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

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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A novel exo-beta-mannanase (1,4-beta-D-mannan mannobiohydrolase) was isolated from the culture fluid of strain No. F-25 of Aeromonas hydrophila subspecies anaerogenes, and purified about 4,000-fold by ammonium sulfate precipitation and successive co.umn chromatographies. The final enzyme preparation appeared to be homogeneous on polyacrylamide gel electrophoresis. The enzyme hydrolyzed the beta-1,4-mannan link in polysaccharides of three or more beta-1,4-linked D-mannose units. The enzyme had a molecular weight of 64,000, pI of 5.9, pH optimum of 6.0, and was stable in a pH region of 5.0 to 8.5 and at temperatures below 45 degrees C. The Km values of the enzyme were 5.1 X 10(-4) M for mannotriose, 2.4 X 10(-4) M for mannotetraose and 1.3 X 10(-4) M for mannopentaose. The enzyme attacked codium and coffee mannans to give only mannobiose. Mannobiosyl- and mannotetraosyl-mannitol were hydrolyzed to produce mannobiose and mannitol, while mannobiose and mannosylmannitol were released from mannotriosylmannitol. The enzyme did not act on mannobiose, p-nitrophenyl-beta-D-mannoside, konjac glucomannan, or guar gum galactomannan. Furthermore, the enzyme catalyzed a transglycosylation reaction.

A search was undertaken for bacteria which degrade chondroitin sulfate in nature and to find bacteria with a usefully high rate of chondroitinase (ChSase) productivity. First, 253 ChSase-producing bacteria were obtained from aquatic and land environments in Japan by aerobic and anaerobic screening methods. Identification according to Bergey's Manual of Determinative Bacteriology or Bain and Shewan (1968) permitted assignment of the majority of the isolates to seven genera, Aeromonas, Vibrio, Flavobacterium, Beneckea, Proteus, Micrococcus, and Arthrobacter. Next, ChSase productivities of all the isolates were compared with those of two established ChSase-producing stock strains, Proteus vulgaris NCTC 4636 and Flavobacterium heparinum ATCC 13125. As a result, special attention was given to production by a strain of Aeromonas sp. of large quantities of extracellular ChSase-AC. None of the isolates from the current study displayed significant ChSase-ABC productivity. Finally, ChSase-AC was prepared from the culture fluid of the Aeromonas strain by fractional precipitation with ammonium sulfate, chromatography on phospho-cellulose and diethylaminoethyl-cellulose, and gel filtration on Sephadex G-200. It was concluded that the Aeromonas strain may represent a profitable source of the enzyme ChSase-AC.

A simple turbidimetric method was developed to detect alginate degradation. Bacteria were grown in alginate-containing media, and culture fluids were mixed with an acidic albumin solution. Failure to develop a white turbidity indicated an alginate degrader. The method showed alginate degradation by Vibrio alginolyticus ATCC 17749, in contrast to prior descriptions.