title: Molecular size characterization and kinetics studies on hydrolysis of pullulan by pullulanase in an entangled alginate medium.
abstract: The behavior of a hydrolytic enzyme (pullulanase) toward its substrate (pullulan) in the presence of a nonsubstrate (alginate), both below and above the critical entanglement concentration (C*), was studied. The hydrolysis kinetics were studied with the enzyme and alginate concentrations varied using two main methods: a colorimetric assay of the reducing extremities (RE), which allowed the number-average molar masses (Mn) of the oligosaccharides to be determined, and size exclusion chromatography with on-line, multiangle light scattering, viscometer, and differential refractive index detectors, which allowed the average molar masses, Mn and Mw, of the oligosaccharides during hydrolysis to be determined. Free pullulanase acts via an "endo" process. The presence of alginate slows the hydrolysis kinetics, particularly when the alginate concentration is greater than the C*. These results were confirmed by the evolution of the kinetic parameters (KM, Vmax) obtained via isothermal titration calorimetry (ITC). The amount of oligosaccharides produced is not dependent on the alginate concentration, and the endo enzyme behavior is not modified by the entanglement in the medium. These observations were also confirmed by ITC analysis in the presence of degraded alginate (without entanglement). Our results correlated with the substrate diffusion in entangled media. The pullulanase reaction in the presence of alginate is shown to be diffusion-dependent.
authors.affiliation: Normandie Université, France.
authors.forename: Ghina
authors.initials: G
authors.lastname: Ali
authors.forename: Christophe
authors.initials: C
authors.lastname: Rihouey
authors.forename: Véronique
authors.initials: V
authors.lastname: Larreta-Garde
authors.forename: Didier
authors.initials: D
authors.lastname: Le Cerf
authors.forename: Luc
authors.initials: L
authors.lastname: Picton
mesh_terms: D000464:Alginates / Q000737:chemistry*; D002151:Calorimetry; D002850:Chromatography, Gel; D005936:Glucans / Q000737:chemistry*; D020723:Glucuronic Acid / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006603:Hexuronic Acids / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism
chemical_list: D000464:Alginates; D005936:Glucans; D006603:Hexuronic Acids; D009844:Oligosaccharides; D020723:Glucuronic Acid; C009109:pullulan; D006026:Glycoside Hydrolases; C100181:pullulanase