Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Constitutive Expression of Arylsulfatase from Pseudoalteromonas carageenovora in E. coli and Its Application to Preparation of Agarose

E. coli에서 Pseudoalteromonas carageenovora 유래 Arylsulfatase의 구성적 발현과 Agarose 제조에의 응용

  • Kim, Mi-Jin (Department of Biotechnology and Bioengineering, Department of Biomaterial Control, Dong-Eui University) ;
  • Jang, Yhon-Hwa (Department of Biotechnology and Bioengineering, Department of Biomaterial Control, Dong-Eui University) ;
  • Sung, Moon-Hee (BioLeaders Corp) ;
  • Kim, Yeon-Hee (Department of Biotechnology, Osaka University) ;
  • Nam, Soo-Wan (Department of Biotechnology and Bioengineering, Department of Biomaterial Control, Dong-Eui University)
  • 김미진 (동의대 생명공학과 바이오물질제어학과) ;
  • 장연화 (동의대 생명공학과 바이오물질제어학과) ;
  • 성문희 ((주)바이오리더스) ;
  • 김연희 (오사카대학 응용생물공학과) ;
  • 남수완 (동의대 생명공학과 바이오물질제어학과)
  • Published : 2007.03.28
Download PDF
⟨ Previous Next ⟩

Abstract

The arylsulfatase gene (astA, 984 bp ORF) from Pseudoalteromonas carrageenovora genome was amplified by PCR and subcloned into the pHCE-IA vector, in which the hyper consitutive expression (HCE) promoter from the D-amino acid aminotransferase (D-AAT) gene of Geobacillus toevii was employed. The transformant cell, Escherichia coli BL21 (DE3)/pHCE-AST, on LB agar plate containig 4-methylumbelliferyl sulfate, showed an intense fluorescence at 360 nm, indicating that 4-methylumbelliferone was liberated by desulfatate activity. When BL21 (DE3)/pHCE-AST was grown on LB media containing 0.4% glucose or 0.4% glycerol, the arylsulfatase activity was higher at glycerol rather than at glucose. On 2% glycerol medium, the arylsulfatase activity reached 15.0 unit/ml, which was 2.6-fold higher expression level than that with 1% glycerol. The DNA ladder in agarose prepared from agar by this recombinant enzyme revealed similar resolution and migration patterns with a commercial agarose. This results suggests that arylsulfatase overexpressed in E. coli could be applicable to the economic production of electrophoretic-grade agarose.

Pseudoalteromonas carrageenovora 유래의 arylsulfatase 유전자는 PCR로 증폭한 후 Geobacillus toebii의 D-amino acid aminotransferase(D-ATT) 유전자 유래의 구성적 발현 promoter를 함유하는 pHCE-IA vector로 subcloning 하였다. 4-Methylumbelliferyl sulfate가 포함된 LB 평판배지 상에서 자란 형질전환체 Escherichia coli BL2l (DE3)/pHCE-AST는 360 nm상에서 4-methylumbellifrrone에 의한 강한 형광을 보였고, 이는 대장균에서 arylsulfatase가 활성형으로 생산되었음을 의미하였다. E. coli BL21 (DE3)/pHCE-AST를 0.4% glycerol 또는 0.4% glucose가 포함된 LB 배지로 배양했을 때 arylsulfatase활성은 glycerol이 포함된 배지에서 활성이 더 높게 나타났다. 2% glycerol이 포함된 LB배지에서 arylsulfatase 활성은 약 15.0 unit/ml에 달했으며, 이는 1% glycerol을 첨가해서 배양했을 때보다 2.6배 이상의 높은 발현 수준이였다. 재조합 arylsulfatase 효소로 제조된 agarose와 시판용 agarose를 DNA markers를 이용해서 전기영동 성능을 비교했을 때 우수한 이동성과 분리능을 보였다. 본 연구의 결과, E. coli에서 과발현 생산된 arylsulfatase 효소를 이용하여 전기영동용 고순도 agarose생산 공정에 적용 가능함을 확인하였다.

