Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Gene Cloning, Expression, and Characterization of a $\beta$-Agarase, AgaB34, from Agarivorans albus YKW-34

  • Fu, Xiao Ting (College of Food Science and Engineering, Ocean University of China) ;
  • Pan, Cheol-Ho (Natural Products Research Center, KIST Gangneung Institute) ;
  • Lin, Hong (College of Food Science and Engineering, Ocean University of China) ;
  • Kim, Sang-Moo (Faculty of Marine Bioscience and Technology, Kangnung National University)
  • Received : 2008.01.10
  • Accepted : 2008.07.16
  • Published : 2009.03.31
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Abstract

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

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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, C. H. 1937. Acetylation of agar-like substance of Gelidium amansii. J. Chem. Soc. Japan 58: 1338-1350
  3. 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
  4. Bradford, M. M. 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  5. Dong, J., Y. Tamaru, and T. Araki. 2007. A unique $\beta$-agarase, AgaA, from a marine bacterium, Vibrio sp. strain PO-303. Appl. Microbiol. Biotechnol. 74: 1248-1255 https://doi.org/10.1007/s00253-006-0781-z
  6. 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
  7. Fu, X. T., H. Lin, and S. M. Kim. 2007. Purification and characterization of a Na+/K+ dependent alginate lyase from turban shell gut Vibrio sp. YKW-34. Enzyme Microb. Technol. 41: 828-834 https://doi.org/10.1016/j.enzmictec.2007.07.003
  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. Fujimoto, Z., H. Mizuno, A. Kuno, S. Yoshida, H. Kobayashi, and I. Kusakabe. 1997. Crystallization and preliminary X-ray crystallographic study of Streptomyces olivaceoviridis E-86 $\beta$-xylanase. J. Biochem. 121: 826-828 https://doi.org/10.1093/oxfordjournals.jbchem.a021659
  10. 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
  11. Hamer, G. K., S. S. Bhattacharjee, and W. Yaphe. 1977. Analysis of the enzymic hydrolysis products of agarose by $13_{C}$-n.m.r. spectroscopy. Carbohydr. Res. 54: C7-Cl0 https://doi.org/10.1016/S0008-6215(00)80567-4
  12. Hogset, A., O. R. Blingsmo, O. Saether, V. T. Gautvik, E. Holmgren, M. Hartmanis, et al. 1990. Expression and characterization of a recombinant human parathyroid hormone secreted by Escherichia coli employing the staphylococcal protein A promoter and signal sequence. J. Biol. Chem. 265:7338-7344
  13. Jam, M., D. Flament, J. Allouch, P. Potin, L. Thion, 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. Biochem. J. 385: 703-713 https://doi.org/10.1042/BJ20041044
  14. Kirimura, K., N. Masuda, Y. Iwasaki, H. Nakagawa, R. Kobayashi, and S. Usami. 1999. Purification and characterization of a novel $\beta$-agarase from an alkalophilic bacterium, Alteromonas sp. E-1. J. Biosci. Bioeng. 87: 436-441 https://doi.org/10.1016/S1389-1723(99)80091-7
  15. 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
  16. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  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. Lineweaver, H. and D. Burk. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56: 658-666 https://doi.org/10.1021/ja01318a036
  19. Nelson, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375-380
  20. 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
  21. Ohta, Y., Y. Hatada, Y. Nogi, M. Miyazaki, Z. Li, M. Akita, et al. 2004. Enzymatic properties and nucleotide and amino acid sequences of a thermostable $\beta$-agarase from a novel species of deep-sea Microbulbifer. Appl. Microbiol. Biotechnol. 64: 505-514 https://doi.org/10.1007/s00253-004-1573-y
  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 Microbulbifer-like isolate. Appl. Microbiol. Biotechnol. 66: 266-275 https://doi.org/10.1007/s00253-004-1757-5
  23. Pascal, J. M., P. J. Day, A. F. Monzingo, S. R. Ernst, J. D. Robertus, R. Iglesias, et al. 2001. 2.8-$\AA$ crystal structure of a nontoxic type-II ribosome-inactivating protein, ebulin l. Proteins 43:319-326 https://doi.org/10.1002/prot.1043
  24. Potin, P., C. Richard, C. Rochas, and B. Kloareg. 1993. Purification and characterization of the $\alpha$-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. Eur. J. Biochem. 214: 599-607 https://doi.org/10.1111/j.1432-1033.1993.tb17959.x
  25. Rosenberg, M. and D. Court. 1979. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet. 13: 319-353 https://doi.org/10.1146/annurev.ge.13.120179.001535
  26. Shen, S. H., P. Chretien, L. Bastien, and S. N. Slilaty. 1991. Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli. J. Biol. Chem. 266: 1058-1063
  27. Sugano, Y., I. Terada, M. Arita, and M. Noma. 1993. Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107. Appl. Environ. Microbiol. 59: 1549-1554
  28. Talmadge, K. and W. Gilbert. 1982. Cellular location affects protein stability in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 79:1830-1833 https://doi.org/10.1073/pnas.79.6.1830
  29. Takahara, M., D. W. Hibler, P. J. Barr, J. A. Gerlt, and M. Inouye. 1985. The ompA signal peptide directed secretion of staphylococcal nuclease A by Escherichia coli. J. Biol. Chem. 260: 2670-2674
  30. Wilson, K. 1997. Preparation of genomic DNA from bacteria, pp. 2.4.1-2.4.5. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (eds.), Current Protocols in Molecular Biology., John Wiley & Sons Inc., New York
  31. Wu, S. C., T. N. Wen, and C. L. Pan. 2005. Algal oligosaccharidelysates 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
  32. Yamabhai, M., S. Emrat, S. Sukasem, P. Pesatcha, N. Jaruseranee, and B. Buranabanyat. 2008. Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems. J. Biotechnol. 133: 50-57 https://doi.org/10.1016/j.jbiotec.2007.09.005
  33. Yi, K. W. and I. S. Shin. 2006. Isolation of marine bacterium decomposing sea tangle (Laminaria japonica) to single cell detritus. Korean J. Food Sci. Technol. 38: 237-240 (in Korean)

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