Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Molecular Cloning and Nucleotide Sequence of Xylanase gene (xynT) from Bacillus alcalophilus AX2000.

Bacillus alcalophilus AX2000 유래 xylanase 유전자 (XynT)의 Cloning과 염기서열 분석

  • 박영서 (경원대학교 생명공학부)
  • Published : 2005.10.01
Download PDF
⟨ Previous Next ⟩

Abstract

A gene coding for xylanase from alkali-tolerant Bacillus alcalophilus AX2000 was cloned into Escherichia coli $DH5\alpha$ using pUC19. Among 2,000 transformants, one transformant showed clear zone on the detection agar plate containing oat-spells xylan. Its recombinant plasmid, named pXTY99, was found to carry 7.0 kb insert DNA fragment. When the nucleotide sequence of the cloned xylanase gene (xynT) was determined, xynT gene was found to consist of 1,020 base-pair open reading frame coding for a poly-peptide of 340 amino acids with a deduced molecular weight of 40 kDa. The coding sequence was preceded by a putative ribosome binding site, and the transcription initiation signals. The deduced amino acid sequence of xylanase is similar to those of the xylanases from Bacillus sp. Nl37 and B. stearothermophilus 21 with $61\%$ and $59\%$ identical residues, respectively.

Xylanase를 생산하는 알칼리 내성 Bacillus alcalophilus AX2000의 chromosomal DNA로부터 xylanase 유전자를 cloning하여 그 염기배열 순서를 결정한 다음 이로부터 유전자 발현에 관련된 구조를 분석하였다. Xylanase 유전자의 cloning을 위해 제한효소 PstI으로 절단한 B. alcalophilus AX2000의 chromosomal DNA와 pUC19을 ligation 시켜 E. coli $DH5\alpha$에 형질전환시킨 후 형질전환체 중에서 xylanase 활성을 나타내는 재조합 plasmid pXTY99를 분리하였다. 재조합 plasmid pXTY99은 pUC19의 PstI 부위 내에 7kb의 외래 DNA가 삽입 되 었다. Cloning된 xylanase 유전자(xynT)의 염기배열을 분석한 결과 유전자의 크기는 1,020 bp이었고 이는 340개의 아미노산으로 구성된 분자량 40 kDa의 poly-peptide를 coding하고 있었다. 이 염기배열은 AUG 개시 codon으로부터 각각 259와 282 base상류에 TACAAT의 -10 box와 GTTCACA인 -35 box로 추정되는 염기배열이 존재하였으며 ribosome 결합부위가 존재하였다. B. alcalophilus AX2000의 xylanase와 아미노산배열의 유사성이 가장 높은 xylanase는 Bacillus sp. N137과 B. stearothemophilus 21 유래의 xylanase로 각각 $61\%$와 $59\%$의 유사성을 나타내었다.

Keywords

References

  1. Kulkarni, N., A. Shendye, M. Rao. 1999. Molecular and biotechological aspects of xylanases. FEMS Microbiol. Rev. 23, 411-456 https://doi.org/10.1111/j.1574-6976.1999.tb00407.x
  2. McCarthy, A. A., D. D. Morris, P. J. Bergquist and E. N. Baker. 2000. Structure of XynB, a highly thermostable beta-1,4-xylanase from Dictyogiomus thermophilum Rt46B.1, at 1.8 A resolution. Acta Crystallogr. D. BioI. Crystallogr. 11, 1367-1375 https://doi.org/10.2210/pdb1f5j/pdb
  3. Moran C. P. Jr, N. Lang, S. F. LeGrice, G. Lee, M. Stephens, A. J. Sonenshein, J. Pero and R. Losick. 1982. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol. Gen. Genet. 186, 339-346 https://doi.org/10.1007/BF00729452
  4. Park, Y. S. and T. Y. Kim. 2003. Isolation of Bacillus alcalaphilus AX2000 producing alkaline xylanase and its enzyme production, Korean J. Microbol. Biotechnol. 31, 157-164
  5. Sambrook, J. and D. Russell. 2001. Molecular cloning - A laboratory manual. 3rd eds., CSHL Press. Cold Spring Harbor, New York
  6. Shorenstein, R. G., R. Losick. 1973. Comparative size and properties of the sigma subunits of ribonucleic acid polymerase from Bacillus subtilis and Escherichia coli. J. Biol. Chem. 248, 6170-6173
  7. Somogyi, M. 1952. Notes on sugar determinations. J. Biol. Chem. 195, 19-23
  8. Timell, T. E. 1965. Wood hemicelluloses: Part II. Carbohydr. Chem. 20, 409-483
  9. Timell. T. E. 1967. Recent progress in the chemistry of wood hemicelluloses. Wood Sci. Technol. 1, 45-70 https://doi.org/10.1007/BF00592255
  10. Velikodvorskaya, T. V., I. Y. Volkov, V. T. Vasilevko, V. V. Zverlov and E. S. Piruzian. 1997. Purification and some properties of Thermotoga neapolitana thermostable xylanase B expressed in E. coli cells. Biochemistry 62, 66-70
  11. Wakarchuk, W. W., R. J. Campbell, W. J. Sung, J. Davoodi, M. Taguchi M. 1994. Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase. Protein Sci. 3, 467-475 https://doi.org/10.1002/pro.5560030312
  12. Warren, R. A. J. 1996. Microbial hydrolysis of polysaccharides. Annu. Rev. Microbial. 50, 183-212 https://doi.org/10.1146/annurev.micro.50.1.183
  13. Wong, K. K. Y. and J. N. Saddler. 1992. Applications of hemicellulases in the food, feed, and pulp and paper industries, In: Hemicellulose and hemicellulases (Coughlen, P. P. and G. P. Hazlewood, eds), pp. 127-143, Portland Press, London
  14. Wong, K. K. Y., J. U. J. Tan and J. N. Saddler. 1988. Multiplicity of ${\beta}$-1,4-xylanase in microorganism: Functions and applications. Microbial. Rev. 52, 305-317
  15. Xie, H., H. J. Gilbert, S. J. Charnock, G. J. Davies, M. P. Williamson, P. J. Simpson, S. Raghothama, C. M. Fontes, F. M. Dias, J. M. Ferreira and D. N. Bolam. 2001. Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding. Biochemistry 40, 9167-9176 https://doi.org/10.1021/bi0106742

Cited by

  1. Characterization and pH-dependent substrate specificity of alkalophilic xylanase from Bacillus alcalophilus vol.39, pp.10, 2012, https://doi.org/10.1007/s10295-012-1159-0