Protoplast preparation from Laminaria japonica with recombinant alginate lyase and cellulase.
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 18.2 U/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 × 10(3) protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 × 10(6) protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 μm, 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.
Inoue
Akira
A
Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate Hokkaido, 041-8611, Japan.
Mashino
Chieco
C
Kodama
Teina
T
Ojima
Takao
T
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D007795:Laminaria / Q000201:enzymology* / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011523:Protoplasts / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; C074775:CelA endoglucanase; D002480:Cellulase; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase
Mar Biotechnol (NY). 1999 Jul;1(4):407-410
10489421
Carbohydr Res. 2003 Nov 21;338(24):2841-52
14667705
Biochem J. 1982 May 15;204(2):449-55
7115340
Plant Cell Rep. 1996 Sep;15(12):892-5
24178269
J Protein Chem. 1996 Nov;15(8):709-19
9008294
J Biol Chem. 1949 Nov;181(1):149-51
15390401
Microbiol Mol Biol Rev. 2002 Sep;66(3):506-77, table of contents
12209002
Annu Rev Plant Physiol Plant Mol Biol. 1998 Jun;49:281-309
15012236
Carbohydr Res. 2006 Aug 14;341(11):1809-19
16697989
Plant J. 1994 Feb;5(2):157-72
8148875
Mol Cell Biol. 1985 Dec;5(12):3610-6
3915782
Planta. 1988 Apr;174(1):25-9
24221413
Eur J Biochem. 2003 Feb;270(4):771-8
12581217
Eur J Biochem. 1983 Sep 1;135(1):123-6
6309516
Annu Rev Microbiol. 2000;54:289-340
11018131
Mol Microbiol. 1994 Jul;13(2):219-28
7984103
Anal Biochem. 1976 May 7;72:248-54
942051
Hoppe Seylers Z Physiol Chem. 1967 Jan;348(1):37-50
5592400
Biochim Biophys Acta. 1977 Jan 25;490(1):27-34
836873