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The beta-agarase-d gene (agaD) from a marine bacterium, Vibrio sp. strain PO-303, was cloned and expressed in Escherichia coli. The gene consists of 1,362 bp and encodes a protein of 453 amino acids with a predicted molecular weight of 50,824. The full length of agarase-d consists of a signal peptide, a glycoside hydrolase family 16 catalytic module (CM), and a carbohydrate binding module (CBM). The full length of agarase-d without the signal peptide (rAgaDDeltafull), the catalytic module (rAgaDCM), or the CBM (rAgaDCBM) was expressed in E. coli as recombinant proteins. rAgaDCM exhibited higher enzyme activity (63.6 units/mg) than rAgaDDeltafull (1.20 units/mg) against agarose. rAgaDCM hydrolyzed agar and porphyran to several oligosaccharides and acted on neoagarohexaose to produce neoagarotetraose and neoagarobiose, but did not act on neoagarotetraose. rAgaDCBM bound to agarose.

Differences in the mineral contents of cultured red sea bream of different growth stages, localities, and culture methods were investigated, and a comparison was also made between those fish and thewild ones. The following results were obtained. (1) The contents of P, Fe, Mg, Na and K were nearly constant regardless of growth. The Ca content slightly decreased with growth. (2) The content of P in the meat was slightly higher in the cultured fish than in the wild fish. On the other hand, that in viscera was higher in the latter than in the former. The Fe content was high inthe meat of cultured fish. There were no appreciable differences in the contents of Ca, Na, and Mg in the meat between cultured and wild fish. (3) Fish raised on moist pellets containing more Na amount than fish raised on sardines, whereas the Ca content was high in the latter. (4) No remarkable difference was seen in the mineral contents between fish raised in a marifarm and fish cultivated in net cages. (5) The mineral contents in the meat of the analyzed fish differed in value from those listed in the standard tables of food composition in Japan.

High cellulase activity was detected in the extract of marine gammarid amphipods inhabiting sea weed beds. The cellulase was partially purified by ammonium sulfate precipitation and successive column chromatographies. One of the two fractions separated by Sephadex G-100 gel chromatography was C₁-cellulase (correspond to avicelase, E. C. 3. 2. l. 91), which hydrolyzed more insoluble cellulose, avicel and filterpaper, than soluble cellulose, carboxymethylcellulose. The other was Cx-enzyme (correspond to CMCase, E. C. 3. 2. 1.4), which cleaved more soluble carboxymethylcellulose. Both enzymes had pH optimum of around 5.5, and was fairly stable in a pH region of 5.0 to 8.0 for 20 hr at 30℃ and was completely stable below 40℃ for 30 min-incubation at pH 7.0. C₁-and Cx-enzymes had molecular weights of approximately 34,000 and 26,000, respectively. These cellulase were estimated to originate from gammarids in the sbsence of cellulolytic symbiotic bacteria.

The agaA gene encoding beta-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)-like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40 degrees C and pH 7.5, with a specific activity of 16.4 units mg(-1), a K (m) of 1.10 mg ml(-1), and a V (max) of 22.5 micromol min(-1) mg(-1) for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.

The β-agarase C gene ( agaC ) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. β-Agarase C was identified as the first β-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.