Cloning of $\beta$-Glucosidase Gene from Streptomyces coelicolor A3(2) and Characterization of the Recombinant $\beta$-Glucosidase Expressed in Escherichia coli

Streptomyces coelicolor A3(2)로 부터 $\beta$-Glucosidase 유전자 클로닝 및 재조합 효소의 특성

  • Kim, Jae-Young (Department of herbal medicine, Basic Science Institute, Hoseo University) ;
  • Kim, Bong-Kyu (BMIC, Division of Bioscience and Biotechnology, Konkuk University) ;
  • Yi, Yong-Sub (Department of herbal medicine, Basic Science Institute, Hoseo University) ;
  • Kang, Chang-Soo (Department of Biological Science, Hoseo University) ;
  • Ahn, Joong-Hoon (BMIC, Division of Bioscience and Biotechnology, Konkuk University) ;
  • Lim, Yoong-Ho (BMIC, Division of Bioscience and Biotechnology, Konkuk University)
  • 김재영 (호서대학교 한방화장품과학과 기초과학연구소) ;
  • 김봉규 (건국대학교 생명공학과 생명/분자정보학센터) ;
  • 이용섭 (호서대학교 한방화장품과학과 기초과학연구소) ;
  • 강창수 (호서대학교 생명과학과) ;
  • 안중훈 (건국대학교 생명공학과 생명/분자정보학센터) ;
  • 임융호 (건국대학교 생명공학과 생명/분자정보학센터)
  • Received : 2009.01.12
  • Accepted : 2009.05.25
  • Published : 2009.06.28


The $\beta$-glucosidase gene from Streptomyces coelicolor A3(2) was cloned and expressed in Escherichia coli. The ORF consisted of 1377 nucleotides encoding 51 kDa in a predicted molecular weight. Effects of pH indicated that the $\beta$-glucosidase showed similar activity using $\alpha$-pNPG($\rho$-nitrophenyl-$\alpha$-D-glucopyranoside), $\beta$-pNPG($\rho$-nitrophenyl-$\beta$-D-glucopyranoside), and $\beta$-pNPF($\rho$-nitrophenyl-$\beta$-D-fucopyranoside) at range of pH 3 to 10, and high activity using $\beta$-pNPGA ($\rho$-nitrophenyl-$\beta$-D-galactopyranoside) from pH 5 to 10, especially, 3.3 times higher activity at pH 9. Effects of temperature indicated that the $\beta$-glucosidase showed low activity using $\alpha$-pNPG, $\beta$-pNPG, and $\beta$-pNPF from $20^{\circ}C$ to $70^{\circ}C$, and increased activity using $\beta$-pNPGA from $30^{\circ}C$ to $50^{\circ}C$, 1.8 times higher activity at $50^{\circ}C$ than at $30^{\circ}C$. According to activity determination of other substrates, the enzyme was active on daidzin, genistin, and glycitin, inactive on esculin and apigenin-7-glucose. The EDTA and DTT as reducing agents inhibited $\beta$-glucosidase activity, but SDS and mercaptoethanol did not inhibit. Monovalent or divalent metal ions such as $MnSO_4$, $CaCl_2$, KCl, and $MgSO_4$ did not inhibited $\beta$-glucosidase activity. $CuSO_4$ and NaCl showed low inhibition, and $ZnSO_4$ inhibited 3.3 times higher than control.


$\beta$-glucosidase;deglycosylation;Streptomyces coelicolor A3(2);substrate specificity


