Advertisement

Extremophiles

, Volume 13, Issue 1, pp 121–129 | Cite as

A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp.

  • Tohru Kobayashi
  • Kohsuke Uchimura
  • Masayuki Miyazaki
  • Yuichi Nogi
  • Koki Horikoshi
Original Paper

Abstract

A high-alkaline, salt-activated alginate lyase is produced by Agarivorans sp. JAM-A1m from a deep-sea sediment off Cape Nomamisaki on Kyushu Island, Japan. Purified to homogeneity, as judged by SDS-PAGE, the enzyme (A1m) had a molecular mass of approximately 31 kDa. The optimal pH was around 10 in glycine–NaOH buffer, and the activity was increased to 1.8 times by adding 0.2 M NaCl. However, when the optimal pH in the presence of 0.2 M NaCl was shifted to pH 9.0, the activity was more than 10 times compared with that at pH 9 in the absence of NaCl. A1m showed the optimal temperature at around 30°C and was stable to incubation between pH 6 and 9. The enzyme degraded favorably mannuronate–guluronate and guluronate-rich fragments in alginate. Shotgun cloning and sequencing of the gene for A1m revealed a 930-bp open reading frame, which encoded a mature enzyme of 289 amino acids (32,295 Da) belonging to polysaccharide lyase family 7. The deduced amino acid sequence showed the highest similarity to that of a Klebsiella enzyme, with only 54% identity.

Keywords

Alginate lyase PL family 7 Alkaline enzyme Deep sea Agarivorans 

Notes

Acknowledgments

We are grateful to the captain and crew of the R/V Natushima and the ROV Hyper Dolphin operation team, for their technical support in sampling. We also thank the chief scientist, Prof. K. Kubokawa of the Ocean Research Institute, University of Tokyo, as well as all scientists aboard the NT05-12 cruise. We thank Dr. S. Aihara and Dr. H. Minegishi of Bio-Nano Electronics Research Center, Toyo University, for the IR spectra.

