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Thermotoga maritima로부터 고온성 β-glucosidase (BgIB)의 클로닝과 필수아미노산 잔기의 확인

Cloning and Identification of Essential Residues for Thermostable β-glucosidase (BgIB) from Thermotoga maritima

  • 홍수영 (경상대학교 응용생명과학부) ;
  • 조계만 (경상대학교 응용생명과학부) ;
  • 김용희 (경상대학교 응용생명과학부) ;
  • 홍선주 (경상대학교 응용생명과학부) ;
  • 조수정 (진주산업대학교 미생물공학과) ;
  • 조용운 (진주산업대학교 미생물공학과) ;
  • 김훈 (순천대학교 생물환경화학과) ;
  • 윤한대 (경상대학교 농업생명과학연구원)
  • Hong, Su-Young (Division of Applied Life Science, Gyeongsang National University) ;
  • Cho, Kye-Man (Division of Applied Life Science, Gyeongsang National University) ;
  • Kim, Yong-Hee (Division of Applied Life Science, Gyeongsang National University) ;
  • Hong, Sun-Joo (Division of Applied Life Science, Gyeongsang National University) ;
  • Cho, Soo-Jeong (Department of Microbiological Engineering, Chinju National University) ;
  • Cho, Yong-Un (Department of Microbiological Engineering, Chinju National University) ;
  • Kim, Hoon (Department of Agricultural Chemistry, Sunchon National University) ;
  • Yun, Han-Dae (Research Institute of Agriculture & Life Science, Gyeongsang National University)
  • 발행 : 2006.12.01

초록

초고온성 세균인 Thermotoga maritima로부터 ${\beta}-glucosidase$ 유전자를 클로닝한 후 대장균 숙주에서 발현시켰다. 이 효소는 salicin, arbutin, $_pNPG$과 같은 탄소원의 ${\beta}$-글루코시드 결합을 가수분해하였다. 721개의 아미노산을 암호화하는 2,166 bp의 DNA 염기서열로된 유전자이였다. 다른 ${\beta}-glucosidase$ 효소들과 단백질 유사성을 비교한 결과 glycosyl hydrolase family 3에 속하였으며 MUG-nondenaturing PAGE와 SDS-PAGE에 의해 확인된 단백질의 크기는 약 81 kDa이었다. 효소활성은 pH 7.0, $80^{\circ}C$에서 가장 높은 활성을 나타냈으며 이 효소의 아미노산 서열에 있는 두 개의 아미노산 잔기 (232번 글루탐산과 242번 아스파르트산 잔기)를 알라닌으로 치환시켜 활성이 없어지는 것으로 보아 이 두 잔기가 효소활성에 중요한 역할을 하는 것으로 추정된다.

A hyperthermophilic bacterium Thernotoga maritima produced thermostable ${\beta}-glucosidase$. The gene encoding ${\beta}-glucosidase$ from T. maritima MSB8 was cloned and expressed in Escherichia coli. The en-zyme (BgIB) hydrolyzed ${\beta}-glucosidase$ linkages between glucose and alkyl, aryl of saccharide groups such as salicin, arbutin, and $_pNPG$. The insert DNA contained ORF with 2,166 bp encodes a 721 amino acids (calculated molecular mass of 80,964 and pl of 4.93). The amino a.id sequence of BglB showed the similarity to family 3 glycosyl hydrolases. The molecular weight of the enzyme was estimated to be approximately 81kDa by MUG-nondenaturing PAGE (4-methylumbelliferyl 13-D-glucoside-nondenaturing polyacrylamide gel electophoresis) and SDS-PACE. The ${\beta}-glucosidase$ exhibited maximal activity at pH 7.0 and $80^{\circ}C$. By exchanging two possible residues (Glu-232 and Asp-242) to Ala by site-directed mutagenesis method, it was found that these were essential for enzymatic activity.

