Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus.
Glycoside-hydrolase-family 9 (GHF9) cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal cellulases.
Nishida
Yukiko
Y
Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan.
Suzuki
Ken-ichi
K
Kumagai
Yuya
Y
Tanaka
Hiroyuki
H
Inoue
Akira
A
Ojima
Takao
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007564:Japan; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D047269:Strongylocentrotus / Q000201:enzymology*; D013696:Temperature
D018076:DNA, Complementary; D002480:Cellulase