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Applied Biochemistry and Biotechnology

, Volume 181, Issue 3, pp 961–971 | Cite as

Cloning, Expression, and Biochemical Characterization of a Novel Acidic GH16 β-Agarase, AgaJ11, from Gayadomonas joobiniege G7

  • Subin Jung
  • Byeong-Chul Jeong
  • Soon-Kwang Hong
  • Chang-Ro Lee
Article

Abstract

A novel β-agarase AgaJ11 belonging to the glycoside hydrolase (GH) 16 family was identified from an agar-degrading bacterium Gayadomonas joobiniege G7. AgaJ11 was composed of 317 amino acids (35 kDa), including a 26-amino acid signal peptide, and had the highest similarity (44 % identity) to a putative β-agarase from an agarolytic marine bacterium Agarivorans albus MKT 106. The agarase activity of purified AgaJ11 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ11 activity were determined to be 4.5 and 40 °C, respectively. Notably, AgaJ11 is an acidic β-agarase that was active only at a narrow pH range from 4 to 5, and less than 30 % of its enzymatic activity was retained at other pH conditions. The K m and V max of AgaJ11 for agarose were 21.42 mg/ml and 25 U/mg, respectively. AgaJ11 did not require metal ions for its activity, but severe inhibition by several metal ions was observed. Thin layer chromatography and agarose-liquefying analyses revealed that AgaJ11 is an endo-type β-agarase that hydrolyzes agarose into neoagarohexaose, neoagarotetraose, and neoagarobiose. Therefore, this study shows that AgaJ11 from G. joobiniege G7 is a novel GH16 β-agarase with an acidic enzymatic feature that may be useful for industrial applications.

Keywords

Agar β-agarase GH16 Gayadomonas joobiniege 

Notes

Acknowledgments

This work was supported by the National Institute of Biological Resources (NIBR) funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR201629201) and the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2015M3A6A2065700).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