키워드

참고문헌

  1. Bauer, M. W., E. J. Bylina, R. V. Swanson and R. M. Kelly. 1996. Comparison of a ${\beta}$-glucosidase and a ${\beta}$-mannosidase from the hyperthermophilic archaeon Pyrococcus furiosus. J. Biol. Chem. 271, 23749-23755 https://doi.org/10.1074/jbc.271.39.23749
  2. Bibel, M., C. Brettl, U. Gosslar, C. Kriegshauser and W. Liebl. 1998. Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima. FEMS Microbiol. Lett. 158, 9-15 https://doi.org/10.1111/j.1574-6968.1998.tb12793.x
  3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation 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. Bragger, J. M., R. M. Daniel, T. Coolbear and H. W. Morgan. 1989. Very stable enzymes from extremely thermostable archaebacteria and eubacteria. Appl. Microbiol. Biotechnol. 31, 556-561 https://doi.org/10.1007/BF00270794
  5. Bronnenmeier, K., A. Kern, W. Liebl and W. L. Staudenbauer. 1995. Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials. Appl. Environ. Microbiol. 61, 1399-1407
  6. Bronnenmeier, K. and L. Staudenbauer. 1988. Purification and properties of an extracellular ${\beta}$-glucosidase from the cellulolytic thermophilic Clostridium stercorarium. Appl. Microbiol. Biotechnol. 28, 380-386 https://doi.org/10.1007/BF00268200
  7. Chhabra, S. R., K. R. Shockley, D. E. Ward and R. M. Kelly. 2002. Regulation of endo-acting glycosyl hydrolases in the hyperfhermophilic bacterium Thermotoga maritima grown on glucan- and mannan-based polysaccharides. Appl. Environ. Microbiol. 68, 545-554 https://doi.org/10.1128/AEM.68.2.545-554.2002
  8. Chhabra, S. R., K. R. Shockley, S. B. Conners, K. L. Scott, R. D. Wolfinger and R. M. Kelly. 2003. Carbohydrate-induced differential gene expression patterns in the hyper-thermophilic bacterium Thermotoga maritima. J. Biol. Chem. 278, 7540-7552 https://doi.org/10.1074/jbc.M211748200
  9. Conners, S. B., C. I. Montero, D. A. Comfort, K. R. Shockley, M. R. Johnson, S. R. Chhabra and R. M. Kelly. 2005. An expression-driven approach to the prediction of carbohydrate transport and utilization regulons in the hyperthermophilic bacterium Thermotoga maritima. J. Bacteriol. 187, 7267-7282 https://doi.org/10.1128/JB.187.21.7267-7282.2005
  10. Dan, S., I. Marton, M. Dekel, B. A. Bravdo, S. He, S. Wither 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
  11. Daniel, R. M., D. A. Cowan, H. W. Morgan and M. P. A. Curran. 1982. A correlation between protein thermo-stability and resistance to proteolysis. Biochem. J. 207, 641-644 https://doi.org/10.1042/bj2070641
  12. Davies, G. and B. Henrissat. 1995. Structures and mechanisms of glycosyl hydrolases. Biochem. J. 3, 853-859
  13. Gabelsberger, J., W. Liebl and K. H. Schleifer. 1993. Cloning and characterization of ${\beta}$-galactoside and ${\beta}$-glucoside hydrolyzing enzymes of Thermotoga maritima. FEMS Microbiol. Lett. 109, 131-138
  14. Gabelsberger, J., W. Liebl and K. H. Schleifer. 1993. Purification and properties of recombinant ${\beta}$-glucosidase of the hyperthermophilic bacterium Thermotoga maritima. Appl. Microbiol. Biotechnol. 40, 44-52
  15. Goyal, K., P. Selvakumar and K. Hayashi. 2001. Characterization of a thermostable ${\beta}$-glucosidase (BglB) from Thermotoga maritima showing transglycosylation activity. J. Mol. Cat. B: Enzym. 15, 45-53 https://doi.org/10.1016/S1381-1177(01)00003-0
  16. Goyal, K., Y. -K. Kim, M. Kitaoka and K. Hayashi. 2001. Construction and characterization of chimeric enzymes of the Agrobacterium tumefaciens and Thermotoga maritima ${\beta}$-glucosidases. J. Mol. Cat. B:Enzym. 16, 43-51 https://doi.org/10.1016/S1381-1177(01)00043-1
