title: Substrate specificity of the recombinant alginate lyase from the marine bacteria Pseudomonas alginovora.
abstract: The gene coding for an alginate lyase from the marine bacteria Pseudomonas alginovora X017 was cloned and heterologously expressed in Escherichia coli strains. The protein was produced in inclusion bodies and the active form was obtained by applying a refolding protocol based upon dilution. The biochemical characterization was performed on the active, refolded form of the alginate lyase. The substrate specificity was monitored by NMR. The degradation products were size-fractioned by size exclusion chromatography. The fractions were subsequently analyzed by ESI-MS to determine the molecular weight of the compounds. The structures of the different oligosaccharides were then elucidated by NMR. The enzyme was shown to be only acting on M-M diads. No enzymatic hydrolysis occurred between M-MG, G-MM or G-MG blocks proving that the sequence accounting for the generated oligomers by enzymatic hydrolysis is M-MM. The unsaturated oligosaccharides produced by the alginate lyase were ΔM, ΔMM, ΔMMM, and ΔMMMM indicating that the minimum structure recognized by the enzyme is the M6 oligosaccharide.
authors.affiliation: Department of Chemistry, Swedish University of Agricultural Sciences, PO Box 7015, SE-750 07 Uppsala, Sweden. lena.lundqvist@slu.se
authors.forename: Lena C E
authors.initials: LC
authors.lastname: Lundqvist
authors.forename: Murielle
authors.initials: M
authors.lastname: Jam
authors.forename: Tristan
authors.initials: T
authors.lastname: Barbeyron
authors.forename: Mirjam
authors.initials: M
authors.lastname: Czjzek
authors.forename: Corine
authors.initials: C
authors.lastname: Sandström
mesh_terms: D000464:Alginates / Q000378:metabolism; D003001:Cloning, Molecular; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D058849:Protein Refolding; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D011994:Recombinant Proteins; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase