Advertisement

Applied Biochemistry and Biotechnology

, Volume 173, Issue 7, pp 1703–1716 | Cite as

Production and Characterization of a Novel Thermostable Extracellular Agarase from Pseudoalteromonas hodoensis Newly Isolated from the West Sea of South Korea

  • Won-Jae Chi
  • Jae-Seon Park
  • Dae-Kyung Kang
  • Soon-Kwang Hong
Article

Abstract

A Gram-negative, aerobic, motile, rod-shaped, agarolytic bacterium, designated as H7, was isolated from a coastal seawater sample. This strain grows at pH 6.0–8.0, temperature of 15–40 °C, and at an NaCl concentration of 1–7 % (w/v). Ubiquinone-8 was the predominant respiratory quinone, and the DNA G+C content was 45.82 mol%. Analysis of the 16S rRNA sequence suggests that strain H7 belongs to the genus Pseudoalteromonas. DNA-DNA hybridization analysis showed DNA relatedness of as low as 55.42 and 40.27 % with its nearest phylogenetic neighbors Pseudoalteromonas atlantica IAM12927T and Pseudoalteromonas espejiana NCIMB2127T, respectively, which led us to name H7 Pseudoalteromonas hodoensis sp. nov. The type strain is H7T (=DSM25967T = KCTC23887T). An agarase (AgaA7) was purified to homogeneity from the cell-free culture broth of H7 through many steps of chromatography. Purified AgaA7 had an apparent molecular weight of 35 kDa, with a distinct NH2-terminal sequence of Ala-Asp-Ala-Thr-X-Pro (X, any amino acid) from the reported proteins, implying that it is a novel enzyme. The optimum pH and temperature for agarase activity were 7.0 and 45 °C, respectively. Thin-layer chromatography analysis, mass spectrometry, and enzyme assay using p-nitrophenyl-α/β-D-galactopyranoside revealed that AgaA7 is both an exo- and endo-type β-agarase that degrades agarose into neoagarotetraose, neoagarohexaose, and neoagarooctaose (minor).

Keywords

β-1,4-Agarase Neoagarotetraose Neoagarohexaose Neoagarooctaose Pseudoalteromonas hodoensis 

Notes

Acknowledgments

This work was supported by Grant No. 2012-R1A1B3002174 from the Basic Research Program of the National Research Foundation (KRF) of Korea.

Supplementary material

12010_2014_958_MOESM1_ESM.docx (138 kb)
Fig. S1 (DOCX 137 kb)
12010_2014_958_MOESM2_ESM.docx (89 kb)
Fig. S2 (DOCX 88 kb)
12010_2014_958_MOESM3_ESM.docx (20 kb)
Table S1 (DOCX 20 kb)
12010_2014_958_MOESM4_ESM.docx (18 kb)
Table S2 (DOCX 18 kb)

References

  1. 1.
    Araki, C. (1959). Seaweed polysaccharides. In M. L. Wolfrom (Ed.), Carbohydrate chemistry of substances of biological interest (pp. 15–30). London: Pergamon.Google Scholar
  2. 2.
    van de Velde, F., Knutsen, S. H., Usov, A. I., Rollema, H. S., & Cerezo, A. S. (2002). Trends in Food Science & Technology, 13, 73–92.CrossRefGoogle Scholar
  3. 3.
    Usov, A. I. (1998). Food Hydrocolloids, 12, 301–308.CrossRefGoogle Scholar
  4. 4.
    Hehemann, J. H., Correc, G., Barbeyron, T., Helbert, W., Czjzek, M., & Michel, G. (2010). Nature, 464, 908–912.CrossRefGoogle Scholar
  5. 5.
    Chi, W. J., Chang, Y. K., & Hong, S. K. (2012). Applied Microbiology and Biotechnology, 94, 917–930.CrossRefGoogle Scholar
  6. 6.
    Knutsen, S. H., Myslabodski, D. E., Larsen, B., & Usov, A. I. (1994). Botanica Marina, 37, 163–169.CrossRefGoogle Scholar
  7. 7.
    Araki, T., Hayakawa, M., Zhang, L., Karita, S., & Morishita, T. (1998). Journal of Marine Biotechnology, 6, 260–265.Google Scholar
  8. 8.
    Duckworth, M., & Turvey, J. R. (1969). Biochemical Journal, 113, 139–142.Google Scholar
  9. 9.
    Ohta, Y., Hatada, Y., Miyazaki, M., Nogi, Y., Ito, S., & Horikoshi, K. (2005). Current Microbiology, 50, 212–216.CrossRefGoogle Scholar
  10. 10.
    Hu, Z., Lin, B. K., Xu, Y., Zhong, M. Q., & Liu, G. M. (2008). Journal of Applied Microbiology, 106, 181–190.CrossRefGoogle Scholar
  11. 11.
    Yamaura, I. T., Matsumoto, M., Funatsu, H., & Shigeiri, S. T. (1991). Agricultural and Biological Chemistry, 55, 2531–2536.CrossRefGoogle Scholar
  12. 12.
    Kim, J. H., & Hong, S. K. (2012). Journal of Microbiology and Biotechnology, 23, 1621–1628.CrossRefGoogle Scholar
  13. 13.
    Oh, C., Nikapitiya, C., Lee, Y., Whang, I., Kim, S. J., Kang, D. H., & Lee, J. (2010). Journal of Industrial Microbiology and Biotechnology, 37, 483–494.CrossRefGoogle Scholar
  14. 14.
    Schroeder, D. C., Jaffer, M. A., & Coyne, V. E. (2003). Microbiology, 149, 2919–2929.CrossRefGoogle Scholar
  15. 15.
    Vera, J., Alvarez, R., Murano, E., Carlos, J. C., & Leon, O. (1998). Applied and Environmental Microbiology, 64, 4378–4383.Google Scholar
  16. 16.
    Temuujin, U., Chi, W. J., Lee, S. Y., Chang, Y. K., & Hong, S. K. (2011). Applied Microbiology and Biotechnology, 92, 749–759.CrossRefGoogle Scholar
  17. 17.
    Temuujin, U., Chi, W. J., Chang, Y. K., & Hong, S. K. (2012). Journal of Bacteriology, 194, 142–149.CrossRefGoogle Scholar
  18. 18.
    Chi, W. J., Park, J. S., Kwak, M. J., Kim, J. F., Chang, Y. K., & Hong, S. K. (2013). Journal of Microbiology and Biotechnology, 23, 1509–1518.CrossRefGoogle Scholar
  19. 19.
    Chun, J. S., Lee, J. H., Jung, Y. Y., Kim, M. J., Kim, S. I., Kim, B. K., & Lim, Y. W. (2007). International Journal of Systematic and Evolutionary Microbiology, 57, 2259–2261.CrossRefGoogle Scholar
  20. 20.
    Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). Nucleic Acids Research, 22, 4673–4680.CrossRefGoogle Scholar
  21. 21.
    Hall, T. A. (1999). Nucleic Acids Symposium Series, 41, 95–98.Google Scholar
  22. 22.
    Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Distributed by the author. Department of Genome Sciences, University of Washington, Seatle, USA.Google Scholar
  23. 23.
    Kimura, M. (1983). The neutral theory of molecular evolution. UK: Cambridge Univesity Press.CrossRefGoogle Scholar
  24. 24.
    Komagata, K., & Suzuki, K. (1987). Methods in Microbiology, 19, 161–207.CrossRefGoogle Scholar
  25. 25.
    Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Newark: MIDI Inc.Google Scholar
  26. 26.
    Mesbah, M., Premachandran, U., & Whitman, W. B. (1989). International Journal of Systematic Bacteriology, 39, 159–167.CrossRefGoogle Scholar
  27. 27.
    Miller, G. L. (1959). Analytical Chemistry, 31, 426–428.CrossRefGoogle Scholar
  28. 28.
    Laemmli, U. K. (1970). Nature, 227, 680–685.CrossRefGoogle Scholar
  29. 29.
    Stackebrandt, E., & Goebel, B. M. (1994). International Journal of Systematic Bacteriology, 44, 846–849.CrossRefGoogle Scholar
  30. 30.
    Xu, X. W., Wu, Y. H., Wang, C. S., Gao, X. H., Wang, X. G., & Wu, M. (2010). International Journal of Systematic and Evolutionary Microbiology, 60, 2176–2181.CrossRefGoogle Scholar
  31. 31.
    Khuday, R. A., Stoβer, N. I., Qoura, F., & Antranikian, G. (2008). International Journal of Systematic and Evolutionary Microbiology, 58, 2018–2024.CrossRefGoogle Scholar
  32. 32.
    Gauthier, G., Gauthier, M., & Christen, R. (1995). International Journal of Systematic Bacteriology, 45, 755–761.CrossRefGoogle Scholar
  33. 33.
    Holmström, C., & Kjelleberg, S. (1999). FEMS Microbiology Ecology, 30, 285–293.CrossRefGoogle Scholar
  34. 34.
    Hanefeld, U., Floss, H. G., & Laatsch, H. (1994). Journal of Organic Chemistry, 59, 3604–3608.CrossRefGoogle Scholar
  35. 35.
    Ma, C., Lu, X., Shi, C., Li, J., Gu, Y., Ma, Y., Chu, Y., Han, F., Gong, Q., & Yu, W. (2007). Journal of Biological Chemistry, 282, 3747–3754.CrossRefGoogle Scholar
  36. 36.
    Oh, Y. H., Jung, C., & Lee, J. (2011). Journal of Microbiology and Biotechnology, 21, 818–821.CrossRefGoogle Scholar
  37. 37.
    Wang, J., Jiang, X., Mou, H., & Guan, H. (2004). Journal of Applied Phycology, 16, 333–340.CrossRefGoogle Scholar
  38. 38.
    Wu, S. C., Wen, T. N., & Pan, C. L. (2005). Fisheries Science, 71, 1149–1159.CrossRefGoogle Scholar
  39. 39.
    Kobayashi, R., Takisada, M., Suzuki, T., Kirimura, K., & Usami, S. (1997). Bioscience, Biotechnology, and Biochemistry, 61, 162–163.CrossRefGoogle Scholar
  40. 40.
    Xiao, T. F., & Kim, S. M. (2010). Marine Drugs, 8, 200–218.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Won-Jae Chi
    • 1
    • 3
  • Jae-Seon Park
    • 1
  • Dae-Kyung Kang
    • 2
  • Soon-Kwang Hong
    • 1
  1. 1.Department of Biological Science and BioinformaticsMyongji UniversityYonginSouth Korea
  2. 2.Department of Animal Resources ScienceDankook UniversityCheonanSouth Korea
  3. 3.Life Science R&D Center, Sinil Pharmaceutical Co., Ltd.Chungju-cityKorea

Personalised recommendations