한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제37권2호
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  • Pages.99-104
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  • 2009
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  • 1598-642X(pISSN)
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  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

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)
  • 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)
  • 투고 : 2009.01.12
  • 심사 : 2009.05.25
  • 발행 : 2009.06.28
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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.

키워드

참고문헌

  1. Barras, F., J. P. Chambost, and M. Chippaux. 1984. Cellobiose metabolism in Erwinia: genetic study. Mol. Gen. Genet. 197: 486-490 https://doi.org/10.1007/BF00329947
  2. 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 https://doi.org/10.1038/417141a
  3. 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 https://doi.org/10.1016/0003-2697(76)90527-3
  4. Coutinho, P. M. and B. Henrissat. Carbohydrate-Active Enzymes Server. http://afmb.cnrs-mrs.fr/CAZY
  5. 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 https://doi.org/10.1074/jbc.275.7.4973
  6. 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 https://doi.org/10.3109/07388558709044153
  7. 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
  8. Grabnitz, F. and W. L. Staudenbauer. 1988. Characterization of two $\beta$ glucosidase genes from Clostridium thermocellum. Biotechnol. Lett. 10: 73-78 https://doi.org/10.1007/BF01024629
  9. 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 https://doi.org/10.1111/j.1432-1033.1991.tb16186.x
  10. 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 https://doi.org/10.1111/j.1365-2672.2005.02707.x
  11. 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 https://doi.org/10.1016/0021-9673(85)80009-1
  12. Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280: 309-316
  13. 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 https://doi.org/10.1007/s10529-004-8356-5
  14. 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$-strands four and seven. FEBS Letters 362: 281-285 https://doi.org/10.1016/0014-5793(95)00252-5
  15. Jiresová, M., Z. Dobrov$\'{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 https://doi.org/10.1007/BF02879487
  16. 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 https://doi.org/10.1016/0141-0229(88)90091-9
  17. 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
  18. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685 https://doi.org/10.1038/227680a0
  19. 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 https://doi.org/10.1111/j.1365-2621.2005.tb07194.x
  20. 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 https://doi.org/10.1042/0264-6021:3530117
  21. 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 https://doi.org/10.1111/j.1432-1033.1994.tb19025.x
  22. 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 https://doi.org/10.1016/0167-4838(95)00074-5
  23. 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 https://doi.org/10.1111/j.1432-1033.1990.tb15308.x
  24. Rashid, M. H. and K. S. Siddiqui. 1997. Purification and characterization of a $\beta$-glucosidase from Aspergillus niger. Folia Microbiol. 42: 544-550 https://doi.org/10.1007/BF02815462
  25. 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 https://doi.org/10.1016/0141-0229(87)90100-1
  26. Shewale, J. G. 1982. 3-Glucosidase: its role in cellulase synthesis and hydrolysis of cellulose. Int. J. Biochem. 14: 435-443 https://doi.org/10.1016/0020-711X(82)90109-4
  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. 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 https://doi.org/10.1021/bi002947j
  29. 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 https://doi.org/10.1016/j.procbio.2005.03.044
  30. 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 https://doi.org/10.1007/s10529-008-9715-4
  31. 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 https://doi.org/10.2174/138920306777452367