Purification and Characterization of a Thermostable ${\beta}-Glycosidase$ from Thermus caldophilus GK24

  • Yoo, Jin-Sang (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Han, Ki-Woong (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Kim, Hyun-Kyu (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Kim, Min-Hong (Biotechnology Division, R&D Center, Daesang Co. Ltd.) ;
  • Kwon, Suk-Tae (Department of Genetic Engineering, Sungkyunkwan University)
  • Published : 2000.10.01

Abstract

A ${\beta}-glycosidase$ enzyme with $\beta$-D-fucosidase, ${\beta}-D-galactosidase$, and $\beta$-D-glucosidase activities has been purified from Thermus caldophilus GK24. The enzyme was monomeric with a molecular mass of 49 kDa, as evidenced by SDS-PAGE. The $K_m$ values for p-nitrophenyl ${\beta}-D-fucopyranoside$ (p-NPFuc), p-nitrophenyl ${\beta}-D-galactopyranoside$ (p-NPGal), and p-nitrophenyl ${\beta}-D-glucopyranoside$ (p-NPGlu) were 0.23 mM, 6.25 mM, and 0.28 mM, respectively. The enzyme showed optimal pH ranging between 5.5-6.5 and maximum temperature in the range of $85-90^{\circ}C$ for all the above mentioned activities. The half-life of the enzyme in sodium phosphate buffer (pH 6.0) at $80^{\circ}C$ was approximately 7 h. The p-NPGal hydrolyzing activity of Tca ${\beta}-glycosidase$ was strongly activated by L-histidine, while the p-NPFuc and p-NPGlu hydrolyzing activities of Tca ${\beta}-glycosidase$ were not affected at all by the amino acid. These results suggest differences in the conformation or in the reactive residues at the active site of Tca ${\beta}-glycosidase$.

Keywords

References

  1. J. Mol. Biol. v.271 Crystal structure of the β-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: resilience as a key factor in thermostability Aguilar, C. F.;I. Sanderson;M. Moracci;M. Ciaramella;R. Nucci;M. Rocci;L. H. Pearl
  2. Proteins and Enzymes Bell, J. E.;E. T. Bell
  3. Protein Methods Bollang, D. M.;M. D. Rozycki;S. J. Edelstein;Wiley-Liss(ed);John Wiley(ed);Sons(ed)
  4. J. Biochem v.21 Characterization and kinetics of β-D-gluco/fuco/galactosidase from sheep liver Chinchetru, M. A.;J. A. Cabezas;P. Calvo
  5. Biotech. Bioeng v.26 Some properties of a β-galactosidase from an extremly thermophilic bacterium Cowan, D. A.;R. M. Daniel;A. M. Martin;H. W. Morgan
  6. J. Bacteriol v.102 Comparative study of isoenzyme of bacterial β-galactosidase Erickson, R.;P. Steers Jr.
  7. Biochem. J. v.274 Hisidines, histamines and imidazoles as glycosidase inhibitors Field, R. A.;A. H. Haines;E. J. T. Chrystal;M. C. Luszniak
  8. Biochemical. J. v.280 A classification of glycosyl hydrolases based on amino acid sequence similarities Henrissat, B.
  9. Eur. J. Biochem v.213 purification and characterization of an extremely thermostable β-glucosidase from the hyperthermophilic archaeon Pyrococus furiosus Kengen, S. W.;E. J. Luesink;A. J. M. Stams;A. J. B. Zehnder
  10. J. Microbiol. Biotechnol v.9 Effect of temperature and cabon soruce in the expression of β-galactosidase gene of Lactococcus lactis ssp. lactic ATCC 7962 Kim, T. Y.;J. M. Lee;H. C. Chang;D. K. Chung;J.-H. Lee;J. H. Kim;H. J. Lee
  11. Nature v.27 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Laemmli. U. K.
  12. J. Am. Chem. Soc v.56 The determination of enzyme dissociation contants Lineweaver, H.;P. Burk
  13. J. Biol. Chem v.193 Protein measurement with the follin phenol reagent Lowry, O. H.;N. J. Rosebrough;A. L. Farr;R. J. Randall
  14. Thermophiles Industrial applications of thermostable enzymes Ng, T. K.;R. K. William;T. D. Brock(ed.)
  15. Biotechnol. Appl. Biochem v.17 Exo-glucosidase activity and substrare specificity of the β-glycosidase isolated from the extreme thermophile Sulfolobus solfaricus Nucci, R.;M. Moracci;C. Vaccaro;N. Vespa;M. Rossi
  16. Biosci. Biotech. Biochem v.60 Purification and characterization of β-D-glucosidase (β-D-fucosidase) from Bifidobacterium breve clb acclimated to cellobiose Nunoura, N.;K. Ohdan;T. Yano;K. Yamamoto;H. Kumagai
  17. Biosci. Biotechnol. Biochem v.62 Thermostable β-galactosidase from an extremly thermophile, Thermus sp. A4:Enzyme purification and characterization, and gene cloning and sequencing Ohtsu, N.;H. Motoshima;K. Goto;F. Tsukasaki;H. Matsuzawa
  18. J. Biol. Chem v.254 Glycoprotein nature of yeast alkaline phosphatase: Formation of active enzyme in the presence of tunicamycin Onish, H. R.;J. S. Tkacz;J. O. Lampen
  19. Eur. J. Biochem v.187 Thermostable β-galactosidase from the the archaebacterium Sulfolobus solfataricus Pisani, F. M.;R. Rella;C. Raia;C. Rozzo;R. Nucci;A. Cambacorta;M. De Rosa;M. Rossi
  20. J. Biochem v.91 Heat stable and fructose 1,6-bisphosphate activated L-lactate dehydrogenase from an extremely thermophilic bacterium Taguchi, H.;M. Yanashita;H. Matsuzawa;T. Ohta
  21. Agric, Biol. Chem v.53 A thermostable β-glucosidase isolated from a bacterial species of the genus Thermus Takase, M.;K. Horikoshi
  22. Kor. J. Appl. Microbiol. Biotechnol v.23 Optimal production of thermostable β-galactosidase from Thermus caldophilus GK24 Yoo, J. S.;H. K. Kim;M. J. In;M. H. Kim;S.-T. Kwon
  23. ARch. Biochem. Biophys v.298 Purification and characterization of a stsictly specific β-D-Fucosidase from Aspergillus phoenicis Zeng, Y.-C.;Y. -T. Li;Y.-J. Gu;S.-Z. Zhang