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소 반추위 메타게놈에서 새로운 섬유소분해효소 유전자(cel5C) 클로닝 및 유전산물의 특성

Cloning and Characterization of Cellulase Gene (cel5C) from Cow Rumen Metagenomic Library

  • Kim, Min-Keun (Gyeongsangnam-do Agricultural Research and Extension Service) ;
  • Barman, Dhirendra Nath (Division of Applied Life Science (BK21 Program), Gyeongsang National University) ;
  • Kang, Tae-Ho (Division of Applied Life Science (BK21 Program), Gyeongsang National University) ;
  • Kim, Jung-Ho (Department of Agricultural Chemistry, Sunchon National University) ;
  • Kim, Hoon (Department of Agricultural Chemistry, Sunchon National University) ;
  • Yun, Han-Dae (Division of Applied Life Science (BK21 Program), Gyeongsang National University)
  • 투고 : 2011.12.19
  • 심사 : 2012.02.14
  • 발행 : 2012.04.30

초록

한우의 반추위에서 게놈 DNA를 분리하여 메타게놈 은행을 구축한 다음 섬유소분해효소를 암호화하는 유전자를 클로닝 및 유전자를 선별하였다. 선별된 유전자의 DNA 염기서열 및 아미노산 서열을 분석하고 유전산물의 생화학적인 특성을 조사하였다. $cel$5C 유전자는 1,125 bp로 374개의 아미노산 잔기를 가진 단백질을 암호화하였으며 이 단백질 분자량은 42 kDa이었다. 이 효소의 최적 pH는 4 근방이었으며 최적 온도는 $50^{\circ}C$ 부근이었다. $cel$5C 유전자의 internal primer를 사용하여 인공적으로 배양할 수 있는 49종의 반추세균에서 분리한 게놈 DNA을 주형으로 PCR 분석한 결과 해당하는 밴드를 확인할 수 없었다. Cel5C는 현재로서는 배양할 수 없는 반추 미생물로 추정된다.

A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $E.$ $coli$ EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with $Sau$3AI, ligated into the $Bam$HI site of the pBluescript II SK+ vector, and transformed into $E.$ $coli$ $DH5{\alpha}$. We obtained a 1.5 kb insert DNA, designated $cel$5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from $E.$ $coli$ $DH5{\alpha}$, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and $50^{\circ}C$. We examined whether the $cel$5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the $cel$5C gene came from the unidentified cow rumen bacteria.

