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Streptomyces coelicolor A3(2)로 부터 $\beta$-Glucosidase 유전자 클로닝 및 재조합 효소의 특성
Cloning of $\beta$-Glucosidase Gene from Streptomyces coelicolor A3(2) and Characterization of the Recombinant $\beta$-Glucosidase Expressed in Escherichia coli

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  );
  • 초록

    Streptomyces coelicolor A3(2)의 $\beta$ -glucosidase 유전자를 분리하여 대장균에서 발현하여 특성을 조사하였다. 최적 활성을 나타내는 온도는 pH 5에서는 $20^{\circ}C$ , pH 6에서는 $60^{\circ}C$ 에서 높은 활성을 나타냈다. pH에 따른 활성은 pH 3 이하와 pH 9 이상의 범위에서는 낮은 활성을 나타냈으며 pH 7에서 가장 높은 활성을 나타냈다. $\alpha$ -pNPG( $\rho$ -nitrophenyl- $\alpha$ -D-glucopyranoside), $\beta$ -pNPG ( $\rho$ -nitrophenyl- $\beta$ -D-glucopyranoside), $\beta$ -pNPF( $\\rho$ -nitrophenyl- $\beta$ -D-fucopyranoside)는 pH 3-10까지 비슷한 활성을 나타냈으며, $\alpha$ -pNPG가 pH 7에서 다소 높은 활성을 보였다. $\beta$ -pNPGA는 pH 5-9까지 높은 활성을 나타냈으며, 특히 pH 9에서 3배 이상의 높은 활성을 나타냈다. 기질 $\alpha$ -pNPG, $\beta$ -pNPG, $\beta$ -pNPF의 온도에 따른 활성변화는 $\beta$ -pNPF의 활성이 $60^{\circ}C$ 에서 증가하였고, $\beta$ -pNPGA는 $30-50^{\circ}C$ 까지 활성이 증가하여 $50^{\circ}C$ 에서 최대활성을 나타내었다. 당화 flavonoid를 이용한 기질특이성의 상대활성은 daidzin, glycitin, genistin, 순으로 나타났으며 esculin과 apigenin-7-glucose는 기질로 사용하지 않았다. $\beta$ -Glucosidase 활성은 EDTA, DTT에 의해 억제되었으며, $MnSO_4$ , $CaCl_2$ , KCl, $MgSO_4$ 에 의해 증가하였고, 특히 Mn이온에 의해 증가하였다. $CuSO_4$ , NaCl에 의해 효소활성이 저해되었으며, 특히 $ZnSO_4$ 의 경우 효소활성이 강하게 억제되었다.


    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.


  • 주제어

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

  • 참고문헌 (31)

    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. Jiresova, 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. http://afmb.cnrs-mrs.fr/CAZY 
    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 

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