Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

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

Identification and Characterization of Alginate Lyase Producing Pseudomonas sp. N7151-6

Alginate Lyase 생산 균주 Pseudomonas sp. N7151-6의 분리 및 특성

  • Lee, Jae-Hyung (Department of Biomaterial Control, Dong-Eui University) ;
  • Bae, Min-Ji (Department of Biotechnology & Bioengineering, Dong-Eui University) ;
  • Kim, Yang-Choon (Gijang Local Products Co., Ltd.) ;
  • Nam, Soo-Wan (Department of Biomaterial Control, Dong-Eui University)
  • 이재형 (동의대학교 바이오물질제어학과) ;
  • 배민지 (동의대학교 생명공학과) ;
  • 김양춘 (기장물산(주)) ;
  • 남수완 (동의대학교 바이오물질제어학과)
  • Published : 2009.12.28
Download PDF
⟨ Previous Next ⟩

Abstract

A Gram-negative, alginate lyase-producing bacterium was isolated from the Haeundae Coast, Korea. The isolated strain N7151-6 produced alginate lyase. The optimal temperature and pH for growth were found to be $30^{\circ}C$ and pH 8.0, respectively. This strain can be grown at the NaCl concentration of 0-7% (w/v). Analysis of 16S rDNA sequence and physiological profiling indicated that the strain N7151-6 belonged to Pseudomonas sp. The enzyme alginate lyase produced by Pseudomonas sp. N7151-6 was partially purified by ultrafiltration (MWCO= 30 kDa). The optimum pH and temperature for the activity of the purified enzyme were found to be 7.0 and $30^{\circ}C$, respectively. The enzyme was stable at the pH range of 5.0-9.0 and temperature range of $23-30^{\circ}C$. The total activity of alginate lyase produced was reached about 110 unit/L.

해운대 연안에서 그람 음성균이면서 알긴산 분해효소를 생산하는 세균을 분리하였다. 분리된 N7151-6 균주의 성장을 위한 최적 온도는 $30^{\circ}C$, 최적 pH는 8.0으로 조사되었다. 또한 0-7%(w/v) NaCl 농도에서도 성장 가능하다. 16S rDNA 염기 서열 분석과 생화학적 분석에 의해 이 균주는 Pseudomonas 속으로 동정되어 Pseudomonas sp. N7151-6으로 명명하였다. Pseudomonas sp. N7151-6에서 생산하는 알긴산 분해효소를 한외여과(ultrafilteration; MWCO=30 kDa) 방법에 의해 부분정제하였다. 분리된 효소의 최적 pH는 7.0으로 최적 온도는 $30^{\circ}C$로 조사 되었다. pH 5.0에서 9.0까지 이 효소는 안정하였으며, $23^{\circ}C$에서 $37^{\circ}C$까지의 범위에서도 안정성을 보여주었다. 알긴산 분해효소의 전체 활성은 110 unit/L이었다.

