Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Characterization of the arfA Gene from Bacillus stearothermophilus No. 236 and Its Protein Product, $\alpha$-L-Arabinofuranosidase

  • Published : 2004.06.01
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Abstract

The $\alpha$-L-arabinofuranosidase (Arfase) gene of Bacillus stearothermophilus No. 236 was cloned and sequenced. The ORF of the gene, designated arfA, encoded a 507 -residue polypeptide with calculated molecular mass of 57 kDa. The Arfase produced by a recombinant Escherichia coli strain containing the arfA gene was purified to apparent homogeneity and characterized. The molecular mass of the Arfase determined by SDS-PAGE was 60 kDa. However, according to gel filtration, it was estimated to be approximately 190 kDa. These results indicated that the functional form of the Arfase is trimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and $55^{\circ}C$, respectively. The half-life of the enzyme at $60^{\circ}C$ was about 6 h. Kinetic experiments at $45^{\circ}C$ with pNPM (p-nitrophenyl $\alpha$-L-arabinofuranoside) as a substrate gave the $K_m and V_{max}$ values of 1.19 mM and 26.1 U/ mg, respectively. When the enzyme was combined with Bacillus stearothermophilus No. 236 endoxylanase and $\beta$-xylosidase, it hydrolyzed arabinoxylan into L-arabinose and xylose more efficiently than Arfase alone. This synergistic effect suggested that the complete hydrolysis of xylan with large amounts of arabinose side chains required Arfase as well as endoxylanase and $\beta$-xylosidase.

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References

  1. Adv. Carbohydr. Chem. v.14 Structural chemistry of the hemicellulose Aspinall,G.O. https://doi.org/10.1016/S0096-5332(08)60228-3
  2. Trends Biotechnol. v.3 Microbial xylanolytic systems Biely,P. https://doi.org/10.1016/0167-7799(85)90004-6
  3. Carbohydr. Res. v.101 The degradation of isolated hemicellulose and lignin-hemicellulose complexes by cell-free rumen hemicellulase Brice,R.E.;L.H.Morrison https://doi.org/10.1016/S0008-6215(00)80797-1
  4. J. Microbiol. Biotechnol. v.5 Nucleotide sequence analysis of an endo-xylanase gene (xynA) from Bacillus stearothermophilus Cho,S.G.;Y.J.Choi
  5. J. Biol. Chem. v.277 Detailed kinetic analysis and identification of the nucleophile in α-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase Dalia,S.;V.Belakhov;D.Solmon;G.Shoham;T.Baasov https://doi.org/10.1074/jbc.M208285200
  6. ACS Symp. Ser. v.399 Biodegradation of the hetero-1,4-linked xylans Dekker,R.F.H. https://doi.org/10.1021/bk-1989-0399.ch045
  7. J. Microbiol. Biotechnol. v.11 Carbon catabolite repression of expression of the xylanase α gene of Bacillus stearothermophilus No. 236 Ha,G.S.;I.D.Choi;Y.J.Choi
  8. Adv. Carbohydr. Chem. Biochem. v.42 L-arabinosidases Kaji,A.
  9. Appl. Environ. Microbiol. v.64 Purification and substrate specificities of two α-L-arabinofuranosidase from Aspergillus awamori IFO 4033 Kaneko,S.;M.Arimoto;M.Ohba;H.Kobayashi;T.Ishii;I.Kusakabe
  10. Kor. J. Appl. Microbiol. Bioeng. v.25 Nucleotide sequence of the est-gene coding for Bacillus stearothermophilus acetyxylan esterase Lee,J.S.;T.J.Choi
  11. J. Biol. Chem. v.193 Protein measurement with folin phenol reagent Lowry,O.H.;N.J.Rasebrough;A.L.Farr;R.J.Randall
  12. Anal. Chem. v.31 Use of dinitrosalicylic acid reagent for determination of reducing sugar Miller,G.L. https://doi.org/10.1021/ac60147a030
  13. Kor. J. Appl. Microbiol. Bioeng. v.20 Purification and characterization of exo-xylanase from Escherichia coli cells harboring the recombinant plasmid pMG1 Mun,A.R.;Y.J.Choi
  14. J. Biol. Chem. v.240 The release of enzymes from E. coli by osmotic shock and during the formation of spheroplasts Neu,H.C.;L.A.Heppel
  15. Kor. J. Appl. Microbiol. Bioeng. v.20 Molecular cloning and expression of Bacillus stearothermophilus β-D-xyosidase grne in E. coli Oh,S.W.;S.S.Park;Y.I.Park;Y.J.Choi
  16. Kor. J. Appl. Microbiol. Bioeng. v.17 Production of xylanase by Bacillus stearothermophilus Song,H.S.;Y.J.Choi
  17. J. Microbiol. Biotechnol. v.5 Synergism among endo-xylanase, β-xylosidase, and acetyl xylan esterase from Bacillus stearothermophilus Suh,J.H.;Y.J.Choi
  18. Crit. Rev. Biochem. v.17 Xylanolytic enzymes from fungi and bacteria Sunna,A.;G.Antranikian
  19. Wood Sci. Technol. v.1 Recent progress in the chemistry of wood hemicellulose Timell,T.E. https://doi.org/10.1007/BF00592255
  20. Biochem. J. v.290 The xylan-degrading enzyme system of Talasomyces emersonii: Novel enzyme with activity against aryl β-D-xylosides and unsubstituted xylans Tuohy,M.G.;J.Puls;M.Claeyssens;M.Vrsanskas;M.P.Coughlan https://doi.org/10.1042/bj2900515
  21. The Carbohydrates v.2a Hemicellulose Whistler,R.L.;E.L.Richards
  22. Chemtech v.13 Hemicellulose Wilkie,K.C.B.
  23. Microbiol. Rev. v.52 Multiplicity of β-1,4-xylanase in microorganism: Functions and applications Wong,K.K.T.;L.U.L.Tan;J.N.Saddler
  24. FEMS Microbiol. Lett. v.164 Nucleotide sequence of arfB of Clostridium stercorarium, and prediction of catalytic residues of alpha arabinofuranosidases based on local similarity with several families of glycosyl hydrolases Zverlov,V.V.;W.Liebl;M.Bachleitner;W.H.Schwarz