키워드

참고문헌

  1. Alrenbuchner, J. 1993. A new ${\lambda}RES$ vector with a built-in Tn 1721-encoded excision system. Gene 123, 63-68. https://doi.org/10.1016/0378-1119(93)90540-J
  2. An, J. M., Kim, Y. K., Lim, W. J., Hong, S. Y., An, C. L., Shin, E. C., Cho, K. M., Choi, B. R., Kang, J. M., Lee, S. M., Kim, H. and Yun, H. D. 2005. Evaluation of a novel bifunctional xylanase-cellulase constructed by gene fusion. Enzyme Microb. Technol. 36, 989-995. https://doi.org/10.1016/j.enzmictec.2005.01.030
  3. Birsan, C. P., Johnson, M., Joshi, A., MacLeod, L., McIntosh, V., Menem, M., Nitz, D. R., Rose, D., Tull, W. W. Wakarchuck, Q. Wang, R. A. J. Warren, White, A. and Withers, S. G. 1998. Mechanisms of cellulases and xylanases. Biochem. Soc. Trans. 26, 156-160.
  4. Cho, S. J. and Yun, H. D. 2005. Cloning of ${\alpha}$-amylase gene from unculturable bacterium using cow rumen metagenome. J. Life Sci. 15, 1013-1021. https://doi.org/10.5352/JLS.2005.15.6.1013
  5. Cho, K. M., Shin, E. C, Lim, W. J., Hong, S. Y., Choi, B. R., Kang, J. M., Lee, S. M., Kim, Y. H., Cho, S. J., Kim, H. and Yun, H. D. 2006. 16S rDNA analysis of bacterial diversity in three fractions of cow rumen. J. Microbiol. Biotechnol. 16, 92-101.
  6. Daniel, R. 2004. The soil metagenome-a rich resource for the discovery of novel natural products. Curr. Opin. Biotechnol. 15, 199-204. https://doi.org/10.1016/j.copbio.2004.04.005
  7. Ferrer, M., Golyshina, O. V., Chernikova, T. N., Khachane, A. N., Reyes-Duarte, D., Martins Dos Santos, V. A. P., Strompl, C., Elborough, K., Jarvis, G., Neef, A., Yakimov, M. M., Timmis, K. N. and Golyshin, P. N. 2005. Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environ. Microbiol. 7, 1996-2010. https://doi.org/10.1111/j.1462-2920.2005.00920.x
  8. Fields, M. W., Russell, J. B. and Wilson, D. B. 1998. The role of ruminal carboxymethyl cellulases in the degradation of ${\beta}$-glucans from cereal grain. FEMS Microbiol. Ecol. 27, 261-268.
  9. Henrissat, B., Teeri, T. T. and Warren, R. A. J. 1998. A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants. FEBS Lett. 425, 352-354. https://doi.org/10.1016/S0014-5793(98)00265-8
  10. Hess, M., Sczyrba, A., Egan, R., Kim, H. T., Chokhawala, W., Schroth, S. Luo, G., Clark, D. S., Chen, F., Zhang, T., Mackie, R. I., Pennacchio, L. A., Tringe, S. G., Visel, A., Woyke, T., Wang, Z. and Rubin, E. M. 2011. Metagenomic discovery of biomass-degrading genes and genomes from cow rumen. Science 331, 463-467. https://doi.org/10.1126/science.1200387
  11. Hristov, A. N., McAllister, T. A. and Cheng, K. J. 1998. Effect of dietary or abomasal supplementation of exogenous polysaccharide- degrading enzymes on rumen fermentation and nutrient digestibility. J. Anim. Sci. 76, 3146-3156.
  12. Hristov, A. N., McAllister, T. A. and Cheng, K. J. 1998. Stability of exogenous polysaccharide-degrading enzymes in the rumen. Anim. Feed. Sci. Technol. 76, 161-168. https://doi.org/10.1016/S0377-8401(98)00217-X
  13. Hungate, R. E. 1966. The rumen and its microbe. Academic Press, Inc., New York.
  14. Islam, S. A., Kim, M. K., Math, R. K., Reddy, S. R., Kim, E. J., Kim, J., Kim, H. and Yun, H. D. 2010. Cloning and characterization of a novel carboxylesterase gene from cow rumen metagenomic library. J. Life Sci. 20, 1306-1313. https://doi.org/10.5352/JLS.2010.20.9.1306
  15. Kudo, H., Cheng, K. J. and Costerton, J. W. 1987. Electron microscopic study of the methyl cellulose-mediated detachment of cellulolytic rumen bacteria from cellulose fibers. Can. J. Microbiol. 33, 267-272. https://doi.org/10.1139/m87-045
  16. Kuriki, R., Okada, S. and Imanaka, T. 1988. New type of pullulanase from Bacillus strearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis. J. Bacteriol. 170, 1554-1559.
  17. Lam, T. B. T., Iiyama, K. and Stone, B. A. 1990. Primary and secondary walls of grasses and other forage plants: taxonomic and structural considerations. In Akin D. E., Ljungdahl, L. G., Wilson, J. R. and Harris, P. J. (eds.), Microbial and Plant Opportunities to Improve Lignocellulose Utilization by Ruminants, pp. 43-69, Elsevier Science Publishers, London.
