Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning, Expression, and Characterization of a Glycoside Hydrolase Family 118 ${\beta}$-Agarase from Agarivorans sp. JA-1

  • Lee, Dong-Geun (Major in Bioscience and Biotechnology, Graduate School, Silla University) ;
  • Jeon, Myong Je (Major in Bioscience and Biotechnology, Graduate School, Silla University) ;
  • Lee, Sang-Hyeon (Major in Bioscience and Biotechnology, Graduate School, Silla University)
  • Received : 2012.09.11
  • Accepted : 2012.09.20
  • Published : 2012.12.28
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Abstract

We report a glycoside hydrolase (GH)-118 ${\beta}$-agarase from a strain of Agarivorans, in which we previously reported recombinant expression and characterization of the GH-50 ${\beta}$-agarase. The GH comprised an open reading frame of 1,437 base pairs, which encoded a protein of 52,580 daltons consisting of 478 amino acid residues. Assessment of the entire sequence showed that the enzyme had 97% nucleotide and 99% amino acid sequence similarities to those of GH-118 ${\beta}$-agarase from Pseudoalteromonas sp. CY24, which belongs to a different order within the same class. The gene corresponding to a mature protein of 440 amino acids was inserted, recombinantly expressed in Escherichia coli, and purified to homogeneity with affinity chromatography. It had maximal activity at $35^{\circ}C$ and pH 7.0 and had 208.1 units/mg in the presence of 300 mM NaCl and 1 mM $CaCl_2$. More than 80% activity was maintained after 2 h exposure to $35^{\circ}C$; however, < 40% activity remained at $45^{\circ}C$. The enzyme hydrolyzed agarose to yield neoagarooctaose as the main product. This enzyme could be useful for industrial production of functional neoagarooligosaccharides.

