Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning of Agarase Gene from Non-Marine Agarolytic Bacterium Cellvibrio sp.

  • Ariga, Osamu (School of Environmental Science and Engineering, Kochi University of Technology) ;
  • Inoue, Takayoshi (School of Environmental Science and Engineering, Kochi University of Technology) ;
  • Kubo, Hajime (School of Environmental Science and Engineering, Kochi University of Technology) ;
  • Minami, Kimi (School of Environmental Science and Engineering, Kochi University of Technology) ;
  • Nakamura, Mitsuteru (School of Environmental Science and Engineering, Kochi University of Technology) ;
  • Iwai, Michi (School of Environmental Science and Engineering, Kochi University of Technology) ;
  • Moriyama, Hironori (Kochi Prefectural Industrial Technology Center) ;
  • Yanagisawa, Mitsunori (International Development Engineering, Graduate School of Engineering, Tokyo Institute of Technology) ;
  • Nakasaki, Kiyohiko (International Development Engineering, Graduate School of Engineering, Tokyo Institute of Technology)
  • Received : 2012.02.09
  • Accepted : 2012.05.15
  • Published : 2012.09.28
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Abstract

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

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References

  1. Aoki, T., T. Araki, and M. Kitamikado. 1990. Purification and characterization of a novel ${\beta}$-agarase from Vibrio sp. AP-2. Eur. J. Biochem. 187: 461-465. https://doi.org/10.1111/j.1432-1033.1990.tb15326.x
  2. Araki, T., M. Hayakawa, Z. Lu, S. Karita, and T. Morishita. 1998. Purification and characterization of agarases from a marine bacterium, Vibrio sp. PO-303. J. Marine Biotechnol. 6: 260-265.
  3. Ajisaka, K., S. Agawa, S. Nagumo, K. Kurato, T. Yokoyama, K. Arai, and T. Miyazaki. 2009. Evaluation and comparison of the antioxidative potency of various carbohydrates using different methods. J. Agric. Food Chem. 57: 3102-3107. https://doi.org/10.1021/jf804020u
  4. Birnboim, H. C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513-1523. https://doi.org/10.1093/nar/7.6.1513
  5. Buttner, M. J., I. M. Fearnley, and M. J. Bibb. 1987. The agarase gene (dagA) of Streptomyces coelicolor A3 (2): Nucleotide sequence and transcriptional analysis. Mol. Gen. Genet. 209: 101-109. https://doi.org/10.1007/BF00329843
  6. Chen, H. M. and X. J. Yan. 2005. Antioxidant activities of agaro-oligosaccharides with different degrees of polymerization in cell-based system. Biochim. Biophys. Acta 1722: 103-111. https://doi.org/10.1016/j.bbagen.2004.11.016
  7. Chen, H., X. Yan, P. Zhu, and J. Lin. 2006. Antioxidant activity and hepatoprotective potential of agaro-oligosaccharides in vitro and in vivo. Nutr. J. 31: 1-12.
  8. Ekborg, N. A., L. E. Taylor, A. G. Longmire, B. Henrissat, R. M. Weiner, and S. W. Hutcheson. 2006. Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40. Appl. Environment. Microbiol. 72: 3396-3405. https://doi.org/10.1128/AEM.72.5.3396-3405.2006
  9. Fu, W., B. Han, D. Duan, W. Liu, and C. Wang. 2008. Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6. J. Ind. Microbiol. Biotechnol. 35: 915-922. https://doi.org/10.1007/s10295-008-0365-2
  10. Hosoda, A., M. Sakai, and S. Kanazawa. 2003. Isolation and characterization of agar-degrading Paenibacillus spp. associated with the rhizosphere of spinach. Biosci. Biotechnol. Biochem. 67: 1048-1055. https://doi.org/10.1271/bbb.67.1048