  17. Hansen, T., B. Schlichting and P. Schonheit. 2002. Clucose-6-phosphate dehydrogenase from the hyper-thermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzyme. FEMS Microbiol. Lett. 216, 249-253 https://doi.org/10.1111/j.1574-6968.2002.tb11443.x
  18. Harbey, A. J., M. Hrmova, R. D. Gori, J. N. Varghese and G. B. Fincher. 2000. Comparative modeling of the three dimentional structures of family 3 glycoside hydrolases. Proteins 41, 257-269 https://doi.org/10.1002/1097-0134(20001101)41:2<257::AID-PROT100>3.0.CO;2-C
  19. Jiang, Z., Y. Zhu, L. Li, X. Yu, I. Kusakabe, M. Kitaoka and K. Hayashi. 2004. Transglycosylation reaction of xylanase B from the hyperthermophilic Thermotoga maritima with the ability of synthesis of tertiary alkyl ${\beta}$-D-xylobio-sides and xylosides. J. Biotechnol. 114, 125-134 https://doi.org/10.1016/j.jbiotec.2004.05.007
  20. Kim, J. H., B. R. Lee and M. Y. Pack. 1998. Overproduction and secretion of ${\beta}$-glucosidase in Bacillus subtilis. J. Microbiol. Biotechnol. 8, 141-145
  21. Legler, G., M. L. Sinnott and S. G. Withers. 1980. Catalysis by ${\beta}$-glucosidase A3 of Aspergillus wentii. J. Chem. Soc. Perkin Trands 2, 1376-1383
  22. Li, Y. K., J. Chir and F. Y. Chen. 2001. Catalytic mechanism of a family 3 ${\beta}$-glucosidase and mutagenesis study on residue Asp-247. Biochem. J. 355, 835-840 https://doi.org/10.1042/bj3550835
  23. Libel, W., J. Gabelsberger and K. H. Schleifer. 1994. Comparison amino acid sequence analysis of Thermotoga maritima ${\beta}$-glucosidase (BgIA) deduced from the nucleotide sequence of the gene indicates distant relationship between ${\beta}$-glucosidases of the BGA family and other families of ${\beta}$-1,4-glycosyl hydrolases. Mol. Gen. Genet. 242, 111-115
  24. Lim, W. J., S. K. Ryu, S. R. Park, M. K. Kim, C. L. An, S. Y. Hong, E. C. Shin, J. R. Lee, Y. P. Lim and H. D. Yun. 2005. Cloning of celC, third cellulase gene, from Pectobacterium carotovorum subsp. carotovorum LY34 and its comparison to those of Pectobacterium sp. J. Microbiol. Biotechnol. 15, 302-309
  25. Lim, W. J., S. R. Park, C. L. An, J. Y. Lee, S. Y. Hong, E. C. Shin, E. J. Kim, J. O. Kim, H. Kim and H. D. Yun. 2003. Cloning and characterization of a thermostable intracellular ${\alpha}$-amylase gene from the hyperfhermophilic bacterium Thermotoga maritima MSB8. Res. Microbiol. 154, 681-687 https://doi.org/10.1016/j.resmic.2003.09.005
  26. Nelson, K. E., R. A. Clayton, S. R. Gill, M. L. Gwinn, R. J. Dodson, D. H. Haft and E. K. Hickey. 1999. Evidence for lateral gene transfer between archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399, 323-329 https://doi.org/10.1038/20601
  27. Nguyen, T. N., A. D. Ejaz, M. A. Brancieri, A. M. Mikula, K. E. Nelson, S. R. Gill and K. M. NolL 2004. Whole-genome expression profiling of Thermotoga maritima in response to growth on sugars in a chemostat. J. Bacteriol. 186, 4824-4828 https://doi.org/10.1128/JB.186.14.4824-4828.2004
  28. Park, J. N., H. O. Kim, D. J. Shin, H. J. Kim, H. B. Lee, S. B. Chun and S. Bae. 2001. Cloning of a Paenibacillus sp. endo-${\beta}$-1,4-glucanase gene and its coexpression with the Endomyces fibuliger ${\beta}$-glucosidase gene in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 11, 685-692
  29. Park, S. R, W. J. Lim, M. K. Kim, S. Y. Hong, E. C. Shin, E. J. Kim, J. R. Lee, J. G. Woo, H. Kim and H. D. Yun. 2004. Analysis of cel and pel genes from Pectobacterium chrysanthemi PY35 for relatedness to pathogenecity. J. Microbiol. Biotechnol. 14, 1047-1051
  30. Ruttersmith, L. D. and R. M. Daniel. 1991. Thermostable cellobiohydrolase from the thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1. Purification and properties. Biochem. J. 277, 887-890 https://doi.org/10.1042/bj2770887