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References

  1. Allouch, J., M. Jam, W. Helbert, T. Barbeyron, B. Kloareg, B. Henrissat, and M. Czjzek. 2003. The three-dimensional structures of two ${\beta}$-agarases. J. Biol. Chem. 278: 47171-47180. https://doi.org/10.1074/jbc.M308313200
  2. Araki, T., Z. Lu, and T. Morishita. 1998. Optimization of parameters for isolation of protoplasts from Gracilaria verrucosa (Rhodophyta). J. Mar. Biotechnol. 6: 193-197.
  3. Belas, R., D. Bartlett, and S. Michael. 1988. Cloning and gene replacement mutagenesis of a Pseudomonas atlantica agarase gene. Appl. Environ. Microbiol. 54: 30-37.
  4. Dong, J., S. Hashikawa, T. Konishi, Y. Tamaru, and T. Araki. 2006. Cloning of the novel gene encoding ${\beta}$-agarase C from a marine bacterium, Vibrio sp. strain PO-303, and characterization of the gene product. Appl. Environ. Microbiol. 72: 6399-6401. https://doi.org/10.1128/AEM.00935-06
  5. Dong, J., Y. Tamaru, and T. Araki. 2007. Molecular cloning, expression, and characterization of a ${\beta}$-agarase gene, agaD, from a marine bacterium, Vibrio sp. strain PO-303. Biosci. Biotechnol. Biochem. 71: 38-46. https://doi.org/10.1271/bbb.60304
  6. Du, Z. J., G. Q. Lv, A. P. Rooney, T. T. Miao, Q. Q. Xu, and G. J. Chen. 2011. Agarivorans gilvus sp. nov. isolated from seaweed. Int. J. Syst. Evol. Microbiol. 61: 493-496. https://doi.org/10.1099/ijs.0.019810-0
  7. Fu, X. T., C. H. Pan, H. Lin, and S. M. Kim. 2009. Gene cloning, expression, and characterization of a ${\beta}$-agarase, AgaB34, from Agarivorans albus YKW-34. J. Microbiol. Biotechnol. 19: 257-264.
  8. Fu, X. T., H. Lin, and S. M. Kim. 2008. Purification and characterization of a novel ${\beta}$-agarase, AgaA34, from Agarivorans albus YKW-34. Appl. Microbiol. Biotechnol. 78: 265-273. https://doi.org/10.1007/s00253-007-1303-3
  9. Giordano, A., G. Andreotti, A. Tramice, and A. Trincone. 2006. Marine glycosyl hydrolases in the hydrolysis and synthesis of oligosaccharides. Biotechnol. J. 1: 511-530. https://doi.org/10.1002/biot.200500036
  10. Hehemann, J. H., G. Correc, T. Barbeyron, W. Helbert, M. Czjzek, and G. Michel. 2010. Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature 464: 908-912. https://doi.org/10.1038/nature08937
  11. Hu, Z., B.-K. Lin, Y. Xu, M. Q. Zhong, and G.-M. Liu. 2009. Production and purification of agarase from a marine agarolytic bacterium Agarivorans sp. HZ105. J. Appl. Microbiol. 106: 181-190. https://doi.org/10.1111/j.1365-2672.2008.03990.x
  12. Jam, M., D. Flament, J. Allouch, P. Potin, L. Yhion, B. Kloareg, et al. 2005. The endo-${\beta}$-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: Two paralogue enzymes with different molecular organizations and catalytic behaviours. J. Biochem. 385: 703-713. https://doi.org/10.1042/BJ20041044
  13. Jang, M. K., S. W. Lee, D. G. Lee, N. Y. Kim, K. H. Yu, H. J. Jang, et al. 2010. Enhancement of the thermostability of a recombinant beta-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis. Biotechnol. Lett. 32: 943-949. https://doi.org/10.1007/s10529-010-0237-5
  14. Kim, H. T., S. Lee, K. H. Kim, and I. G. Choi. 2012. The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production. Bioresour. Technol. 107: 301-306.
  15. Kohno, T., H. Kitagawa, and T. Hiraga. 1990. Production of hetero-oligosaccharides, pp. 87-105. In Gijutsu, Kenkyu, Kukami (eds.). Shokuhin Sangyo Bioreactor System: Jissen Bioreactor. Shokuhin Kagaku Shimbunsa, Tokyo, Japan.
  16. Kurahashi, M. and A. Yokota. 2004. Agarivorans albus gen. nov., sp. nov., a ${\gamma}$-proteobacterium isolated from marine animals. Int. J. Syst. Evol. Microbiol. 54: 693-697. https://doi.org/10.1099/ijs.0.02778-0
  17. Lee, D. G., G. T. Park, N. Y. Kim, E. J. Lee, M. K. Jang, Y. G. Shin, et al. 2006. Cloning, expression, and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate. Biotechnol. Lett. 28: 1925-1932. https://doi.org/10.1007/s10529-006-9171-y
  18. 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
  19. Lee, S. W., D. G. Lee, M. K. Jang, M. J. Jeon, H. J. Jang, and S. H. Lee. 2011. Improvement in the catalytic activity of ${\beta}$-agarase AgaA from Zobellia galactanivorans by site-directed mutagenesis. J. Microbiol. Biotechnol. 21: 1116-1122. https://doi.org/10.4014/jmb.1107.07001
  20. Ma, C., X. Lu, C. Shi, J. Li, Y. Gu, Y. Ma, et al. 2007. Molecular cloning and characterization of a novel ${\beta}$-agarase, AgaB, from marine Pseudoalteromonas sp. CY24. J. Biochem. 282: 3747-3754.
  21. Ohta, Y., Y. Hatada, S. Ito, and K. Horikoshi. 2005. High-level expression of a neoagarobiose-producing ${\beta}$-agarase gene from Agarivorans sp. JAMB-A11 in Bacillus subtilis and enzymic properties of the recombinant enzyme. Biotechnol. Appl. Biochem. 41: 183-191. https://doi.org/10.1042/BA20040083
  22. 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
  23. Sambrook, J., E. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A laboratory manual. 2nd Ed., pp. 23-38. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
  24. Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195: 19-23.
  25. 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.
  26. Yu, R., J. Graf, and B. F. Smets. 2008. An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments. J. Microbiol. Methods 72: 235-240. https://doi.org/10.1016/j.mimet.2007.12.001
  27. Yun, E., M. Shin, J.-J. Yoon, Y. Kim, I.-G. Choi, and K. Kim. 2011. Production of 3,6-anhydro-L-galactose from agarose by agarolytic enzymes of Saccharophagus degradans 2-40. Process Biochem. 46: 88-93. https://doi.org/10.1016/j.procbio.2010.07.019
  28. Wolfrom, M. L. 1959. Carbohydrate chemistry of substances of biological interest. Proceedings of the 4th International Congress of Biochemistry. Pergamon Press, London.
  29. Wu, S. C., T. N. Wen, and C. L. Pan. 2005. Algaloligosaccharide lysates prepared by two bacterial agarases stepwise hydrolyzed and their anti-oxidative properties. Fish. Sci. 71: 1149-1159. https://doi.org/10.1111/j.1444-2906.2005.01075.x

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