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Biotechnology and Bioprocess Engineering

, Volume 23, Issue 5, pp 525–531 | Cite as

Cloning, Expression, and Characterization of a Thermotolerant β-agarase from Simiduia sp. SH-4

  • Jae-Deog Kim
  • Dong-Geun Lee
  • Sang-Hyeon Lee
Research Paper
  • 3 Downloads

Abstract

The gene coding for a thermotolerant β-agarase from an isolated Simiduia sp. SH-4 was cloned, recombinantly expressed, and characterized after purification. This gene was sequenced after cassette mediated polymerase chain reaction and composed of an open reading frame of 1,809 base pairs, encoding a protein of 66.2 kilodaltons comprising of 602 amino acid residues. The amino acids sequence showed 74% homology with β-agarase of Simiduia agarivorans. A new β-agarase gene corresponding to mature protein of 577 amino acids was recombinantly expressed and purified by chitin bead column to homogeneity. The maximal specific activity was 505.07 U/mg at 50oC in Tris/HCl (pH 6.0) buffer. Recombinant β-agarase hydrolyzed agar into neoagarotetraose (57%) and neoagarohexaose (43%). It generated products from melted and non-melted powder agar and agarose at 30-50oC, meaning cheap agar materials could be used with energy- and costsavings. Thus, recombinant β-agarase could be used for industrial production of neoagarotetraose and neoagarohexaose.

Keywords

β-agarase cloning expression purification Simiduia sp. 

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References

  1. 1.
    Lee, D. G., G. T. Park, N. Y. Kim, E. J. Lee, M. K. Jang, Y. G. Shin, and S. J. Kim (2006) Cloning, expreßsion, and characterization of a glycoside hydrolase family 50 ß-agarase from a marine Agarivorans isolate. Biotechnol. Lett. 28: 1925–1932.CrossRefGoogle Scholar
  2. 2.
    Kobayashi, R., M. Takisada, T. Suzuki, K. Kirimura, and S. Usami (1997) Neoagarobiose as a novel moisturizer with whitening effect. Biosci. Biotechnol. Biochem. 61: 62–63.Google Scholar
  3. 3.
    Hong, S. J., J.-H. Lee, E. J. Kim, H. J. Yang, Y. -K. Chang, J.-S. Park, and S.-K. Hong (2017) In vitro and in vivo investigation for biological activities of neoagarooligosaccharides prepared by hydrolyzing agar with ß-agarase. Biotechnol. Bioproc. Eng. 22: 489–496.CrossRefGoogle Scholar
  4. 4.
    Zhang, N., E. Hou, J. Song, J. Li, Q. Tang, and X, Mao (2017) Neoagarotetraose-modulated gut microbiota and alleviated gut inflammation in antibiotic treatment mice. Food Agric. Immunol. 28: 1–16.CrossRefGoogle Scholar
  5. 5.
    Sugano, Y., T. Matsumoto, and M. Noma (1994) Sequence analysis of the agaB gene encoding a new beta-agarase from Vibrio sp. strain JT0107. Biochim. Biophys. Acta 17: 105–108.CrossRefGoogle Scholar
  6. 6.
    Lee, M. H., J. H. Jang, G. Y. Yoon, S. J. Lee, M. G. Lee, T. H. Kang, and Y. M. Park (2017) Neoagarohexaose-mediated activation of dendritic cells via Toll-like receptor 4 leads to stimulation of natural killer cells and enhancement of antitumor immunity. BMB Rep. 50: 263.CrossRefGoogle Scholar
  7. 7.
    Zhang, N., X. Mao, R. W. Li, E. Hou, Y. Wang, C. Xue, and Q. Tang (2017) Neoagarotetraose protects mice against intense exercise-induced fatigue damage by modulating gut microbial composition and function. Mol. Nutr. Food Res. 61: 1600585.CrossRefGoogle Scholar
  8. 8.
    Lee, D. G., M. K. Jang, O. H. Lee, N. Y. Kim, S. A. Ju, and S. H. Lee (2008) Over-production of a glycoside hydrolase family 50 ß-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product. Biotechnol. Lett. 30: 911–918.Google Scholar
  9. 9.
    Lee, S., D. G. Lee, M. K. Jang, M. J. Jeon, H. J. Jang, and S. H. Lee (2011) Improvement in the catalytic activity of ß-agarase AgaA from Zobellia galactanivorans by site-directed mutagenesis. J. Microbiol. Biotechnol. 21: 1116–1122.CrossRefGoogle Scholar
  10. 10.
    Kim, J. D., S. J. Lee, J. G. Jo, D. G. Lee, and S. H. Lee (2016) Characterization of ß-agarase from isolated Simiduia sp. SH-4. J. Life Sci. 26: 453–459.CrossRefGoogle Scholar
  11. 11.
    Hong, J. C., J. B. Hong, J. J. Park, W. J. Chi, M. C. Kim, Y. K. Chang, and S. K. Hong (2011) Production of agarase from a novel Micrococcus sp. GNUM-08124 strain isolated from the East Sea of Korea. Biotechnol. Bioproc. Eng. 16: 81–88.CrossRefGoogle Scholar
  12. 12.
    Tawara, M., A. Sakatoku, R. E. Tiodjio, D. Tanaka, and S. Nakamura (2015) Cloning and characterization of a novel agarase from a newly isolated bacterium Simiduia sp. strain TM-2 able to degrade various seaweeds. Appl. Biochem. Biotechnol. 177: 610–623.Google Scholar
  13. 13.
    Miller, G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426–428.CrossRefGoogle Scholar
  14. 14.
    Laemmli, U. K. (1970) SDS-page Laemmli method. Nature 227: 680–685.CrossRefGoogle Scholar
  15. 15.
    Lin, S. Y., W. Y. Shieh, J. S. Chen, and S. L. Tang (2013) Complete genome sequence of Simiduia agarivorans SA1T, a marine bacterium able to degrade a variety of polysaccharides. Genome Announc. 1: e00039–12.Google Scholar
  16. 16.
    Jang, M. K., S. W. Lee, D. G. Lee, N. Y. Kim, K. H. Yu, H. J. Jang, S. Kim, A. Kim, and S. H. Lee (2010) Enhancement of the thermostability of a recombinant beta-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis. Biotechnol. Lett. 32: 943–949.CrossRefGoogle Scholar
  17. 17.
    Koti, B. A., M. Shinde, and J. Lalitha (2013) Repeated batch production of agaroligosaccharides from agarose by an amberlite IRA-900 immobilized agarase system. Biotechnol. Bioproc. Eng. 18: 333–341.CrossRefGoogle Scholar
  18. 18.
    Jonnadula, R. and S. C. Ghadi (2011) Purification and characterization of ß-agarase from seaweed decomposing bacterium Microbulbifer sp. strain CMC-5. Biotechnol. Bioproc. Eng. 16: 513–519.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Green-Chemistry Convergence Engineering, Graduate SchoolSilla UniversityBusanKorea
  2. 2.Department of Bioscience, Graduate SchoolSilla UniversityBusanKorea

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