Kinetic study on enzymatic degradation of biodegradable polyesters

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Enzymatic degradation of biopolyesters was investigated by a quartz crystal microbalance (QCM) and an atomic force microscope (AFM). In the case of enzymatic degradation of a melt-crystallized film of poly[(R)-3-hydroxybutyrate] (P(3HB)) by PHB depolymerase from Ralstonia pickettii T1, the erosion rate was not constant during the degradation due to heterogeneity of the film composed of an amorphous region and a crystalline region. Characteristic profiles were also observed for dependence of the erosion rate on depolymerase concentrations. The rate increased with enzyme concentrations to reach a maximum value at 1.0 μg/mL, and then decreased at higher enzyme concentrations. This dependence can be explained in terms of mutual steric hindrance among adsorbed enzyme molecules. The characteristic dynamic adsorption behavior of PHB depolymerase was also revealed. In contrast to the melt-crystallized P(3HB) film, the erosion rate of an amorphous poly [L-lactide] (PLLA) film by proteinase K was constant during degradation, indicating that the PLLA film is homogeneously amorphous. The erosion rate increased with enzyme concentration and remained constant under the conditions of [proteinase K] > 100 μg/mL. The erosion rate was proportional to the adsorbed amount of the enzymes. Adsorption of proteinase K was irreversible despite lack of the substrate-binding domain, so that the enzyme molecules on the film could be directly observed by AFM. Transformation of the molecules caused by packing in high density on the surface was observed at higher enzyme concentrations. The "footprint" of the individual proteinase K molecule on the PLLA film after enzymatic degradation suggests that the enzyme moves on the surface to hydrolyze the film around its. The differences between proteinase K and the PHB depolymerase might result from whether these enzymes possess the substrate-binding domain or not.