caa a22 4500 445318945 CHVBK 20180317142657.0 cr unu---uuuuu 170323e20110401xx s 000 0 eng 10.1007/s10126-010-9290-2 doi (NATIONALLICENCE)springer-10.1007/s10126-010-9290-2 Protoplast Preparation from Laminaria japonica with Recombinant Alginate Lyase and Cellulase [Elektronische Daten] [Akira Inoue, Chieco Mashino, Teina Kodama, Takao Ojima] Functional recombinant abalone alginate lyase (rHdAly) and β-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2U/mg for rHdAly and rHdEG66, respectively, was comparable to its native form at 30°C. Purified rHdAly and rHdEG66 showed the highest specific activity both at 35°C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (<1 × 103 protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 × 106 protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6μm, these cells were mostly derived from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase, cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts of the epidermal layer, which are known to be able to be regenerated. Springer Science+Business Media, LLC, 2010 Alginate lyase nationallicence Cellulase nationallicence Abalone nationallicence Brown algae nationallicence Laminaria japonica nationallicence Protoplast nationallicence Inoue Akira Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 041-8611, Hakodate, Hokkaido, Japan aut Mashino Chieco Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 041-8611, Hakodate, Hokkaido, Japan aut Kodama Teina Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 041-8611, Hakodate, Hokkaido, Japan aut Ojima Takao Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 041-8611, Hakodate, Hokkaido, Japan aut Marine Biotechnology Springer-Verlag 13/2(2011-04-01), 256-263 1436-2228 13:2<256 2011 13 10126 https://doi.org/10.1007/s10126-010-9290-2 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10126-010-9290-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Inoue Akira Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 041-8611, Hakodate, Hokkaido, Japan aut NATIONALLICENCE 700 1- Mashino Chieco Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 041-8611, Hakodate, Hokkaido, Japan aut NATIONALLICENCE 700 1- Kodama Teina Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 041-8611, Hakodate, Hokkaido, Japan aut NATIONALLICENCE 700 1- Ojima Takao Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 041-8611, Hakodate, Hokkaido, Japan aut NATIONALLICENCE 773 0- Marine Biotechnology Springer-Verlag 13/2(2011-04-01), 256-263 1436-2228 13:2<256 2011 13 10126 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer