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 oligosacchandes 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 so confirmed by ITC an 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.