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Isolation of an Agarase-producing Persicobacter sp. DH-3 and Characterization of its β-agarase

Agarase를 생산하는 Persicobacter sp. DH-3의 분리 및 β-agarase의 특성

  • Heo, Da-Hye (Department of Green-Chemistry Convergence Engineering, Graduate School, Silla University) ;
  • Lee, Dong-Geun (Major in Pharmaceutical Engineering, Division of Bioindustry, Silla University) ;
  • Lee, Sang-Hyeon (Department of Green-Chemistry Convergence Engineering, Graduate School, Silla University)
  • 허다혜 (신라대학교 일반대학원 그린화학융합공학과) ;
  • 이동근 (신라대학교 제약공학과) ;
  • 이상현 (신라대학교 일반대학원 그린화학융합공학과)
  • Received : 2018.10.12
  • Accepted : 2018.10.26
  • Published : 2019.02.28

Abstract

The purpose of this study was to isolate a new marine agarase-producing bacterium. Agarase can hydrolyze agar and agarose to produce agarooligosaccharides or neoagarooligosaccharides, which possess many physiological functions. Strain DH-3 was isolated from seawater collected from the coast of Yeosu at Jeollanam province, Korea. A 16S rDNA sequence analysis showed this strain to be Persicobacter sp. DH-3. Extracellular agarase was prepared from culture media of Persicobacter sp. DH-3 and used for characterization. Relative activities at 20, 30, 40, 50, 60, and $70^{\circ}C$ were 50, 55, 70, 100, 90, and 50%, respectively. Relative activities at pH 5, 6, 7, and 8 were 75, 100, 90, and 75%, respectively. The enzyme showed maximum activity at $50^{\circ}C$ in a 20 mM Tris-HCl buffer at pH 6. This enzyme could be useful, as agar is in liquid state at $50^{\circ}C$. Agarase activities were maintained at 80% or more for 2 hr at 20, 30, and $40^{\circ}C$. Thin layer chromatography analysis suggested that Persicobacter sp. DH-3 produced extracellular ${\beta}$-agarases as it hydrolyzed agarose to produce neoagarohexaose and neoagarotetraose. In addition, zymogram analysis confirmed that Persicobacter sp. DH-3 produces at least three agar-degrading enzymes with molecular weights of 45, 70, and 140 kDa. Therefore, it is expected that agarases from Persicobacter sp. DH-3 could be used to produce functional neoagarooligosaccharides.

이 연구의 목적은 agarase를 생산하는 새로운 해양 박테리아를 분리하는 것이다. Agarase는 agarose를 가수분해하여 다양한 생리활성을 가지는 agarooligosaccharides 또는 neoagarooligosaccharides를 생성할 수 있다. 전라남도의 여수연안에서 채취한 해수로부터 DH-3 균주를 분리하였다. DH-3 균주는 16S rDNA 서열분석을 통하여 Persicobacter sp. DH-3로 명명하였다. 세포 외로 분비된 효소는 Persicobacter sp. DH-3의 배양액으로부터 얻었으며, 특성연구에 사용하였다. 20, 30, 40, 50, 60 및 $70^{\circ}C$에서의 온도별 상대활성은 각각 50, 55, 70, 100, 90 및 50%였다. pH 5, 6, 7 및 8에서의 pH별 상대활성은 각각 75, 100, 90, 75%였다. 본 효소는 20 mM Tris-HCl 완충액에서 pH 6.0 및 $50^{\circ}C$에서 최대활성을 나타내었다. 본 효소는 한천이 액체상태로 존재하는 $50^{\circ}C$에서 최대활성을 나타내었으므로 산업적으로 유용한 효소로 판단된다. 본 효소는 20, 30 및 $40^{\circ}C$에서 2시간 동안 열처리하여도 80% 이상의 잔존활성을 보였다. TLC 분석결과, Persicobacter sp. DH-3 균주의 한천분해효소는 네오한천올리고당인 neoagarohexaose와 neoagarotetraose를 생성하였으므로 ${\beta}$-agarase로 확인되었다. Zymogram 분석결과, Persicobacter sp. DH-3는 적어도 45, 70 및 140 kDa 크기의 3종류 이상의 한천분해효소를 생산하는 것을 확인할 수 있었다. 그러므로, Persicobacter sp. DH-3 유래 agarase는 기능성을 가진 neoagarooligosaccharides의 생산에 활용될 수 있을 것으로 기대된다.

Keywords

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Fig. 1. Phylogenetic tree based on almost complete 16S rDNA sequence comparing strain DH-3 with other bacteria. The numbers at the branch are bootstrap values and numbers in parenthesis are accession numbers at NCBI.

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Fig. 2. The growth curve (□) of Persicobacter sp. DH-3 and the production of agarase (■) in the presence of 0.2% (w/v) agar. The highest level of agarase was reached at the period of lag phase.

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Fig. 3. Effects of reaction temperatures on agarase activities. The reactions were carried out at 20, 30, 40, 50, 60 and 70℃ in 1 ml of 20 mM Tris-HCl (pH 6.0) buffer and 0.5 ml of enzyme solution for 30 min.