References

  1. Boyen C, Bertheau Y, Barbeyron T, Kloareg B (1990a) Preparation of guluronate lyase from Pseudomonas alginovora for protoplast isolation in Laminaria. Enzyme Microb Technol 12:885–890CrossRefGoogle Scholar
  2. Boyen C, Kloareg B, Polne-Fuller M, Gibor A (1990b) Preparation of alginate lyases from marine molluscs for protoplast isolation in brown algae. Phycologia 29:173–181Google Scholar
  3. Brown BJ, Preston JF (1991) l-guluronan-specific alginate lyase from a marine bacterium associated with Sargassum. Carbohydr Res 211:91–102PubMedCrossRefGoogle Scholar
  4. Evans LR, Linker A (1973) Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacteriol 116:915–924PubMedGoogle Scholar
  5. Gacesa P (1988) Alginates. Carbohydr Polym 8:161–182CrossRefGoogle Scholar
  6. Gorin PAJ, Spencer JFT (1966) Exocellular alginic acid from Azotobacter vinelandii. Can J Chem 44:993–998CrossRefGoogle Scholar
  7. Haug A, Larsen B, Smidsrød O (1966) A study of the constitution of alginic acid by partial acid hydrolysis. Acta Chem Scand 20:183–190CrossRefGoogle Scholar
  8. Haug A, Larsen B, Smidsrød O (1967) Studies on the sequence of uronic acid residues in alginic acid. Acta Chem Scand 21:691–704CrossRefGoogle Scholar
  9. Horikoshi K, Akiba T (1982) Alkalophilic microorganisms. Japan Scientific Societies Press, Tokyo, p 137Google Scholar
  10. Iwamoto Y, Araki R, Iriyama K, Oda T, Fukuda H, Hayashida S, Muramatsu T (2001) Purification and characterization of bifunctional alginate lyase from Alteromonus sp. strain No. 272 and its action on saturated oligomeric substrates. Biosci Biotechnol Biochem 65:133–142PubMedCrossRefGoogle Scholar
  11. Kawamoto H, Horibe A, Miki Y, Kimura T, Tanaka K, Nakagawa T, Kawamukai M, Matsuda H (2006) Cloning and sequencing analysis of alginate lyase genes from the marine bacterium Vibrio sp. O2. Mar Biotechnol 8:481–490PubMedCrossRefGoogle Scholar
  12. Kitamikado M, Tseng CH, Yamaguchi K, Nakamura T (1992) Two types of alginate lyases. Appl Environ Microbiol 58:2474–2478PubMedGoogle Scholar
  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  14. Lange B, Wingender J, Winkler UK (1989) Isolation and characterization of an alginate lyase from Klebsiella aerogenes. Arch Microbiol 152:302–308PubMedCrossRefGoogle Scholar
  15. Matsubara Y, Kawada R, Iwasaki K, Oda T, Muramatsu T (1998) Extracellular poly (α-l-guluronate) lyase from Corynebacterium sp.: purification, characterization, and conformational properties. J Protein Chem 17:29–36PubMedCrossRefGoogle Scholar
  16. Min KH, Sasaki SF, Kashiwabara Y, Nishizawa K (1977) Substrate specificity of endo-polyguluronide lyase of Pseudomonas sp. J Biochem 81:547–553PubMedGoogle Scholar
  17. Muramatsu T, Hashimoto H, Takahashi T (1984) Physicochemical characteristics and conformational features of alginate lyase isozymes form Turbo cornutus. Biosci Biotechnol Biochem 48:79–85Google Scholar
  18. Osawa T, Matsubara Y, Muramatsu T, Kimura M, Kakuta Y (2005) Crystal structure of the alginate (poly-α-l-guluronate) lyase from Corynebacterium sp. at 1.2 Å resolution. J Mol Biol 345:1111–1118PubMedCrossRefGoogle Scholar
  19. Pereira L, Sousa A, Coelho H, Amado AM, Ribeiro-Claro PJA (2003) Use of FTIR, FT-Raman and 13C-NMR spectroscopy for identification of some seaweed phycocolloids. Biomol Eng 20:223–228PubMedCrossRefGoogle Scholar
  20. Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629PubMedCrossRefGoogle Scholar
  21. Sartori C, Finch DS, Ralph B (1997) Determination of the cation content of alginate thin films by FT i.r. spectroscopy. Polymer 38:43–51CrossRefGoogle Scholar
  22. Shirai T, Kobayashi T, Ito S, Horikoshi K (2008) Chapter IV-Alkaline adaptation of proteins. In: Siddiqui KS, Thomas T (eds) Protein adaptation in extremophiles. Nova Science Publishers Inc, Hauppauge, NY, pp 105–141Google Scholar
  23. Suda K, Tanji Y, Hori K, Unno H (1999) Evidence for a novel Chlorella virus-encoded alginate lyase. FEMS Microbiol Lett 180:45–53PubMedCrossRefGoogle Scholar
  24. Sugimura I, Sawabe T, Ezura Y (2000) Cloning and sequence analysis of Vibrio halioticoli genes encoding three types of polyguluronate lyase. Mar Biotechnol 2:65–73PubMedGoogle Scholar
  25. Suzuki H, Suzuki K, Inoue A, Ojima T (2006) A novel oligoalginate lyase from abalone, Haliotis discus hannai, that releases disaccharide from alginate polymer in an exolytic manner. Carbohydr Res 341:1809–1819PubMedCrossRefGoogle Scholar
  26. Svanem BIG, Strand WI, Ertesvåg H, Skjåk-Bræk G, Hartmann M, Barbeyron T, Valla S (2001) The catalytic activities of the bifunctional Azotobacter vinelandii mannuronan C-5-epimerase and alginate lyase AlgE7 probably originate from the same active site in the enzyme. J Biol Chem 276:31542–31550PubMedCrossRefGoogle Scholar
  27. Weissbach A, Hurwitz J (1959) The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli B. J Biol Chem 234:705–709PubMedGoogle Scholar
  28. Wong TY, Preston LA, Schiller NL (2000) Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Ann Rev Microbiol 54:289–340CrossRefGoogle Scholar
  29. Xiao L, Han F, Yang Z, Lu XZ, Yu WG (2006) A novel alginate lyase with high activity on acetylated alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03. World J Microbiol Biotechnol 22:81–88CrossRefGoogle Scholar
  30. Yamasaki M, Moriwaki S, Miyake O, Hashimoto W, Murata K, Mikami B (2004) Structure and function of a hypothetical Pseudomonas aeruginosa protein PA1167 classified into family PL-7. J Biol Chem 279:31863–31872PubMedCrossRefGoogle Scholar
  31. Yamasaki M, Ogura K, Hashimoto W, Mikami B, Murata K (2005) A structural basis for depolymerization of alginate by polysaccharide lyase family-7. J Mol Biol 352:11–21PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2008

Authors and Affiliations

  • Tohru Kobayashi
    • 1
  • Kohsuke Uchimura
    • 1
  • Masayuki Miyazaki
    • 1
  • Yuichi Nogi
    • 1
  • Koki Horikoshi
    • 1
  1. 1.Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology (JAMSTEC)YokosukaJapan

Personalised recommendations