  11. Joon, Y., C. R. Han, W. C. Kim, D. Y. Jun, I. K. Rhee, and Y. H. Kim. 2010. Isolation of a novel freshwater agarolytic Cellvibrio sp. KY-YJ-3 and characterization of its extracellular ${\beta}$-agarase. J. Microbiol. Biotechnol. 20: 1378-1385. https://doi.org/10.4014/jmb.1007.07010
  12. Kidby, D. K. and D. J. Davidson. 1973. A convenient ferricyanide estimation of reducing sugars in the nanomole range. Anal. Biochem. 55: 321-325. https://doi.org/10.1016/0003-2697(73)90323-0
  13. Kobayashi, R., M. Takisada, T. Suzuki, K. Kirimura, and S. Usami. 1997. Neoagarobiose as a novel moisturizer with whitening effect. Biosci. Biotechnol. Biochem. 61: 162-163. https://doi.org/10.1271/bbb.61.162
  14. 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 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
  15. Mergaert, J., D. Lednicka, J. Goris, M. C. Cnockaert, P. D. Vos, and J. Swings. 2003. Taxonomic study of Cellvibrio strains and description of Cellvibrio ostraviensis sp. nov., Cellvibrio fibrivorans sp. nov. and Cellvibrio gandavensis sp. nov. Int. J. Syst. Evol. Microbiol. 53: 465-471. https://doi.org/10.1099/ijs.0.02316-0
  16. Meulen, H. J., W. Harder, and H. Veldkamp. 1975. Isolation and characterization of Cytophaga flevensis sp. nov., a new agarolytic flexibacterium. Antonie Van Leeuwenhoek 41: 431-447. https://doi.org/10.1007/BF02565087
  17. Ohta, Y., Y. Hatada, Y. Nogi, Z. Li, S. Ito, and K. Horikoshi. 2004. Cloning, expression, and characterization of a glycoside hydrolase family 86 beta-agarase from a deep-sea Microbulbiferlike isolate. Appl. Microbiol. Biotechnol. 66: 266-275. https://doi.org/10.1007/s00253-004-1757-5
  18. Pell, G., L. Szabo, S. J. Charnock, H. Xie, T. M. Gloster, G. J. Davies, and H. J. Gilbert. 2004. Structural and biochemical analysis of Cellvibrio japonicus xylanase 10C: How variation in substrate-binding cleft influences the catalytic profile of family GH-10 xylanases. J. Biol. Chem. 279: 11777-11788. https://doi.org/10.1074/jbc.M311947200
  19. Saito, H. and K. I. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72: 619-629. https://doi.org/10.1016/0926-6550(63)90386-4
  20. Sambrook, J., E. F. Frisch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  21. Sugano, Y., I. Terada, M. Arita, M. Noma, and T. Matsumoto. 1993. Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107. Appl. Environ. Microbiol. 59: 1549-1554.
  22. Sugano, Y., T. Matsumoto, H. Kodama, and M. Noma. 1993. Cloning and sequencing of agaA, a unique agarase 0107 gene from a marine bacterium, Vibrio sp. strain JT0107. Appl. Environ. Microbiol. 59: 3750-3756.
  23. Wynne, E. C. and J. M. Pemberton. 1986. Cloning of a gene cluster from Cellvibrio mixtus which codes for cellulase, chitinase, amylase, and pectinase. Appl. Environ. Microbiol. 52: 1362-1367.
  24. Xiao, T. F., L. Hong, and S. M. Kim. 2008. Purification and characterization of a novel b-agarase, AgaA34, from Agarivorans albus YKW-34. Appl. Microbiol. Biotechnol. 78: 265-273. https://doi.org/10.1007/s00253-007-1303-3
  25. Zhang, W. W. and L. Sun. 2007. Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp. strain V134. Appl. Environ. Microbiol. 73: 2825-2831. https://doi.org/10.1128/AEM.02872-06
  26. Zhong, Z., A. Toukdarian, D. Helinski, V. Knauf, S. Sykes, J. E. Wilkinson, et al. 2001. Sequence analysis of a 101-kilobase plasmid required for agar degradation by a Microscilla isolate. Appl. Environ. Microbiol. 67: 5771-5779. https://doi.org/10.1128/AEM.67.12.5771-5779.2001

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