P-Agarase was purified from the culture fluid of a porphyran-decomposing marine bacterium (strain AP-2) by ammonium sulfate precipitation, successive column chromatography and DNase and RNase treatment. The final enzyme preparation appeared to be homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 20 kDa, a pH optimum of 5.5, and was stable in the pH region 4.0-9.0 and at temperatures below 45' C.The P-agarase was a novel endo-type enzyme which hydrolyzed neoagarotetraose, larger neoagarooligosaccharides and agar to give neoagarobiose [3,6-anhydro-a-~-galactopyranosyl-(l + 3)-u-galactose] as the predominant product, The enzyme did not act on K-carrageenan.According to the criteria of Bergey's Manual of Systematic Bacteriology, the strain was assigned to the genus Vibrio.Agar and porphyran, sulfated polysaccharides, are distributed in the cell wall of several species of red algae [I]. There have been some reports on agarases from agar-decomposing bacteria, especially on the extracellular fl-agarase from Cytophaga sp. and Pseudomonas atlantica [2 ~ 41. The enzymes from these microorganisms hydrolyzed agar and porphyran to give neoagarotetraose as the predominant product. The existence of another type of intracellular agarase (p-neoagarotetraose hydrolase or P-agarase 11) from P. atluntica was suggested by Morrice et al., but the properties or mechanism of action have not been fully investigated [3 -51.In a previous work, we isolated a porphyran-degrading bacterium (strain AP-2) from an alga [6]. The strain AP-2 produces three extracellular agarases. One of them hydrolyzes neoagarotetraose and larger saccharides to give neoagarobiose, whereas the other two agarases act on hexoses or larger saccharides in the same way as the known P-agarases [4, 71. This paper describes the purification and characterization of the novel P-agarase from the strain AP-2. MATERIALS AND METHODS OrganismThe organism (strain AP-2) was isolated from an alga collected in the coastal sea of the Fukuoka Prefecture of Japan in 1985 [6]. The organism was grown on a slant medium, pH 7.4, composed of 0.5% peptone, 0.1 % yeast extract, 0.2% porphyran, 3.0% NaCl and 1.5% agar, stored at 4'C in a

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The extracellular alginate lyases were purified from Vibrio harveyi AL-128 and V. alginolyticus ATCC 17749. The former enzyme appears to be specific for ai-1,4 bonds involving L-guluronate units in alginate, whereas the latter exhibits specificity for 1-1,4 bonds involving D-mannuronate units. The molecular weights of the enzymes were estimated to be 57,000 and 47,000, and they had isoelectric points of 4.3 and 4.6, respectively. The enzyme from strain AL-128 was most active at NaCI concentrations of 0.3 to 1.0 M. Optimum activity of the enzyme from strain ATCC 17749 was found in the presence of 5 to 10 mM CaCl2.

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