Keywords

References

  1. Akagawa-Matsushita, M., M. Matsuo, Y. Koga, and K. Yamasato. 1992. Alteromonas atlantica sp. nov. and Alteromonas carrageenovora sp. nov., bacteria that decompose algal polysaccharides. Int. J. Syst. Bacteriol. 42: 621-627 https://doi.org/10.1099/00207713-42-4-621
  2. Allan, G., P. G. Johnson, Y. Lay, and K. V. Sarkanen. 1971. Marine polymers; Part 1. A new procedure for the fractionation of agar. Carbohydr. Res. 17: 234-236 https://doi.org/10.1016/S0008-6215(00)81565-7
  3. Araki, C. 1937. Agar-agar. III. Acetylation of the agar-like substance of Gelidium amansii(L). J. Chem. Soc. Japan. 58: 1338-1350
  4. Barbeyron, T., P. Potin, C. Richard, O. Collin, and B. Kloareg. 1995. Arylsulphatase from Alteromonas carrageenovora. Microbiology 141: 2897-2904 https://doi.org/10.1099/13500872-141-11-2897
  5. Beil, S., H. Kehrli, J. Peter, W. Staudenmann, A. M. Cook, T. Leisinger, and M. A. Kertesz. 1995. Purification and characterization of the agaropectin sulfatase synthesized by Psedomonas aeruginosa PAO during growth in sulfate-free medium and cloning of the arylsulfatase gene (atsA). Eur. J. Biochem. 229: 385-394 https://doi.org/10.1111/j.1432-1033.1995.0385k.x
  6. De Hostos, E. L., R. K. Togasaki, and A. Grossman. 1988. Purification and biosynthesis of a derepressible periplasmic arylsulfatase from Chlamydomonas reinharditii. J. Cell Biol. 106: 29-37 https://doi.org/10.1083/jcb.106.1.29
  7. Delisle, G. and F. H. Milazzo. 1970. The isolation of arylsulphatase isoenzymes from pseudomonas aeruginosa. Biochim. Biophys. Acta 212: 505-508 https://doi.org/10.1016/0005-2744(70)90258-5
  8. Do, J. R. 1997. Extraction and purification of agar from Gelidium amansii. J. Kor. Fish. Soc. 30: 423-427
  9. Do, J. R. and S. W. Oh. 1999. Preparation of agarose from Gelidium amansii for gel electrophoresis using various purification methods and its resolution characteristics for DNA. Kor. J. Food Sci. Technol. 31: 110-114
  10. Do, J. R., J. H. Park, and K. S. Jo. 1998. A manufacturing technique of agar with strong gelling ability from Gelidium amansii. J. Kor. Fish. Soc. 31: 673-676
  11. Dodgson, K. S. and R. G Price. 1963. A note on the deternimation of the ester sulfate content of sulfated polysaccarides. Biochem. J. 84: 350-356
  12. Duckworth, M. and W. Yaphe. 1971. The structure agar. Part 1. The fractionation of a complex mixture of polysaccarides. Carbohydr. Res. 16: 189-197 https://doi.org/10.1016/S0008-6215(00)86113-3
  13. Fielding, A. H. and G Russell. 1974. The Competitive properties of marine algae in culture. J. Ecol. 62: 689-698 https://doi.org/10.2307/2258949
  14. Flemenger, G., B. Solomon, T. Wolf, and E. Hadas. 1990. Effect of polyethylene glycol on the non-specific adsorption of proteins to Eupergit C and agarose. J. Chromatogr. 510: 271-279 https://doi.org/10.1016/S0021-9673(01)93761-6
  15. Guiseley, K. B. 1970. The relationship between methoxyl content and gelling temperature of agarose. Carbohydr. Res. 13: 247-256 https://doi.org/10.1016/S0008-6215(00)80831-9
  16. Guiseley, K. B., F. H. Kirpatrick, R. B. Provonchee, M. M. Dumais, and S. Nochumson. 1993. A further fractionation of agarose. Hydrobiologia 260: 505-511 https://doi.org/10.1007/BF00049063