Supported by : 농촌진흥청


  1. Bentley, S. D., K. F. Chater, A. M. Cerdeno-Tarraga, G. L. Challis, N. R. Thomson, K. D. James, D. E. Harris, M. A. Quail, H. Kieser, D. Harper. et al. 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature. 417: 141-147
  2. Enari, T. M. and M. L. Niku-paavola. 1987. Enzymatic hydrolysis of cellulose: Is the current theory of the mechanism of hydrolysis valid? Crit. Rev. Biotechnol. 5: 67-87
  3. Grabnitz, F. and W. L. Staudenbauer. 1988. Characterization of two $\beta$ glucosidase genes from Clostridium thermocellum. Biotechnol. Lett. 10: 73-78
  4. Gunata, Y. Z., C. L. Bayonove, R. L. Baumes, and R. E. Cordonier. 1985. The aroma of grapes I. Extraction and determination of free and glycosidically bound fractions of some grape aroma components. J. Chromatogr. 331: 83-90
  5. Jang, H. D. and K. S. Chang. 2005. Thermostable cellulases from Streptomyces sp.: scale-up production in a 50-l fermenter. Biotechnol. Lett. 27: 239-242
  6. Kadam, S., A. L. Demain, J. Millet, P. Biguin, and J. P. Aubert. 1988. Molecular cloning of a gene for a thermostable $\beta$-glucosidase from Clostridium thermocellum into Escherichia coli. Enzyme Microb. Technol. 10: 9-13
  7. Perez-Pons, J. A., A. Cayetano, X. Rebordosa, J. Lloberas, A. Guasch, and E. Querol. 1994. A beta-glucosidase gene (bgl3) from Streptomyces sp. strain QM-B814. Molecular cloning, nucleotide sequence, purification and characterization of the encoded enzyme, a new member of family 1 glycosyl hydrolases. Eur. J. Biochem. 223: 557-565
  8. Vallmitjana, M., M. Ferrer-Navarro, R. Planell, M. Abel, C. Aus$\'{\i}$n, E. Querol, A. Planas, and J. A. P$\'{e}$rez-Pons. 2001. Mechanism of the family 1 beta-glucosidase from Streptomyces sp: catalytic residues and kinetic studies. Biochemistry. 40: 5975-5982
  9. Yoon, J. J., C. J. Cha, Y. S. Kim, and W. Kim. 2008. Degradation of cellulose by the major endoglucanase produced from the brown-rot fungus Fomitopsis pinicola. Biotechnol Lett. 30: 1373-1378
  10. Grabnitz, F., M. Seiss, K. P. Rucknagel, and W. L. Staudenbauter. 1991. Structure of the β-glucosidase gene A of Clostridium thermocellum. Eur. J. Biochem. 200: 301-309
  11. Kaur, J., B. S. Chadha, B. A. Kumar, G. S. Kaur, and H. S. Saini. 2007. Purification and characterization of $\beta$-glucosidase from Melanocarpus sp. MTCC3922. Electronic Journal of Biotechnology 10: 260-270
  12. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254
  13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685
  14. Otieno, D. O., J. F. Ashton, and N. P. Shah. 2005. Stability of $\beta$-glucosidase activity produced by Bifidobacterium and Lactobacillus spp. in fermented soymilk during processing and storage. J. Food Sci. 70: 236-241
  15. Dan, S., I. Marton, M. Dekel, B. Bravdo, S. He, S. G. Withers, and O. Shoseyov. 2000. Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger $\beta$-Glucosidase. J. Biol. Chem. 275: 4973-4980
  16. Parry, N. J., D. E. Beever, I. Vandenberghe, J. Van Beeumen, and M. K. Bhat. 2001. Biochemical characterization and mechanism of action of a thermostable $\beta$-glucosidase purified from Thermoascus aurantiacus. Biochem. J. 353: 117-127
  17. Grimaldi, A., E. Bartowsky, and V. Jiranek. 2005. Screening of Lactobacillus spp. and Pediococcus spp. for glucosidase activities that are important in oenology. J. Appl. Microbiol. 99: 1061-1069
  18. Shewale, J. G. 1982. 3-Glucosidase: its role in cellulase synthesis and hydrolysis of cellulose. Int. J. Biochem. 14: 435-443
  19. Pisani, F. M., R. Rella, C. Raia, C. Rozzo, R. Nucci, A. Cambacorta, D. M. Rosa, and M. Rossi. 1990. Thermostable $\beta$-galactosidase from the archaebacterium Sulfolobus solfataricus purification and properties. Eur. J. Biochem. 187: 321-328
  20. Romaniec, M. P. M., K. Davidson, and G. P. Hazlewood, 1987. Cloning and expression in Escherichia coli of Clostridium thermocellum DNA encoding beta-glucosidase activity. Enzyme Microb. Technol. 9: 474-478
  21. Rashid, M. H. and K. S. Siddiqui. 1997. Purification and characterization of a $\beta$-glucosidase from Aspergillus niger. Folia Microbiol. 42: 544-550
  22. Barras, F., J. P. Chambost, and M. Chippaux. 1984. Cellobiose metabolism in Erwinia: genetic study. Mol. Gen. Genet. 197: 486-490
  23. Zamocky, M., R. Ludwig, C. Peterbauer, B. M. Hallberg, C. Divne, P. Nicholls, and D. Haltrich. 2006. Cellobiose dehydrogenase a flavocytochrome from wood-degrading, phytopathogenic and saprotropic fungi. Curr. Protein Pept. Sci. 7: 255-280
  24. P$\'{e}$rez-Pons, J. A., X. Rebordosa, and E. Querol. 1995. Properties of a novel glucose-enhanced beta-glucosidase purified from Streptomyces sp. (ATCC 11238). Biochim. Biophys. Acta. 1251: 145-153
  25. Wen, Z., W. Liao, and S. Chen. 2005. Production of cellulase/ $\beta$-glucosidase by the mixed fungi culture Trichoderma reesei and Aspergillus phoenicis on dairy manure. Process Biochem. 40: 3087-3094
  26. Jiresová, M., Z. Dobrov$\'{a}$, J. N$\'{a}$prstek, P. Rysav$\'{y}$, and J. Janecek. 1983. Induction of beta-D-glucosidase in Streptomyces granaticolor. Folia Microbiol. (Praha) 28: 379-385
  27. Tamas J., K. Krisztina, S. Zsolt, and R. Kati. 2003. Production of $\beta$-glucosidases in mixed culture of Aspergillus niger BKMF 1305 and Trichoderma reesei RUT C30. Food Technol. Biotechnol. 41: 49-53
  28. Estruch, J. J., D. Chriqui, K. L. Grossmann, J. Schell, and A. Spena. 1991. The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates. EMBO J. 10: 2889-2895
  29. Coutinho, P. M. and B. Henrissat. Carbohydrate-Active Enzymes Server.
  30. Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280: 309-316
  31. Jenkins, J. L., L. L. Leggio, G. Harris, and R. Pickersgill. 1995. $\beta$-Glucosidase, $\beta$-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes wit 8-fold $\beta$$\beta$/$\alpha$$\alpha$ architecture and with two conserved glutamates near the carboxy-terminal ends of $\beta$$\beta$-strands four and seven. FEBS Letters 362: 281-285