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Journal of Applied Phycology

, Volume 20, Issue 5, pp 633–640 | Cite as

Preparation of protoplasts from Laminaria japonica using native and recombinant abalone alginate lyases

  • Akira Inoue
  • Mayu Kagaya
  • Takao Ojima
Article

Abstract

Laminaria japonica protoplasts were released with high yields using the abalone alginate lyase HdAly in combination with a cellulase and chelating agents. Addition of EDTA at concentrations higher than 10 mM to Laminaria thalli which had been preincubated with HdAly and Cellulase Onozuka, dramatically improved the yield of protoplasts. EDTA was far more effective than EGTA, indicating that chelating divalent metal ions such as Mg2+ and Sr2+ in addition to Ca2+ is a key factor for high-yield production of Laminaria protoplasts. Protoplasts had a mean diameter of 27 μm, suggesting that most protoplasts were derived from cortical cells rather than epidermal layer cells. Recombinant HdAly (rHdAly) was produced from a cDNA clone in the Sf9 insect cell expression system. rHdAly had substantially the same enzymatic properties and protoplast-producing ability as did native HdAly. The optimal conditions for high yield production of protoplasts from Laminaria using native and recombinant HdAlys were investigated.

Keywords

Abalone Alginate lyase Brown algae Laminaria japonica Protoplast 

Notes

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists (B), No. 17780164, and a 21st century COE program grant of the international COE of "Marine Bio-Manipulation Frontier for Food Production" both from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and the Akiyama Foundation.

References

  1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  2. Butler DM, Østgaard K, Boyen C, Evans LV, Jensen A, Kloareg B (1989) Isolation conditions for high yields of protoplasts from Laminaria saccharina and L. digitata (Phaeophyceae). J Exp Bot 40:1237–1246CrossRefGoogle Scholar
  3. Ducreux G, Kloareg B (1988) Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae). Planta 174:25–29CrossRefGoogle Scholar
  4. Fisher DD, Gibor A (1987) Production of protoplasts from the brown alga, Sargassum muticum (Yendo) Fensholt (Phaeophyta). Phycologia 26:488–495Google Scholar
  5. Jothisaraswathi S, Babu B,Rengasamy R (2006) Seasonal studies on alginate and its composition II: Turbinaria conoides (J.Ag.) Kütz. (Fucales, Phaeophyceae). J Appl Phycol 18:161–166CrossRefGoogle Scholar
  6. Kain JM (1971) Synopsis of biological data on Laminaria hyperborea. FAO Fish Synop 84:1–74Google Scholar
  7. Kajiwara T, Hatanaka A, Fujimura T, Kawai T, Irie M (1988) Isolation of protoplasts from marine brown algae Dictyotaceae plants. Nippon Suisan Gakkaishi 547:1255Google Scholar
  8. Kloareg B, Quatrano RS (1987a) Enzymatic removal of the cell walls from zygotes of Fucus distichus (L.) Powell (Phaeophyta). Hydrobiologia 151/152:123–129CrossRefGoogle Scholar
  9. Kloareg B, Quatrano RS (1987b) Isolation of protoplasts from zygotes of Fucus distichus (L.) Powell (Phaeophyta). Plant Sci 50:189–194CrossRefGoogle Scholar
  10. Kloareg B, Polne-Fuller M, Gibor, A (1989) Mass production and regeneration of protoplasts from Macrocystis pyrifera. Plant Sci 62:105–112CrossRefGoogle Scholar
  11. Lane CE, Mayes C, Druehl LD, Saunders GW (2006) A multi-gene molecular investigation of the kelp (Laminariales, Phaeophyceae) supports substantial taxonomic re-organization. J Phycol 42:493–512CrossRefGoogle Scholar
  12. Matsumura W, Yasui H, Yamamoto H (2000) Mariculture of Laminaria japonica (Laminariales, Phaeophyceae) using protoplast regeneration. Phycol Res 48:169–176CrossRefGoogle Scholar
  13. Park JT, Johnson MJ (1949) A submicrodetermination of glucose. J Biol Chem 181:149–151PubMedGoogle Scholar
  14. Polne-Fuller M, Gibor A (1987) Calluses and callus-like growth in seaweeds, induction and culture. Hydrobiologia 151/152:131–138CrossRefGoogle Scholar
  15. Porzio MA, Pearson AM (1977) Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochim Biophys Acta 490:27–34PubMedGoogle Scholar
  16. Saga N (1984) Isolation of protoplasts from edible seaweeds. Bot Mag Tokyo 97:423–427CrossRefGoogle Scholar
  17. Saga N, Sakai Y (1984) Isolation of protoplasts from Laminaria and Porphyra. Bull Jap Soc Sci Fish 50:1085Google Scholar
  18. Saga N, Polne-Fuller M, Gibor A (1986) Protoplasts from seaweed: production and fusion. Beih Nova Hedwigia 83:37–43Google Scholar
  19. Saraswathi SJ, Babu B, Rengasamy R (2003) Seasonal studies on the alginate and its biochemical composition I: Sargassum polycystum (Fucales), Phaeophyceae. Phycol Res 51:240–243CrossRefGoogle Scholar
  20. Sawabe T, Ezura Y, Kimura T (1993) Application of an alginate lyase from Alteromonas sp. for isolation of protoplasts from a brown algae Laminaria japonica. Bull Jap Soc Sci Fish 59:705–709Google Scholar
  21. Sawabe T, Ezura Y (1996) Regeneration from Laminaria japonica Areschoug (Laminariales, Phaeophyceae) protoplasts isolated with bacterial alginase. Plant Cell Rep 15:892–895CrossRefGoogle Scholar
  22. Shimizu E, Ojima T, Nishita K (2003) cDNA cloning of an alginate lyase from abalone, Haliotis discus hannai. Carbohydr Res 338:2841–2852PubMedCrossRefGoogle Scholar
  23. Suzuki K, Ojima T, Nishita K (2003) Purification and cDNA cloning of a cellulase from abalone Haliotis discus hannai. Eur J Biochem 270:771–778PubMedCrossRefGoogle Scholar
  24. Tokuda H, Kawashima Y (1988) Protoplast isolation and culture of a brown alga, Undaria pinnatifida. In: Stadler T, Mollion J, Verdus MC, Karamanos Y, Morvan H, Christiaen D (eds) Algal biotechnology. Elsevier Applied Science, London, pp 151–159Google Scholar
  25. Wakabayashi T, Kuboi T, Tuboi T, Kaji M, Hara M (1999) Preparation of high yields of algal protoplasts using buccal juice of sea hare and commercial cellulase. Mar Biotechnol 1:407–410PubMedCrossRefGoogle Scholar
  26. Yoon HS, Lee JY, Boo SM, Bhattacharya D (2001) Phylogeny of Alariaceae, Laminariaceae, and Lessoniaceae (Phaeophyta) based on plastid-encoded RuBisCo spacer and nuclear-encoded ITS sequence comparisons. Mol Phylogenet Evol 21:231–243PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries SciencesHokkaido UniversityHakodateJapan

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