References

  1. 1.
    Chi, W. J., Chang, Y. K., & Hong, S. K. (2012). Agar degradation by microorganisms and agar-degrading enzymes. Applied Microbiology and Biotechnology, 94, 917–930.CrossRefGoogle Scholar
  2. 2.
    Duckworth, M., & Yaphe, W. (1972). The relationship between structures and biological properties of agars. In K. Nisizawa (Ed.), Proceedings of the 7th international seaweed symposium (pp. 15–22). New York: Halstead Press.Google Scholar
  3. 3.
    Morrice, L. M., McLean, M. W., Long, W. F., & Williamson, F. B. (1983). Porphyran primary structure. An investigation using β-agarase I from Pseudomonas atlantica and 13C-NMR spectroscopy. European Journal of Biochemistry, 133, 673–684.CrossRefGoogle Scholar
  4. 4.
    Hehemann, J. H., Correc, G., Barbeyron, T., Helbert, W., Czjzek, M., & Michel, G. (2010). Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature, 464, 908–912.CrossRefGoogle Scholar
  5. 5.
    Chi, W. J., Park da, Y., Seo, Y. B., Chang, Y. K., Lee, S. Y., & Hong, S. K. (2014). Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1. Applied Microbiology and Biotechnology, 98, 4545–4555.CrossRefGoogle Scholar
  6. 6.
    Hsu, P. H., Wei, C. H., Lu, W. J., Shen, F., Pan, C. L., & Lin, H. T. (2015). Extracellular production of a novel endo-β-agarase AgaA from Pseudomonas vesicularis MA103 that cleaves agarose into neoagarotetraose and neoagarohexaose. International Journal of Molecular Sciences, 16, 5590–5603.CrossRefGoogle Scholar
  7. 7.
    Liao, L., Xu, X. W., Jiang, X. W., Cao, Y., Yi, N., Huo, Y. Y., Wu, Y. H., Zhu, X. F., Zhang, X. Q., & Wu, M. (2011). Cloning, expression, and characterization of a new β-agarase from Vibrio sp. strain CN41. Applied and Environmental Microbiology, 77, 7077–7079.CrossRefGoogle Scholar
  8. 8.
    Dong, J., Tamaru, Y., & Araki, T. (2007). A unique β-agarase, AgaA, from a marine bacterium, Vibrio sp. strain PO-303. Applied Microbiology and Biotechnology, 74, 1248–1255.CrossRefGoogle Scholar
  9. 9.
    Park, Y., Chi, W. J., Park, J. S., Chang, Y. K., & Hong, S. K. (2015). Cloning, expression, and biochemical characterization of a GH16 β-agarase AgaH71 from Pseudoalteromonas hodoensis H7. Applied Biochemistry and Biotechnology, 175, 733–747.CrossRefGoogle Scholar
  10. 10.
    Temuujin, U., Chi, W. J., Chang, Y. K., & Hong, S. K. (2012). Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo- and endo-type β-agarase-producing neoagarobiose. Journal of Bacteriology, 194, 142–149.CrossRefGoogle Scholar
  11. 11.
    Temuujin, U., Chi, W. J., Lee, S. Y., Chang, Y. K., & Hong, S. K. (2011). Overexpression and biochemical characterization of DagA from Streptomyces coelicolor A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose. Applied Microbiology and Biotechnology, 92, 749–759.CrossRefGoogle Scholar
  12. 12.
    Han, W., Cheng, Y., Wang, D., Wang, S., Liu, H., Gu, J., Wu, Z., & Li, F. (2016). Biochemical characteristics and substrate degradation pattern of a novel exo-type β-agarase from the polysaccharide-degrading marine bacterium Flammeovirga sp. strain MY04. Applied Microbiology and Biotechnology, 82, 4944–4954.Google Scholar
  13. 13.
    Hehemann, J. H., Correc, G., Thomas, F., Bernard, T., Barbeyron, T., Jam, M., Helbert, W., Michel, G., & Czjzek, M. (2012). Biochemical and structural characterization of the complex agarolytic enzyme system from the marine bacterium Zobellia galactanivorans. The Journal of Biological Chemistry, 287, 30571–30584.CrossRefGoogle Scholar
  14. 14.
    Cui, F., Dong, S., Shi, X., Zhao, X., & Zhang, X. H. (2014). Overexpression and characterization of a novel thermostable β-agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01(T). Marine Drugs, 12, 2731–2747.CrossRefGoogle Scholar
  15. 15.
    Lee, D. G., Jeon, M. J., & Lee, S. H. (2012). Cloning, expression, and characterization of a glycoside hydrolase family 118 β-agarase from Agarivorans sp. JA-1. Journal of Microbiology and Biotechnology, 22, 1692–1697.CrossRefGoogle Scholar
  16. 16.
    Li, G., Sun, M., Wu, J., Ye, M., Ge, X., Wei, W., Li, H., & Hu, F. (2015). Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH. Applied Microbiology and Biotechnology, 99, 10019–10029.CrossRefGoogle Scholar
  17. 17.
    Li, J., Sha, Y., Seswita-Zilda, D., Hu, Q., & He, P. (2014). Purification and characterization of thermostable agarase from Bacillus sp. BI-3, a thermophilic bacterium isolated from hot spring. Journal of Microbiology and Biotechnology, 24, 19–25.CrossRefGoogle Scholar
  18. 18.
    Ohta, Y., Hatada, Y., Miyazaki, M., Nogi, Y., Ito, S., & Horikoshi, K. (2005). Purification and characterization of a novel α-agarase from a Thalassomonas sp. Current Microbiology, 50, 212–216.CrossRefGoogle Scholar
  19. 19.
    Zhu, Y., Zhao, R., Xiao, A., Li, L., Jiang, Z., Chen, F., & Ni, H. (2016). Characterization of an alkaline β-agarase from Stenotrophomonas sp. NTa and the enzymatic hydrolysates. International Journal of Biological Macromolecules, 86, 525–534.CrossRefGoogle Scholar
  20. 20.
    Tawara, M., Sakatoku, A., Tiodjio, R. E., Tanaka, D., & Nakamura, S. (2015). Cloning and characterization of a novel agarase from a newly isolated bacterium Simiduia sp. strain TM-2 able to degrade various seaweeds. Applied Biochemistry and Biotechnology, 177, 610–623.CrossRefGoogle Scholar
  21. 21.
    Chi, W. J., Park, J. S., Kwak, M. J., Kim, J. F., Chang, Y. K., & Hong, S. K. (2013). Isolation and characterization of a novel agar-degrading marine bacterium, Gayadomonas joobiniege gen, nov, sp. nov., from the Southern Sea, Korea. Journal of Microbiology and Biotechnology, 23, 1509–1518.CrossRefGoogle Scholar
  22. 22.
    Araki, C. (1959). Seaweed polysaccharides. In M. L. Wolfrom (Ed.), Carbohydrate chemistry of substances of biological interests (pp. 15–30). London: Pergamon Press.Google Scholar
  23. 23.
    Potin, P., Richard, C., Rochas, C., & Kloareg, B. (1993). Purification and characterization of the α-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. European Journal of Biochemistry, 214, 599–607.CrossRefGoogle Scholar
  24. 24.
    Ha, S. C., Lee, S., Lee, J., Kim, H. T., Ko, H. J., Kim, K. H., & Choi, I. G. (2011). Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40. Biochemical and Biophysical Research Communications, 412, 238–244.CrossRefGoogle Scholar
  25. 25.
    Hehemann, J. H., Smyth, L., Yadav, A., Vocadlo, D. J., & Boraston, A. B. (2012). Analysis of keystone enzyme in agar hydrolysis provides insight into the degradation (of a polysaccharide from) red seaweeds. The Journal of Biological Chemistry, 287, 13985–13995.CrossRefGoogle Scholar
  26. 26.
    Sugano, Y., Kodama, H., Terada, I., Yamazaki, Y., & Noma, M. (1994). Purification and characterization of a novel enzyme, α-neoagarooligosaccharide hydrolase (α-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107. Journal of Bacteriology, 176, 6812–6818.CrossRefGoogle Scholar
  27. 27.
    Frey, P. A. (1996). The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose. The FASEB Journal, 10, 461–470.Google Scholar
  28. 28.
    Yun, E. J., Lee, S., Kim, H. T., Pelton, J. G., Kim, S., Ko, H. J., Choi, I. G., & Kim, K. H. (2015). The novel catabolic pathway of 3, 6-anhydro-L-galactose, the main component of red macroalgae, in a marine bacterium. Environmental Microbiology, 17, 1677–1688.CrossRefGoogle Scholar
  29. 29.
    Zor, T., & Selinger, Z. (1996). Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Analytical Biochemistry, 236, 302–308.CrossRefGoogle Scholar
  30. 30.
    Segel, I. H. (1976). Enzyme kinetics. In Biochemical calculations. How to solve mathmatical problems in general biochemistry, 2nd edn (pp. 214–229). New York: Wiley.Google Scholar
  31. 31.
    Duckworth, M., & Yaphe, W. (1970). Thin-layer chromatographic analysis of enzymic hydrolysates of agar. Journal of Chromatography, 49, 482–487.CrossRefGoogle Scholar
  32. 32.
    Fu, X. T., & Kim, S. M. (2010). Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Marine Drugs, 8, 200–218.CrossRefGoogle Scholar
  33. 33.
    Oh, C., Nikapitiya, C., Lee, Y., Whang, I., Kim, S. J., Kang, D. H., & Lee, J. (2010). Cloning, purification and biochemical characterization of beta agarase from the marine bacterium Pseudoalteromonas sp. AG4. Journal of Industrial Microbiology & Biotechnology, 37, 483–494.CrossRefGoogle Scholar
  34. 34.
    Yasuike, M., Nakamura, Y., Kai, W., Fujiwara, A., Fukui, Y., Satomi, M., & Sano, M. (2013). Draft genome sequence of Agarivorans albus strain MKT 106 T, an agarolytic marine bacterium. Genome Announcements, 1(4), e00367–e00313.CrossRefGoogle Scholar
  35. 35.
    Oh, C., Nikapitiya, C., Lee, Y., Whang, I., Kang, D. H., Heo, S. J., Choi, Y. U., & Lee, J. (2010). Molecular cloning, characterization and enzymatic properties of a novel β-agarase from a marine isolate Psudoalteromonas sp. AG52. Brazilian Journal of Microbiology, 41, 876–889.CrossRefGoogle Scholar
  36. 36.
    Kobayashi, R., Takisada, M., Suzuki, T., Kirimura, K., & Usami, S. (1997). Neoagarobiose as a novel moisturizer with whitening effect. Bioscience, Biotechnology, and Biochemistry, 61, 162–163.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Subin Jung
    • 1
  • Byeong-Chul Jeong
    • 1
  • Soon-Kwang Hong
    • 1
  • Chang-Ro Lee
    • 1
  1. 1.Department of Biological SciencesMyongji UniversityYonginRepublic of Korea

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