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.
Shimokawa
T
T
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Yoshida
S
S
Kusakabe
I
I
Takeuchi
T
T
Murata
K
K
Kobayashi
H
H
D000464:Alginates / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D016972:Enterobacter cloacae / Q000201:enzymology*; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D011133:Polysaccharide-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000378:metabolism
D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D011135:Polysaccharides, Bacterial; C111091:polyguluronic acid; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C022416:poly-alpha-L-guluronate lyase