Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Molecular Cloning and Analysis of Nucleotide Sequence of Xylanase Gene (xynk) from Bacillus pumilus TX703

Bacillus pumilus TX703 유래 Xylanase 유전자(xynK)의 Cloning과 염기서열 분석

  • 박영서 (경원대학교 식품생물공학과)
  • Published : 2002.04.01
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Abstract

A gene coding for xylanase from thermo-tolerant Bacillus pumilus TX703 was cloned into Escherichia coli DH5 $\alpha$ using pUC19. Among 7,400 transformants, four transformants showed clear zones on the detection agar plates containing oat-spells xylan. One of them which showed highest xylanase activity was selected and its recombinant plasmid, named pXES106, was found to carry 2.24 kb insert DNA fragment. When the nucleotide sequence of the cloned xylanase gene (xynK) was determined, xynK gene was found to consist of 1,227 base-pair open reading frame coding for a polypeptide of 409 amino acids with a deduced molecular weight of 48 kDa. The coding sequence was preceded by a putative ribosome binding site, the transcription initiation signals, and cia-acting catabolite responsive element. The deduced amino acids sequence of xylanase is similar to those of the xylanases from Hordeum vulgare (barley) and Clostridium thermocellum, with 39 and 31% identical residues, respectively. The amino acids sequence of this xylanase was quite different from those of the xylanases from other Bacillus species.

Xylanase를 생산하는 내열성 Bacillus pumilus TX703의 chromosomal DNA로부터 xylanase 유전자를 cloning하여 그 염기배열 순서를 결정한 다음 이로부터 유전자 발현에 관련된 구조를 분석하였다. Xylanase 유전자의 cloning을 위해 제한효소 HindIII로 절단한 B. pumilus TX703의 chromosomal DNA와 pUC19을 ligation시켜 E. coli DH5 $\alpha$에 형질전환시킨 후 형질전환체 중에서 xylanase 활성을 나타내는 재조합 plasmid pXES106을 분리하였다. 재조합 plasmid pXES106은 pUC19의 HindIII 부위 내에 2.24 kb의 외래 DNA가 삽입되었고, 이 plasmid DNA를 분리하여 E. coli DH5 $\alpha$에 재형질전환시킨 결과 vector 내에 xylanase 유전자가 cloning되었음을 확인하였다. Cloning된 유전자의 염기배열을 분석한 결과 이 유전자의 총 크기는 2,187 bp였고 이는 409개기 아미노산을 coding 하는 open reading frame 1,227 bp를 포함하고 있었다. 이 염기배열은 ATG개시 codon으로부터 각각 193과 216 base 상류에 TTTAAT의 -10 box와 TCGAAA인 -35 box로 추정되는 염기배열이 존재하였고 -10 box로부터 7 bp하류에 전사개시점인 A가 위치하고 있었다. 또한, 개시 codon으로부터 432 bp 상류에 공통염기배열과 14개의 염기 중 11개의 염기가 일치하는 TGATGGCGTCGGCA의 catabolite responsive element (CRE)가 존재하였다. B. pumilus TX703의 xylanase와 아미노산배열의 유사성이 가장 높은 xylanase는 Hordeum vulgare의 isozyme X-I이었고 본 xylanase는 208번째와 322번째에 glutamic acid 잔기를 가지고 있어 Clostridium thermocellum, Dictyoglomus thermophilum, Thermotoga neapolitana 등에서 밝혀진 바와 같이 glutamic acid 부위가 xylanase의 활성부위라 여겨진다.

