A bacterial sialyltransferase, named sialyltransferase 0160, was purified from a marine bacterium that had been isolated from seawater from Sagami Bay, Kanagawa. This strain has been identified as Photobacterium damsela, and named P. damsela JT0160. Sialyltransferase 0160 was purified 688-fold to homogeneity from the crude extract of the cells with a yield of 19% using a combination of anion exchange chromatography, hydroxyapatite chromatography, gel filtration chromatography, and affinity chromatography. The purified enzyme migrated as a single band (61 kDa) on sodium dodecyl sulfate-polyacrylamide gel. This sialyltransferase was found to be a beta-galactoside alpha 2,6-sialyltransferase [EC 2.4.99.1] which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6 on the basis of an analysis of the enzymatic reaction products with HPLC, 1H-, 13C-NMR spectroscopy, and fast atom bombardment mass spectroscopy.

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A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.

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