Molecular Size Characterization and Kinetics Studies on Hydrolysis of Pullulan by Pullulanase in an Entangled Alginate Medium Ghina Ali, †,‡,§ Christophe Rihouey, †,‡,§ Ve ́ ronique Larreta-Garde, ∥ Didier Le Cerf,* ,†,‡,§ and Luc Picton †,‡,§ † Normandie Universite ́ , France ‡ Universite ́ de Rouen, Laboratoire Polyme ̀ res Biopolyme ̀ res Surfaces, F-76821 Mont Saint Aignan, France § CNRS UMR 6270 and FR3038, F-76821 Mont Saint Aignan, France ∥ Universite ́ de Cergy-Pontoise, Laboratoire ERRMECE, F-95000 Cergy Pontoise, France 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 (M n ) 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, M n and M w , 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 (K M , V max ) 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. ■ INTRODUCTION In general, enzyme kinetics are followed under homogeneous conditions (concentrations of enzyme and substrate, presence of salt at a ionic strength, pH controlled), in enzymes isolated from living cells. The in vivo and in vitro enzyme properties are not necessarily similar. Unlike in vitro conditions, cells (in vivo) are composed of different compartments and most likely exist in a heterogeneous medium. Polysaccharides, nucleic acids, lipids, and proteins are present in large amounts in the cytosol of living cells and form a complex biocolloid. Enzymes present in the cytoplasm or organelle of living cells interact with other biomacromolecules that have important roles in stabilizing the structure and function of enzymes. 1 In particular, polysacchar- ides enhance the stability of enzymes against pressure and temperature fluctuations. 2,3 The confinement effect of poly- saccharides added to the medium can influence the ability of an enzyme to maintain its structure; they may also inhibit enzyme aggregation or alter enzyme behavior. 2 In addition, the presence of hydrophilic polysaccharides enhances the hydration of enzymes. 4,5 Electrostatic interactions between enzymes and polysaccharides are closely related to the conformation, activity and stability of the enzymes. 1 Some studies have described the effect of substrate diffusion and solution viscosity on enzymatic behavior. The decrease of lysozyme activity or a modification in the selectivity of protease or gliadin has been shown to occur when the viscosity is increased due to a high concentration of glucose, fructose, sucrose, or sorbitol. 6,7 The influence of the self-evolving environment on the activity of β-D-glucosidase was demonstrated and quantified for different initial substrate compositions (octyl-β-D-glucoside) and enzyme contents. 8 Based on literature results, it is of interest to follow the behavior of an enzyme in a viscous medium that can mimic rheological conditions, such as in vivo conditions. We have focused our interest on the enzymatic model system pullulan− pullulanase in an entanglement of a polymer network of alginate, which is obviously not a substrate of the enzyme. The final goal is to determine if the enzyme activity is modified by the entanglement or if some particular associations (e.g., ionic, hydrophobic) are established between the enzyme and the alginate. Pullulan is a neutral, linear, biocompatible, and hydrophilic polysaccharide. It is generally described as a succession of α- (1,6)-linked (1,4)-α-maltotriose. Its structure endows pullulan with numerous interesting properties, such as high flexibility Received: March 12, 2013 Revised: May 16, 2013 Article pubs.acs.org/Biomac © XXXX American Chemical Society A dx.doi.org/10.1021/bm400371r | Biomacromolecules XXXX, XXX, XXX−XXX