  17. Haryonug, P., J. J. Song, S. P. Hong, Y. H. Choi, S. W. Yun, J. H. Kim, S. C. Lee, S. G Lee, and M. H. Sung. 2002. Novel high-level constitutive expression system, pHCE vector, for a convenient and cost-effective soluble production of humal tumor necrosis factor-a. Biotechnol. Lett. 24: 1185-1189 https://doi.org/10.1023/A:1016107230825
  18. Henderson, M. J. and F. H. Milazzo. 1979. Arylsulfatase in salmonella typhymurium: Detection and influence of carbon source and tyramine on its synthesis. J. Bacteriol. 139: 80-87
  19. Hoshi, M. and T. Moriya. 1980. Arylsulfatase of sea-urchin sperm. 2. Arylsulfatase as a lysin of sea-urchins. Dev. Biol. 74: 343-350 https://doi.org/10.1016/0012-1606(80)90436-4
  20. Izumi, K. 1970. A new method for fractionation of agar. Agr. Biochem. 34: 1739-1740
  21. Jansen, H. J., C. A. Hart, J. M. Rhodes, J. R. Saunders, and J. W. Smalley. 1999. A novel mucin-sulphatase activity found in Bukholderia cepacia and Pseudomonas aeruginosa. J. Med. Microbiol. 48: 551-557 https://doi.org/10.1099/00222615-48-6-551
  22. Jeong, K. J., J. H. Choi, W. M. Yoo, K. C. Keum, N. C. Yoo, S. Y. Lee, and M. H. Sung. 2004. Constitutive production of human leptin by fed-batch culture of recombinant rpoS-Escherichia coli. Protein Expr. Purif. 36: 150-156 https://doi.org/10.1016/j.pep.2004.04.007
  23. Kim, J. H., D. S. Byun, J. S. Godber, J. S. Choi, W. C. Choi, and H. R. Kim. 2004. Purification and characterization of arylsulfatase from Sphingomonas sp. AS6330. Appl. Microbiol. Biotechnol. 63: 553-559 https://doi.org/10.1007/s00253-003-1463-8
  24. Lee, H. S., C. Rhee, and H. C. Yang. 1985. A study on th purification by protein precipitants and washing of agar. Kor. J. Food Sci. Technol. 17: 340-344
  25. Lim, J. M., Y. H. Jang, H. R. Kim, Y. T. Kim, T. J. Choi,J. K. Kim, and S. W. Nam. 2004. Overexpression of arylsulfatase in E. coli and its application to desulfatation of agar. J. Microbiol. Biotechnol. 14: 777-782
  26. Mackie, W. and R. D. Preston. 1974. Cell wall and intracellular region polysaccarides. In: Stewart WDP (eds) Algal physiology and biochemistry. Antarct. Sci. 13: 64-65
  27. Melo, M. R. S., J. P. A. Feitosa, A. L. P. Freitas, and R. C. M. de Paula. 2002. Isolation and characterization of soluble sulfated polysaccaride from the red seaweed Gracilaria cornea. Carbohydr. Polym. 49: 491-498 https://doi.org/10.1016/S0144-8617(02)00006-1
  28. Miech, C., T. Dierks, T. Selmer, K. V. Figura, and B. Schmidt. 1998. Arylsulfatase from Klebsiella pneumoniae-carries a formylglycine generated from a serine. J. Biol. Chem. 273: 4835-4837 https://doi.org/10.1074/jbc.273.9.4835
  29. Milanesi, A. A. and J. W. C. Bind. 1972. Lysosomal enzymes in aquatic species II. Distribution and particle properties of thermally acclimated muscle Iysosomes of rainbow trout Salmo gairdeneri. Comp. Biochem. Physiol. 41: 473-491
  30. Qingping, L., Y. L. Shen, D. Z. Wei, and W. Cao. 2006. Optimization of culture on the overproduction of TRAIL in high-ceIl-density culture by recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 71: 184-191 https://doi.org/10.1007/s00253-005-0131-6
  31. Renn, D. 1997. Biotechnology and the red seaweed polysaccharide industry: status, needs and prospects. Trends Biotechnol. 15: 9-14 https://doi.org/10.1016/S0167-7799(96)10069-X
  32. Yoon, H. S. and Y. H. Park. 1984. Studies on the composition of agarose and agartin in agar-agar. Bull Kor. Fish. Soc. 24: 27-33