Keywords

References

  1. Gene v.26 Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli Bernier;R. Jr.;H. Driguez;M. Desrochers https://doi.org/10.1016/0378-1119(83)90036-7
  2. Appl. Environ. Microbiol. v.61 Protein purification and properties of xulanase A from alkali-tolerant Bacillus sp. strain BP-23. Blanco A.;T. Vidal;J. F. Colom;F. I. Pastor
  3. Microbiol. Rev v.45 Cyclic nucleotides in procaryotes Botsford;J. L.
  4. Carbohydr. Res. v.159 Investigation of the structure of a heteroxylan from the outer pericarp (beeswing bran of wheat kernel) Brillouet;J. M.;J. P. Joseleau https://doi.org/10.1016/S0008-6215(00)90009-0
  5. American Society for Microbioligy Biochemistry, Physiology and Molecular Genetics Chambliss;G. H.
  6. Carbohydr. Res. v.129 Lignin-xylan ester linkage in mesta fiber (Hibiscus cannabinus) Das;N. N.;S. C. Das;A. K. Sarkar;A. K. Mukherjee https://doi.org/10.1016/0008-6215(84)85312-4
  7. Mol. Microbiol. v.15 Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria Deutscher;J.;E. Kster;U. Bergstedt;V. Charrier;W. Hillen https://doi.org/10.1111/j.1365-2958.1995.tb02280.x
  8. J. Microbiol. Biotechnol. v.3 Selection and characterization of catabolite repression resistant mutant of Bacillus firmus var. alkalophilus producing cyclodextrin glucanotransferase Do;E. J.;H. D. Shin;C. Kim;Y. H. Lee
  9. Mol. Microbiol. v.11 The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system Duong;F.;C. Soscia;A. Lazdunski;M. Murgier. https://doi.org/10.1111/j.1365-2958.1994.tb00388.x
  10. Apple. Environ. Microbiol v.60 Cloning and DNA sequence of the gene coding for Bacillus stearothermophilus T-6 xylanase Gat;O. A. Lapidot;T. I. Alchana;C. Regueros;Y. Shoham
  11. Nucleic Acids Res. v.9 Codon catalog usage is a genome strategy modulated for gene expressivity Grantham R;C. Gautier;M. Gouy;M. Jacobzone;R. Mercier
  12. J. Bacteriol. v.173 Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism Guzzo;J.;J. M. Pages;F. Duong;A. Lazdunski;M. Murgier https://doi.org/10.1128/jb.173.17.5290-5297.1991
  13. J. Mol. Biol. v.166 Studies on transformation of Escherichia coli with plasmids Hanahan D. https://doi.org/10.1016/S0022-2836(83)80284-8
  14. Mol. Microbiol. v.5 Catagolite repression of α-amy-lase gene expression in Bacillus subtilis involves a transacting gene product homologous to the Escherichia colilacI and gaIR repressors Henkin;T. M.;F. J. Grundy;W. L. Nicholson;G. H. Chambliss https://doi.org/10.1111/j.1365-2958.1991.tb00728.x
  15. Mol. Microbiol. v.15 Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the Gram-positive bacteria? Hueck;C. J;H. Woofgang https://doi.org/10.1111/j.1365-2958.1995.tb02252.x
  16. J. Bacteriol v.161 Identification of the transcriptional suppressor sof-1 as an alteration in the spo0A protein Hoch;J. A.;K. Trach;F. Kawamura;H. Saito
  17. Mol. Biol. Evol. v.2 Codon usage and tRNA content in unicellular and multicellular organisms Ikemura;T.
  18. FEMS Microbiol. Rev. v.23 Molecular and biotechnological aspects of xylanases Kulkarni;N.;A. Shendye;M. Rao. https://doi.org/10.1111/j.1574-6976.1999.tb00407.x
  19. J. Bacteriol. v.171 Cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: Effect of sporulation mutations and catabolite repression resistance mutations on expression Laoide B. M.;D. J. J. McConnell https://doi.org/10.1128/jb.171.5.2443-2450.1989
  20. Kor. J. Apple. Microbiol. Biotechnol. v.25 Nucleotide sequence of the estl gene coding for Bacillus stearothermophilus acetylxylan esterase Lee;J. S.;Y. J. Choi
  21. EMBO J. v.9 Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli alpha-haemolysin Letoffe;S.;P. Delepelaire;C. Wandersman
  22. Mol. Gen. Genet v.201 Genetical and functional organisation of the Escherichia coli haemolysin determinant 2001 Mackman;N.;J. M. Nicaud;L. Gray;I. B. Holland. https://doi.org/10.1007/BF00425672
