Purification and characterzation of the $\alpha$-L-Arabinofuranosidase from Escherichia coli Cells Harboring the Recombinant Plasmid pKMG11

재조합 균주 Escherichia coli가 생산하는 Bacillus stearothermophilus $\alpha$-L-Arabinofuranosidase의 정제 및 특성

  • 엄수정 (고려대학교 자연자원대학 유전공학과) ;
  • 조쌍구 (고려대학교 자연자원대학 유전공학과) ;
  • 최용진 (고려대학교 자연자원대학 유전공학과)
  • Published : 1995.08.01

Abstract

$\alpha $-Arabinofuranosidase was produced by E. coli HB101 haboring the recombinant plasmid pKMG11 which contained the arfI gene of Bacillus stearothermophilus. The maximum production of the enzyme was observed when E. coli HB101 cells were grown at 37$\circ$C for 20 hours in the medium containing 0.5% arabinose, 1.0% tryptone, 0.5% yeast extract, and 1% NaCl. The $\ALPHA $-arabinofuranosidase produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange column chromatography and Sepharose 6B-100 gel filtration. The purified enzyme was most active at 55$\circ$C and pH 6.5. The K$_{m}$ and V$_{max}$ values of the enzyme on $\rho $-nitrophenyl-$\alpha $-arabinofuranoside was determined to be 2.99 mM and 0.43 $\mu $mole/min (319.74 $\mu $mole/min/mg), respectively. The pI value was 4.5. The molecular weight of the native protein was estimated to be 289 kDa. The SDS-polyacrylamide gel clectrophoresis analysis suggested that the functional protein was a trimer of the 108 kDa identical subunits. The N-terminal amino acid sequence of the a-arabinofuranosidase was identified as X-Ser-Thr-Ala-Pro-Arg( \ulcorner )-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe.

Keywords

References

  1. J. Chem. Soc. v.12 Constitution of Xylan from Esparto Grass Chanda,S.K.;E.L.Hirst;J.K.N.Jones;E.G.V.Percival
  2. J. Am. Chem. Soc. v.76 The carbohydrates of Gramineae Ⅱ. The constitution of the hemicelluloses of wheat straw and corn cobs Ehrenthal,I.;R.Montgomery;F.Smith
  3. Trends Biotechnol. v.3 Microbial xylanolytic systems Biely,P.
  4. Carbohydr. Res. v.148 Linkage of p-coumaroyl and feruloyl groups to cell wall polysaccharides of barley straw Muller-Harvey,I.;R.D.Hartley;P.J.Harris;E.H.Curzon
  5. Appl. Environ. Microbiol. v.57 Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with $\beta$-D-xylosidase and $\alpha$-arabinofuranosidase activities Utt,E.A.;C.K.Eddy;K.F.Keshav;L.O.Ingram
  6. FEBS Lett. v.186 Acetyl xylan esterases in fungal cellulolytic systems Biely,P.;J.Pulse;H.Schneider
  7. Bio/technology. v.4 Cooperativity of esterases and xylanases in the enzymatic degradation of acetyl xylan Biely,P.;C.R.MacKenzie;J.Puls;H.Schneider
  8. Appl. Microbiol. Biotechnol. v.23 Hydrolysis of steamed birchwood hemicellulose by enzymes produced by Trichoderma reesei and Aspergillus awamori Poutanen,K.;J.Puls;M.Linko
  9. Kor. J. Appl. Microbiol. Biotechnol. v.17 Production of xylanase by Bacillus stearothermophilus Song,H.S.;Y.J.Choi
  10. Kor. J. Appl. Microbial. Bioeng. 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
  11. Kor. J. Appl. Microbial. Bioeng. 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
  12. 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
  13. 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
  14. Kor. J. Appl. Microbiol. Biotechnol. v.22 Molecular cloning and expression of the $\alpha$-L-arabinofuranosidase gene of Bacillus stearothermophilus in Escherichia coli Eom,S.J.;H.S.Kim;S.G.Cho;Y.J.Choi
  15. Anal. Chem. v.31 Use of dinitrosalicylic acid reagent for determination of reducing sugar Miller,G.L.
  16. J. Biol. Chem. v.193 Protein measurement with Folin phenol reagent Lowry,O.H.;N.J.Rasebrough;A.L.Farr;R.J.Randall
  17. Nature. v.227 Cleavage of structure proteins during the assembly of the head of bacteriophage T4 Laemmli,U.K.
  18. Protein Methods Bollag,D.M.;S.J.Edelstein
  19. Protein Methods Bollag,D.M.;S.J.Edelstein
  20. Biochemistry Stryer,L.
  21. Biochim. Biophys. Acta. v.207 Purification, crystallization and amino acid composition of $\alpha$-L-arabinofuranosidase from Aspergillus niger Kaji,A.;K.Tagawa
  22. Plant Physiol. v.63 Structure of plant cell walls. Ⅸ. Purification and partial characterization of a wall degrading end-arabanase and an arabinosidase from Bacillus subtilis Weinstein,L.;P.Albersheim
  23. Appl. Environ. Microbiol. v.58 Purification and characterization of an $\alpha$-arabinofuranosidase from Butyrivibrio fibrisolvens GS113 Hespell,R.B.;P.J.O'bryan
  24. Appl. Environ. Microbiol. v.47 $\alpha$-Arabinofuranosidase from Ruminococcus albus 8: purification and possible role in hydrolysis of alfalfa cell wall Greve,J.C.;J.M.Labavitch;R.E.Hungate
  25. Can. J. Microbiol. v.33 Purification and characterization of an $\alpha$-arabinofuranosidase from Clostridium acetobutylicum ATCC 824 Lee,S.F.;C.W.Forsberg
  26. Agric. Biol. Chem. v.46 An $\alpha$-L-arabinofuranosidase from Streptomyces purpurascens IFO 3389 Komae,K.;A.Kafi;M.Sato
  27. Agric. Biol. Chem. v.45 An $\alpha$-L-arabinofuranosidase produced by wild-type Streptomyces sp. no. 17-1 Kaji,A.;M.Sato;Y.Tsutsui
  28. Biochem. J. v.245 Purification and mechanistic properties of an extracellular $\alpha$-L-arabinofuranosidase from Monillinia fructigena Kelly,M.A.;M.L.Sinnott;M.Herrchen