In-situ atomic force microscopy observation of enzymatic degradation in poly(hydroxyalkanoic acid) thin films: normal and constrained conditions.

The enzymatic degradation of lamellar crystals in poly(hydroxyalkanoic acid) thin films has been visualized by using in-situ dynamic force mode (tapping mode) atomic force microscopy (AFM) in buffer solution. It was found that poly(hydroxybutyric acid) (PHB) depolymerase from Ralstonia pickettii T1 degraded the thin surface layers formed at room temperature first, and that lamellar crystals formed at the crystallization temperature (110 degrees C) were eroded from the crystallographic a-axis to show splintered morphologies at the tips of the crystals. In some cases, lamellar crystals were hydrolyzed from the crystallographic b-axis, resulting in the formation of small crevices. These results suggest that disordered molecular chain-packing regions exist in the crystal along the crystallographic a- and b-axes, and that enzymatic degradation predominantly occurs from these defective regions. In addition, cantilever-tip-induced enzymatic degradation was carried out in the presence of PHB depolymerase. A concave area was artificially formed on the stacked lamellar crystals by the AFM tip. In-situ AFM observation has revealed that enzymatic degradation proceeds along both the longitudinal and lateral directions of the lamellae. At the same time, the PHB depolymerase preferentially eroded the concave area along the crystallographic c-axis. These results demonstrated that the PHB depolymerase predominantly degrades the less-ordered molecular chain-packing regions in the crystals.