Toshio Takeuchi

SOME PROPERTIES AND ACTION MODE OF (1 4)-ALPHA -L-GULURONAN LYASE FROM ENTEROBACTER CLOACAE M-1

An intracellular alginate lyase was purified from Enterobacter cloacae M-1 by successive fractionation on Q Sepharose FF, SP Sepharose FF, and Sephacryl S-200 HR. The purified enzyme gave a single band on SDS-PAGE and isoelectric focusing. The enzyme easily degraded polyguluronate and produced unsaturated oligoguluronic acids with a wide range of dp. The major end product of the enzyme reaction on polyguluronate was unsaturated triuronic acid. The pattern of oligoguluronic acids (dp 2-9) generated with the enzyme was investigated by fluorophore-assisted carbohydrate electrophoresis. The enzyme was not capable of degrading oligoguluronic acids having dp < 4. The degradation rate of heptaguluronic acid by this enzyme remarkably increased, compared with that of hexaguluronic acid, and heptaguluronic acid had a single preferential point of cleavage by this enzyme. On the basis of the cleavage pattern of oligoguluronic acids, the number of subsites was estimated to be seven for this enzyme. The catalytic site of the enzyme is located between the second and the third subsites from the non-reducing end.

Isolation and quantification of alginate-derived oligouronic acids by fluorophore-assisted carbohydrate electrophoresis

Abstract Alginate-derived oligouronic acids were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). Oligoguluronic acids and oligomannuronic acids were labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and were separated by polyacrylamide gel electrophoresis. The mobilities of oligoguluronic acids and oligomannuronic acids were much higher than those of maltooligosaccharides, and the best resolution of oligouronic acids having DPs lower than 10 was achieved on 40% polyacrylamide gel. Over the range 0.25 to 4 nmol, the fluorescence intensity of an ANTS-labeled oligouronic acid band could be correlated linearly to the quantity of the oligouronic acid. The distribution of DPs of polyguluronic acid and polymannuronic acid also were estimated by using FACE.