In‐Situ Atomic Force Microscopy Observation of Enzymatic Degradation in Poly(hydroxyalkanoic acid) Thin Films: Normal and Constrained Conditions

Abstract

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.

Microbial synthesis and enzymatic degradation of renewable poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate]

“…Similar enzymatic degradation manner along the crystallographic a-axis has been found on the surface of thin films of other PHAs: poly[(R)-3HB-co-(R)-3-hydroxyvalerate] and poly[(R)-3HB-co-6-hydroxyhexanoate] [11,12]. In some cases, lamellar crystals in the PHA thin films were also eroded at the middle part of the crystal along the crystallographic b-axis, resulting in the formation of small crevices.…”

“…A 400mm long Si cantilever with spring constants of 1.5 N/m was operated in the dynamic force (tapping) mode in phosphate buffer solution. For the AFM observation in buffer solution, the interaction force between the sample surface and cantilever tip was reduced in order to avoid the damage on the lamellar surface by cantilever tip [12]. Simultaneous registration was performed for height and deflection images.…”

Microcantilever sensors for fast analysis of enzymatic degradation of poly (d, l-lactide)

“…As biodegradable polymer films are used in many emerging biomedical applications [10,19] there is a growing need for a fast analysis of degradation behavior of polymer films at the microscopic level. In the last decade, atomic force microscopy (AFM) [28], surface plasmon resonance (SPR) [29] and quartz crystal microbalance (QCM) [7] have shown new approaches to detect biodegradation of PLA in the micro scale. Microcantilevers have already been demonstrated to be highly sensitive sensors for mechanical and thermal characterization of polymers [30].…”

Poly(amino acid)s/polypeptides as potential functional and structural materials

“…The degradation of the fibril-like polymer crystals by enzymes has been reported in the case of lamellar crystals of poly(hydroxyalkanoic acid). [87][88][89][90][91][92] In a crystalline biopolyester, tight and loose chain-packing regions form lamellar crystals. The loose chain-packing region is a disordered crystalline region that degrades faster than the highly crystalline domains.…”