichia coli Cho,S.G.;S.S.Park;Y.I.Park;Y.J.Choi
  11. Bio/technology v.4 Cooperativity of esterases and xylanases in the enzymatic degradation of acetylxylan Biely,P.;C.R.MacKenzie;J.Puls;H.Schneider
  12. Nature(London) v.227 Cleavage of structure proteins during the assembly of the head of bacteriophage T4 Laemmli,U.K.
  13. Trends in Biotechnol. v.3 Microbial xylanolytic systems Biely,P.
  14. Carbohydr. Res. v.148 Linkage of ρ-coumaroyl and feruloyl groups to cell wall polysaccharides of barley straw Muller-Harvey,I.;R.D.Hartley;P.J.Harris;E.H.Curzon
  15. Protein sequence determination P.Edman
  16. Svensk Papperstid. v.76 Positions of the O-acetyl groups in pine glucomannan Lindberg,B.;K.G.Rosell;S.Sevensson
  17. Enzyme Microb. Technol. v.14 Enzymic hydrolysis of water-soluble lignin-carbohydrate complexes from Populus deltoides: Effects of comination of β-mannanases, xylanase, and acetyl xylan esterase Ross,N.W.;K.G.Johnson;C.Braun;C.R.MacKenzie;H.Schneider
  18. Kor. J. Appl. Microbiol. Bioeng. v.21 no.6 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
  19. J. Sci. Food Agric. v.34 Substituent groups liked by alkli-labile bonds to arabinose and xylose residues of legume, grass and cereal straw cell walls and their fate during digestion by rumen microorganisms Chesson,A.;A.H.Gordon;J.A.Lomax
  20. Anal. Chem. v.31 Use of dinitrosalicylic acid reagent for determination of reducing sugar Miller,G.L.
  21. Kor. J. Appl. Microbiol. Biotechnol. v.17 Production of xylanase by Bacillus stearothermophilus Song,H.S.;Y.J.Choi
  22. Agric. BIol. Chem. v.47 Purification of four esterases from Aspergillus niger Iwai,M.;S.Okumura;E.L.de Leal;Y.Tsujisaka
  23. J. Biol. Chem. v.193 Protein measurement with Folin phenol reagent Lowry,O.H.;N.J.Rasebrough;A.L.Farr;R.J.Randall