Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Cloning, Sequencing and Expression of the Gene Encoding a Thermostable β-Xylosidase from Paenibacillus sp. DG-22

Paenibacillus sp. DG-22로부터 열에 안정한 β-xylosidase를 암호화하는 유전자의 클로닝, 염기서열결정 및 발현

  • 이태형 (동국대학교 과학기술대학 생명공학과) ;
  • 이용억 (동국대학교 과학기술대학 생명공학과)
  • Published : 2007.09.30
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Abstract

A genomic DNA library of the bacterium Paenibacillus sp. DG-22 was constructed and the ${\beta}-xylosi-dase-positive$ clones were identified using the fluorogenic substrate $4-methylumbelliferyl-{\beta}-D-xylopyr-anoside$ $({\beta}MUX)$. A recombinant plasmid was isolated from the clone and 4.3-kb inserted DNA was sequenced. The ${\beta}-xylosidase$ gene (xylA) was comprised of a 2,106 bp open reading frame (ORF) en-coding 701 amino acids with a molecular weight of 78,710 dalton and a pI of 5.0. The deduced amino acid sequence of the xylA gene product had significant similarity with ${\beta}-xylosidases$ classified into family 52 of glycosyl hydrolases. The xylA gene was subcloned into the pQE60 expression vector to fuse with six histidine-tag. The recombinant ${\beta}-xylosidase$ $(XylA-H_6)$ was purified to homogeneity by heat-treatment and immobilized metal affinity chromatography. The pH and temperature optima of the $XylA-H_6$ enzyme were pH 5.5-6.0 and $60^{\circ}C$, respectively.

세균인 Paenibacillus sp. DG-22의 유전체 DNA library가 제조되었으며, ${\beta}-xylosidase-$양성 클론이 형광기질인 $4-methylumbelliferyl-{\beta}-D-xylopyranoside$ $({\beta}MUX)$를 사용하여 확인되었다. 이 클론으로부터 재조합 플라스미드가 분리되었고 삽입된 4.3-kb 크기 DNA의 염기서열이 결정되었다. ${beta}-xylosidase$ 유전자는 분자량이 78.710 dal-ton이고 pI가 5.0인 701개의 아미노산을 암호화하는 2,106 염기쌍의 열린해독틀(ORF)로 구성되어있었다. xylA 유전자산물의 추론된 아미노산 서열은 과(family) 52에 속하는 클리코실 가수분해효소로 분류된 ${beta}-xylosidase$들과 상당한 유사성을 가지고 있었다. 이 xylA 유전자에 6개의 히스티딘-꼬리표를 붙이기 위해 pQE60 발현벡터에 다시 클로닝하였다. 재조합 ${beta}-xylosidase$ $(XylA-H_6)$가 열처리와 고정화금속친화성 크로마토그래피(IMAC)에 의해 순수하게 정제되었다. $XylA-H_6$ 효소의 최적 pH와 온도는 각각 pH 5.5-6.0과 $60^{\circ}C$이었다.

Keywords

References

  1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402 https://doi.org/10.1093/nar/25.17.3389
  2. Baba, T., R. Shinke and T. Nanmori. 1994. Identification and characterization of clustered genes for thermostable xylan-degrading enzymes, $\beta$-xylosidase and xylanase, of Bacillus stearothermophilus 21. Appl. Environ. Microbiol. 60, 2252-2258
  3. Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3, 286-290 https://doi.org/10.1016/0167-7799(85)90004-6
  4. 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
  5. Bravman, T., G. Zolotnitsky, S. Shulami, V. Belakhov, D. Solomon, T. Baasov, G. Shoham and Y. Shoham. 2001. Stereochemistry of family 52 glycosyl hydrolases: a $\beta$-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme. FEBS Lett. 495, 39-43 https://doi.org/10.1016/S0014-5793(01)02360-2
  6. Henrissat, B. and A. Bairoch. 1993. New family in the classification of glycosyl hydrolases based on amino acid sequence similarities, Biochem. J. 293, 781-788 https://doi.org/10.1042/bj2930781
  7. Henrissat, B. and A. Bairoch. 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316, 695-696 https://doi.org/10.1042/bj3160695
  8. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685 https://doi.org/10.1038/227680a0
  9. Lee, T. H., P. O. Lim and Y.-E. Lee. 2007. Cloning, characterization, and expression of xylanase A gene from Paenibacillus sp. DG-22 in Escherichia coli. J. Microbiol. Biotechnol. 17, 29-36
  10. Lee, T. H., P. O. Lim and Y.-E. Lee. 2007. Regulation of $\beta$-xylosidase biosynthesis in Paenibacillus sp. DG-22. J. Life Sci. 17, 407-411 https://doi.org/10.5352/JLS.2007.17.3.407
  11. Lee, Y-E. 2004. Isolation and characterization of thermostable xylanase-producing Paenibacillus sp. DG-22. Kor. J. Microbiol. Biotechnol. 32, 22-28
  12. Lee, Y-E. and J. G. Zeikus. 1993. Genetic organization, sequence and biochemical characterization of recombinant $\beta$-xylosidase from Thermoanuercoacterium saccharolyticum strain B6A-RI. J. Gen. Microbiol. 139, 1235-1243 https://doi.org/10.1099/00221287-139-6-1235
  13. Lee, Y-E. and P. O. Lim. 2004. Purification and characterization of two thermostable xylanases from Paenibacillus sp. DG-22. J. Microbiol. Biotechnol. 14, 1014-1021
  14. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3, 208-218 https://doi.org/10.1016/S0022-2836(61)80047-8
  15. Poutanen, K. and J. Puls. 1988. Characteristics of Trichoderma reesei $\beta$-xylosidase and its use in the hydrolysis of solubilized xylans. Appl. Microbiol. Biotechnol. 28, 425-432 https://doi.org/10.1007/BF00268208
  16. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, U.S.A
  17. Sanger, F., S. Nicklen and A. R. Coulson. 1977. DNA sequencing with chain- terminating inhibitors. Pro. Natl. Acad. Sci. USA 74, 5463-5467 https://doi.org/10.1073/pnas.74.12.5463
  18. Suzuki, T., E. Kitagawa, F. Sakakibara, K. Ibata, K. Usui and K. Kawai. 2001. Cloning, expression, and characterization of a family 52 $\beta$-xylosidase gene (xysB) of a multiple-xylanase-producing bacterium, Aeromonas caviae ME-1. Biosci. Biotechnol. Biochem. 65, 487-494 https://doi.org/10.1271/bbb.65.487
  19. Takami, H., K. Nakasone, Y. Takaki, G. Maeno, R. Sasaki, N. Masui, F. Fuji, C. Hirama, Y. Nakamura, N. Ogasawara, S. Kuhara and K. Horikoshi. 2000. Complete genome sequence of the alkaliphilic bacterium Bacillus holodurans and sequence comparison with Bacillus subtilis. Nucleic Acids Res. 28, 4317-4331 https://doi.org/10.1093/nar/28.21.4317
  20. Ward, O. P. and M. Moo-Young. 1989. Enzymatic degradation of cell wall and related plant polysaccharides. Crit. Rev. Biotechnol. 8, 237-274 https://doi.org/10.3109/07388558909148194
  21. Watson, M. E. E. 1984. Compilation of published signal sequences. Nucleic Acids Res. 12, 5145-5164 https://doi.org/10.1093/nar/12.13.5145
  22. Wong, K. K. Y. and J. N. Saddler. 1988. Multiplicity of $\beta$-1,4-xylanase in microorganisms: functions and applications. Microbiol. Rev. 52, 305-317