  31. Ruttersmith, L. D. and R. M. Daniel. 1993. Thermostable ${\beta}$-glucosidase and ${\beta}$-xylosidase from Thermotoga sp. strain FjSS3-B.1. Biochim. Biophys. Acta. 1156, 167-172 https://doi.org/10.1016/0304-4165(93)90132-R
  32. Sambrook, J. and D. W. Russell. 2001. Molecular Cloning:A Laboratory Manual, 3th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  33. Schroder, C., M. Selig and P. Schonheit. 1994. Glucose fermentation to acetate, $CO_2$ and $H_2$ in the hyperthermophilic eubacterium Thermotoga maritima: involvement of the Embden-Meyerhof pathway. Arch. Microbiol. 161, 460-470
  34. Selig, M., K. B. Xavier, H. Santos and P. Schonheit. 1997. Comparative analysis of Embden-Meyerhof and Entner-Doudoroff glycolytic pathways in hyperthermophilic archaea and the bacterium Thermotoga. Arch. Microbiol. 167, 217-232 https://doi.org/10.1007/BF03356097
  35. Simpson, H. D., U. R. Haufler and R. M. Daniel. 1991. An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga. Biochem. J. 277, 413-417 https://doi.org/10.1042/bj2770413
  36. Sinnott, M. L. 1990. Catalytic mechanism of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202 https://doi.org/10.1021/cr00105a006
  37. Stetter, K. O. 1992. Life at the upper temperature border, pp. 195-219 in: J. Van, K. T. T. Van, J. C. Mounolou, J. Schneider, C. McKay (Eds.), Colloque Interdisciplinaire du Comite National de la Recherche Scientifique, Frontiers of Life, Editions Frontieres, Gif-sur-Yvette
  38. Stetter, K. O., G. Fiala, G. Huber and A. Segerer. 1990. Hyperfhermophilic microorganisms. FEMS Microbiol. Rev. 75, 117-124 https://doi.org/10.1111/j.1574-6968.1990.tb04089.x
  39. Teeri, T. T. 1997. Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends Biotechnol. 15, 160-167 https://doi.org/10.1016/S0167-7799(97)01032-9
  40. Vieille, C. and G. J. Zeikus. 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1-43 https://doi.org/10.1128/MMBR.65.1.1-43.2001
  41. Varghese, J. N., M. Hrmova and G. B. Fincher. 1999. Three dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure 7, 179-190 https://doi.org/10.1016/S0969-2126(99)80024-0
  42. Wang, Q., D. Trimbur, R. Graham, R. A. J. Warren and S. G. Withers. 1995. Identification of the acid/base catalyst in Agrobacterium faecalis ${\beta}$-glucosidase by kinetic analysis of mutants. Biochemistry 34, 14554-14562 https://doi.org/10.1021/bi00044a034
  43. Wassenberg, D., W. Liebl and R. Jaenicke. 2000. Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties. J. Mol. Biol. 295, 279-288 https://doi.org/10.1006/jmbi.1999.3367
  44. Yang, M. J., S. H. Jung, E. S. Shin, J. H. Kim, H. D. Yun, S. L. Wong and H. Kim. 2004. Expression of a Bacillus subtilis endoglucanase in protease-deficient Bacillus subtilis strain. J. Microbiol. Biotechnol. 14, 430-434
  45. Yernool, A. D., J. K. Mccarthy, D. E. Eveleigh and J. D. Bok. 2000. Cloning and characterization of the glucooligo-saccharide catabolic pathway ${\beta}$-glucan glucohydrolas and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana. J. Bacteriol. 182, 5172-5179 https://doi.org/10.1128/JB.182.18.5172-5179.2000
  46. Zhengqiang, J., A. Kobayashi, M. M. Ahsan, L. Lite, M. Kitaoka and K. Hayashi. 2001. Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves ${\rho}$-nitrophenyl ${\beta}$-D-xyloside. J. Biosci Bioeng 92, 423-428 https://doi.org/10.1263/jbb.92.423
  47. Zverlov, V. V., I. Y. Volkov, T. V. Velikodvorskaya and W. H. Schwarz. 1997. Thermotoga neapolitana bglB gene, upstream of lamA, encodes a highly thermostable ${\beta}$-glucosidase that is a laminaribiase. Microbiology 143, 3537-3542 https://doi.org/10.1099/00221287-143-11-3537

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