Keywords

References

  1. Anzai, H., N. Uchida, and E. Nishida. 1990. Determination of D-mannuronic to L-guluronic acids ratio in acid hydrolysis under improved conditions. Nippon Suisan Gakkaishi. 56: 73-81 https://doi.org/10.2331/suisan.56.73
  2. Davidson, I. W., I. W. Sutherland, and C. J. Lawson. 1976. Purification and properties of an alginate lyase from a marine bacterium. Biochem. J. 159: 707-713
  3. Dunne, W. M. and F. L. Buckmire. 1985. Partial purification and characterization of a polymannuronic acid depolymerase produced by a mucoid strain of Pseudomonas aeroguinosa isolated from a patient with cystic fibrosis. Appl. Environ. Microbiol. 50: 562-567
  4. Franklin, M. J., C. E. Chitnis, P. Gacesa, A. Sonesson, D. C. White, and D. E. Ohman. 2007. Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase. J. Bacteriol., 186: 4759-4773 https://doi.org/10.1128/JB.186.14.4759-4773.2004
  5. Guven, K. C., Y. Ozsoy, and O. N. Ulutin. 1991. Anticoagulant, fibrinotic and antiaggregant activity of carrageenans and alginic acid. Biotanica. Marina. 34:429-435 https://doi.org/10.1515/botm.1991.34.5.429
  6. Hansen, J. B. and L. K. Nakamura. 1985. Distribution of alginate lyase activity among strains of Bacillus circulans. Appl. Environ. Microbiol. 49: 1019-1021
  7. Haug. A., B. Larsen, and O. Smidsrod. 1966. A study of constitution of alginic acid by partial acid hydrolysis. Acta. Chemica. Scandinabica. 20: 183-190 https://doi.org/10.3891/acta.chem.scand.20-0183
  8. Haug, A., B. Larsen, and O. Smidsrod. 1967. Studies on the sequence of uronic acid residues in alginic acid. Acta. Chemica. Scandinabica. 21: 691-704 https://doi.org/10.3891/acta.chem.scand.21-0691
  9. Hirst, E. L. and D. A. Rees. 1965. The structure of alginic acid. Part V. Isolation and unambiguous characterization of some hydrolysis products of the methylated polysaccharide. J. Chem. Society. 7: 1182-1187 https://doi.org/10.1039/JR9650001182
  10. Iwamoto, Y., R. Araki, K. Iriyam, T. Oda, H. Fukuda, S. Hayashida, and T. Muramatsu. 2001. Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates. Biosci. Biotechnol. Biochem. 65: 133-142 https://doi.org/10.1271/bbb.65.133
  11. Jeong, H. J., S. A. Lee, P. D. Moon, H. J. Na, R. K. Park, J. Y. Um, H. M. Kim, and S. H. Hong. Alginic acid has antianaphylactic effects and inhibits inflammatory cytokine expression via suppression of nuclear factor-kappaB activation. 2006. Clin. Exp. Allergy. 36: 785-794 https://doi.org/10.1111/j.1365-2222.2006.02508.x
  12. Joo, D. S., J. S. Lee, J. J. Park, S. Y. Cho, C. B. Ahn, and E. H. Lee. 1995. Purification and characterization of the intracellular alginase from Vibrio sp. AL-145. Kor. J. Appl. Microbiol. Biotechnol. 23: 432-438
  13. Joo, D. S., S. Y. Cho, and E. H. Lee. 1993. Isolation of alginate-degrading bacteria and production of alginatedegrading activities by the bacteria. Kor. J. Appl. Microbiol. Biotechnol. 21: 207-213
  14. Lanyi, B. 1987. Classical and rapid identification methods for medically important bacteria. pp. 1-67. In Colwell, R.R and R. Grigorova (ed.), Methods in Microbiology, vol. 19, Academic Press Ltd., London
  15. Lee, J. H. and E. Y. Lee. 2003. Isolation of alginatedegrading marine bacteria and characterization of alginase. J. Life Science. 13: 718-722 https://doi.org/10.5352/JLS.2003.13.5.718
  16. Linker, A. and L. R. Evans. 1984. Isolation and characterization of an alginase from mucoid strains of Pseudomonas aeroguinosa. J. Bacteriol. 159: 958-964
  17. Nakagawa A., T. Ozaki, K. Chubachi, T. Hosoyama, T. Okubo, S. Iyobe, and T. Suzuki. 1998. An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase. J. Appl. Microbiol. 84: 328-335 https://doi.org/10.1046/j.1365-2672.1998.00319.x
  18. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428 https://doi.org/10.1021/ac60147a030
  19. Paxman, J. R., J. C. Richardson, P. W. Dettmar, and B. M. Corfe. Alginate reduces the increased uptake of cholesterol and glucose in overweight male subjects: a pilot study. 2008. Nutr. Res. 28: 501-505 https://doi.org/10.1016/j.nutres.2008.05.008
  20. Pecina, A., A. Pascual, and A. Paneque. 1999. Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme. J. Bacteriol. 181: 1409-1414
  21. Penman, A. and G. R. Sanderson. 1972. A method for the determination of uronic acid sequence in alginates. Carbohydrate Res. 25: 273-282 https://doi.org/10.1016/S0008-6215(00)81637-7
  22. Sawabe, T., H. Takahashi, Y. Ezura, and P. Gacesa. 2001. Cloning, sequence analysis and expression of Pseudoalteromonas elyakoii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase. Carbohydr. Res. 335: 11-21 https://doi.org/10.1016/S0008-6215(01)00198-7
  23. Skerman, V. B. D. 1967. A Guide to the Identification of the Genera of Bacteria, 2nd edn. Batimore: Williams & Wilkins
  24. Stevens, R. A. and R. E. Levin. 1977. Purification and characteristics of an alginate from Alginovibrio aquatilis. Appl. Environ. Microbiol. 3: 1156-1161
  25. Tamaoka, J. and K. Komagata. 1984. Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol. Lett. 25: 125-128 https://doi.org/10.1111/j.1574-6968.1984.tb01388.x