HPLC analysis of saturated or unsaturated oligoguluronates and oligomannuronates. Application to the determination of the action pattern of Haliotis tuberculata alginate lyase.
The chromatographic behaviour of various saturated and unsaturated oligouronates obtained by acid or enzymatic degradation of homopolymeric blocks of alginates was investigated by isocratic anion exchange liquid chromatography. This approach was then applied to the determination of the catalytic properties of Haliotis tuberculata alginate lyase. This enzyme presents a high affinity for poly-beta-D-mannuronate blocks, leading to the release of O-(4-deoxy-alpha-L-erythro-hex-4-enopyranosyluronic acid)-(1-->4)-O-(beta-D-mannopyranosyluronic acid)-(1-->4)-O-beta-D-mannopyranuronic acid as the main end reaction product. Kinetic analysis with oligomannuronates of various sizes indicate that the catalytic site of Haliotis tuberculata lyase (abalone) best accommodates an oligomannuronate pentamer. The abalone lyase, however, is also capable of cleaving the G-M linkages of alginate heteropolymeric sequences. In contrast, it does not degrade the G-G nor the M-G diads. This lyase should therefore be referred to as a mannuronate beta-eliminase, indicating that the enzyme performs beta-elimination on mannuronate residues only, from both the M-M and G-M diads of alginates.
Heyraud
A
A
Center de Recherches sur les Macromolécules, Végétales, C.N.R.S., Grenoble, France.
Colin-Morel
P
P
Girond
S
S
Richard
C
C
Kloareg
B
B
D000464:Alginates / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006603:Hexuronic Acids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000031:analogs & derivatives; D008974:Mollusca / Q000201:enzymology*; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D009994:Osmolar Concentration; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D014574:Uronic Acids / Q000032:analysis / Q000737:chemistry*
D000464:Alginates; D006603:Hexuronic Acids; D009844:Oligosaccharides; D014574:Uronic Acids; C007896:guluronic acid; C008324:mannuronic acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase; D008358:Mannose