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Fig. 4. Effects of pHs on the agarase activities. 20 mM Tris-HCl buffer (pH 5.0-8.0) and GTA buffer (pH 8.0-9.0) were used. The reactions were carried out at 50℃ in 1 ml of corresponding buffer and 0.5 ml of enzyme solution for 30 min.

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Fig. 5. Remaining activities of agarase after heat treatments for defined times. The enzyme solutions were pre-incubated at 20 (■), 30 (◆), 40 (▼), 50 (●), 60 (○) and 70℃ (□) for 0, 0.5, 1.0, 1.5, 2.0 hr. The reactions were then carried out at 50℃ in 1 ml of 20 mM Tris-HCl (pH 6.0) buffer containing 0.5 ml of heat-treated enzyme solution for 30 min.

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Fig. 6. Zymogram analysis of agarases. The molecular masses of the enzymes were 45, 70 and 140 kDa. (Lane M size markers; lane E, agarases).

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Fig. 7. TLC analysis of the hydrolyzed products of agarose by agarase. The reactions were carried out at 50℃ in Tris-HCl (pH 6.0) buffer containing 0.2% (w/v) agar and 0.5 ml of enzyme solution for 5, 10, 15, 20, 25 and 30 min. The reaction mixtures were developed by TLC. (G, Dgalactose; NA, neoagarooligosaccharides; NA2, neoagarobiose; NA4, neoagarotetraose; NA6, neoagarohexaose).

References

  1. Chi, W. J., Lim, J. H., Park, D. Y., Kim, M. C., Kim, C. J., Chang, Y. K. and Hong, S. K. 2013. Isolation and characterization of a novel agar degrading bacterium, Alteromonas macleodii subsp. GNUM08120, from red macroalgae. Kor. J. Microbiol. Biotechnol. 41, 8-16. https://doi.org/10.4014/kjmb.1208.08001
  2. Furusawa, G., Leu, N, S., Fuganthi, A. and Amirul, A. A. 2017. Agarolytic bacterium Persicobacter sp. CCB-QB2 exhibited a diauxic growth involving galactose utilization pathway. Microbiologyopen 6, e00405. https://doi.org/10.1002/mbo3.405
  3. Han, W., Liu, S. Z., Liu, H., Wu, Z., Gu, Q. and Li, Y. 2012. Isolation identification and agarose degradation of a polysaccharide-degrading marine bacterium Persicobacter sp. JZB09. Acta Microbiol. Sin. 52, 776-783.
  4. Jang, H. J., Lee, D. G., Lee, S. W., Jeon, M. J., Chun, W. J., Kwon, K. K., Lee, H. S. and Lee, S. H. 2011. Isolation of a marine-derived Flammeovirga sp. mbrc-1 strain and characterization of its agarase. Kor. Soc. Biotechnol. Bioeng. J. 26, 552-556.
  5. Jo, J. G., Lee, S. J., Lee, D. G. and Lee, S. H. 2016. Characterization of agarase produced from the isolated marine bacterium Marinomonas sp. SH-2. J. Life Sci. 26, 198-203. https://doi.org/10.5352/JLS.2016.26.2.198
  6. Kim, J. D., Lee, D. G. and Lee, S. H. 2018. Cloning, expression, and characterization of a thermotolerant ${\beta}$-agarase from Simiduia sp. SH-4. Biotechnol. Bioproc. Eng. 23, 525-531. https://doi.org/10.1007/s12257-018-0072-4
  7. Lee, C. E., Lee, S. J., Lee, D. G. and Lee, S. H. 2016. Isolation of a new agar degrading bacterium, Maribacter sp. SH-1 and characterization of its agarase. Microbiol. Biotechnol. Lett. 44, 156-162. https://doi.org/10.4014/mbl.1511.11007
  8. Lee, D. G., Jang, M. K., Lee, O. H., Kim, N. Y., Ju, S. A. and Lee, S. H. 2008. Over-production of a glycoside hydrolase family 50 ${\beta}$-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product. Biotechnol. Lett. 30, 911-918. https://doi.org/10.1007/s10529-008-9634-4
  9. Lee, D. G., Kim, N. Y., Jang, M. K., Lee, O. H. and Lee, S. H. 2007. Isolation and characterization of a marine bacterium Thalassomonas sp. SL-5 producing ${\beta}$-agarase. J. Life Sci. 17, 70-75. https://doi.org/10.5352/JLS.2007.17.1.070
  10. Lee, D. G., Lee, O. H., Jang, H. J., Jang, M. K., Yoo, K. H. and Lee, S. H. 2008. Isolation and characterization of a marine derived bacterium Glaciecola sp. SL-12 producing ${\beta}$-agarase. J. Life Sci. 18, 58-62. https://doi.org/10.5352/JLS.2008.18.1.058
  11. Lee, S. J., Oh, S. J., Lee, D. G. and Lee, S. H. 2015. Characterization of agarase from an isolated marine bacterium, Simiduia sp. SH-1. J. Life Sci. 25, 1273-1279. https://doi.org/10.5352/JLS.2015.25.11.1273
  12. Lee, S. J., Shin, D. Y., Kim, J. D., Lee, D. G. and Lee, S. H. 2016. Characterization of ${\alpha}$-agarase from Alteromonas sp. SH-1. Kor. Soc. Biotechnol. Bioeng. J. 31, 113-119.