Preparation of protoplasts from Laminaria japonica using native and recombinant abalone alginate lyases
- 204 Downloads
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.
KeywordsAbalone Alginate lyase Brown algae Laminaria japonica Protoplast
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.
- Fisher DD, Gibor A (1987) Production of protoplasts from the brown alga, Sargassum muticum (Yendo) Fensholt (Phaeophyta). Phycologia 26:488–495Google Scholar
- Kain JM (1971) Synopsis of biological data on Laminaria hyperborea. FAO Fish Synop 84:1–74Google Scholar
- 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
- Saga N, Sakai Y (1984) Isolation of protoplasts from Laminaria and Porphyra. Bull Jap Soc Sci Fish 50:1085Google Scholar
- Saga N, Polne-Fuller M, Gibor A (1986) Protoplasts from seaweed: production and fusion. Beih Nova Hedwigia 83:37–43Google Scholar
- 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
- 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