  18. Lee, R. L., Paul, J. W., Willem, H. Z. and Isak, S. P. 2002. Microbial cellulose utilization: Fundamentals and Biotechnology. Microbiol. Mol. Biol. Rev. 66, 506-577. https://doi.org/10.1128/MMBR.66.3.506-577.2002
  19. Levy, I., Shani, Z. and Shoseyov, O. 2002. Modification of polysaccharides and plant cell wall by endo-1,4-${\beta}$-glucanase and cellulose-binding domains. Biomol. Eng. 19, 17-30. https://doi.org/10.1016/S1389-0344(02)00007-2
  20. Lim, W. J., Park, S. R., Cho, S. J., Kim, M. K., Ryu, S. K., Hong, S. Y., Seo, W. T., Kim, H. and Yun, H. D. 2001. Cloning and characterization of an intracellular isoamylase gene from Pectobacterium chrysanthemi PY35. Biochem. Biophys. Res. Commun. 287, 348-354. https://doi.org/10.1006/bbrc.2001.5594
  21. Lorenz, P. and Schleper, C. 2002. Metagenome-a challenging source of enzyme discovery. J. Mol. Catal. B. 20, 13-19. https://doi.org/10.1016/S1381-1177(02)00147-9
  22. McNeil, M., Darvill, A. G., Fry, S. C. and Albersheim, P. 1984. Structure and function of the primary cell wall of plants. Ann. Rev. Biochem. 53, 625-663. https://doi.org/10.1146/annurev.bi.53.070184.003205
  23. Miron, J., Ben-Ghedalia, D. and Morrison, M. 2001. Invited review: adhesion mechanisms of rumen cellulolytic bacteria. J. Dairy Sci. 84, 1294-1309. https://doi.org/10.3168/jds.S0022-0302(01)70159-2
  24. Mittendorf, V. and Thomson, J. A. 1993. Cloning of an endo-(1, 4)-beta-glucanase gene, celA, from the rumen bacterium Clostridium sp. (C. longisporum) and characterization of its product, CelA, in Escherichia coli. J. Gen. Microbiol. 139, 3233-3242. https://doi.org/10.1099/00221287-139-12-3233
  25. Park, S. R., Kim, M. K., Kim, J. O., Cho, S. J., Cho, Y. U. and Yun, H. D. 2000. Cloning and sequencing of cel5Z gene from Erwinia chrysanthemi PY35. Mol. Cells 10, 269-274.
  26. Poole, D. M., Hazlewood, G. P., Laurie, J. I., Barker, P. J. and Gilbert, H. J. 1990. Nucleotide sequence of the Ruminococcus albus SY3 endoglucanase genes celA and celB. Mol. Gen. Genet. 223, 217-223.
  27. Sambrook, J. and Russell, D. W. 2001. Molecular cloning. A Laboratory Manual, 3th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  28. Satoshi, K. and Yasuo, K. 2001. Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FEMS Microbiol. Lett. 204, 361-366. https://doi.org/10.1111/j.1574-6968.2001.tb10911.x
  29. Schloos, P. D. and Handelsman, J. 2003. Biotechnological prospects from metagenomics. Curr. Opin. Biotechnol. 14, 303-310. https://doi.org/10.1016/S0958-1669(03)00067-3
  30. Streit, W. R., Daniel, R. and Jaeger, K. E. 2004. Prospecting for biocatalysts and drugs in the genomes for noncultured microorganisms. Curr. Opin. Biotechnol. 15, 285-290. https://doi.org/10.1016/j.copbio.2004.05.006
  31. Teather, R. and Wood, P. J. 1982. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43, 770-780.
  32. Vazquez-Laslop, N., Lee, J., Hu, R. and Neyfakh, A. A. 2001. Molecular seive mechanism of selective release of cytoplasmic proteins by osmotically shocked Escherichia coli. J. Bacterili. 183, 2399-2404. https://doi.org/10.1128/JB.183.8.2399-2404.2001
  33. Wang, F., Li, F., Chen, G. and Liu, W. 2009. Isolation and characterization of novel cellulase genes from uncultured microorganisms in different environmental niches. Microbiol. Res. 164, 650-657. https://doi.org/10.1016/j.micres.2008.12.002
  34. Weimer, P. J., Waghorn, G. C., Odt, O. L. and Mertens, D. R. 1999. Effect of diet on populations of three species of ruminal cellulolytic bacteria in lactating dairy cows. J. Dairy Sci. 82, 122-134. https://doi.org/10.3168/jds.S0022-0302(99)75216-1

피인용 문헌

  1. Cloning and characterization of thermo-alkalistable and surfactant stable endoglucanase from Puga hot spring metagenome of Ladakh (J&K) vol.103, 2017, https://doi.org/10.1016/j.ijbiomac.2017.05.113