  23. Acta Crystallogr. D. Biol. Crystallogr v.11 Structure of XynB, a highly ther-mostable beta-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1,at 1.8 A resolution McCarthy;A. A.;D. D. Morris;P. L. Bergquist;E. N. Baker
  24. J. Biol. Chem. v.256 Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus bata-lactamase gene McLaughlin;J. R.;C. L. Murray;J. C. Rabinowitz
  25. Mol. Gen. Genet. v.186 Nucleotide sequences that signal the initiation of trascription and translation in Bacillus subtilis Moran C. P. Jr.;N. Lang;S. F. LeGrice;G. Lee;M. Stephens;A. L. Sonenshein;J. Pero;R. Losick. https://doi.org/10.1007/BF00729452
  26. J. Bacteriol. v.172 Purification and properties of ther-mostable sylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain Nanmori T,;T. Watanabe;R. Shinke;A. Kohno;Y. Kawamura https://doi.org/10.1128/jb.172.12.6669-6672.1990
  27. Kor. J. Appl. Microbiol. Biotechnol. v.27 Isolation of xylanase-producing thermo-tolerant Bacillus sp. and its enzyme production Park;Y. S.;M. Y. Kang;H. G. Chang;G. G. Park;J. B. Kang;J. K. Lee;T. K. Oh
  28. Recombinant DNA Techniques-An Introduction Rodriguez;R. L.;R. C. Tait
  29. The New Biologist v.3 A multiplicity of potential carbon catabolite repression mechanisms in prokaryotic and eukaryotic microorganisms Saier;M. H. Jr.
  30. Molecular cloning-A laboratory manual(3rd eds) Sambrook;J.;D. Russell
  31. Phytochemistry v.24 Ether linkage between phenolic acids and lignin franctions from wheat straw Scalbert;A.;B. Monisies;J. Y. Lallemand;E. Guittet;C. Rolando https://doi.org/10.1016/S0031-9422(00)81133-4
  32. Gene v.107 Sequences of three genes specifying xylanases in Streptomyces lividans Shareck;F.;C. Roy;M. Yaguchi;R. Morosoli;D. Kluepfel https://doi.org/10.1016/0378-1119(91)90299-Q
  33. J. Biol Chem. v.248 Comparative size and properties from Bacillus subtilis and Escherichia coli Shorenstein;R. G.;R. Losick
  34. J. Biol. Chem. v.195 Notes on sugar determinations Somogyi;M.
  35. Crit. Rev. Biotechnol. v.17 Xylanolytic enzymes from fungi and bacteria Sunna;A.;G. Antranikian https://doi.org/10.3109/07388559709146606
  36. Appl. Environ. Microbiol. v.61 Cloning and DNA sequencing of xyaA, a gene encoding an endo-beta-1,4-xylanase from an alkalophilic Bacillus strain (N137) Tabernero;C.;J. Sanchez-Torres;P. Perez;R. I. Santamaria
  37. Carbohydr. Chem. v.20 Wood hemicelluloses: Part Ⅱ. Timell;T. E.
  38. Wood Sci. Technol. v.1 Recent progress in the chemistry of wood hemicelluloses Timell;T. E. https://doi.org/10.1007/BF00592255
  39. coli cells. Biochemistry v.62 Purificaition and some properties of Thermotoga neapolitana thermostable xylanase B expressed in D. Velikodvorskaya;T. V.;I. Y. Volkov;V. T. Vasilevko;V. V. Zverlov;E. S. Piruzian
  40. Gene v.19 The pUC plasmids, an M13mp7-derived system for insetion mutagenesis and sequencing with synthetic universal primers Vieira J.;J. Messing https://doi.org/10.1016/0378-1119(82)90015-4
  41. Protein Sci. v.3 Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase Wakachuk;W. W.;R. L. Campbell;W. L. Sung;J. Davoodi;M. Yaguchi https://doi.org/10.1002/pro.5560030312
  42. Annu. Rev. Microbiol. v.50 Microbial hydrolysis of polysaccharides Warren;R. A. J. https://doi.org/10.1146/annurev.micro.50.1.183
  43. Proc. Natl. Acad. Sci. v.87 Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis Weickert M. J.;G. H. Chambliss https://doi.org/10.1073/pnas.87.16.6238
  44. Hemicelolose and Hemicellulases Applications of hemicellulases in the food, feed, and pulp and paper industries Wong;K. K. Y.;J. N. Saddler;Coughlen(ed);P. P.(ed);G. P.Hazlewood(ed)
  45. Microbiol. Rev. v.52 Multiplicity of β-1,4-xylanase in microorganism: Functions and applications Wong;K. K. Y.;L. U. L. Tan;J. N. Saddler
  46. Biochemistry v.40 Clostridium theromcellum Xyn10B carbohydratebinding module 22-2: the role of conserved amino acids in ligand binding 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;L. M. Ferreira;D. N. Bolam. https://doi.org/10.1021/bi0106742