Atomic Force Microscopy Investigation of Poly[(R)-3-hydroxybutyrate] Lamellar Single Crystals: Relationship between Molecular Weight and Enzymatic Degradation Behavior

Murase, Tomohide; Iwata, Tadahisa; Doi, Yoshiharu

doi:10.1002/1616-5195(20011001)1:7<275::aid-mabi275>3.0.co;2-o

Cited by 28 publications

(35 citation statements)

References 14 publications

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“…It seems likely that the parallel arrays are the polymer itself that is being imaged. PHAs form a well‐known lamellar spherulitic microstructure [23–25] and we have observed a structure similar to these parallel arrays in spun‐cast thin films of PHB‐ co ‐V [26]. We further suggest that globular structures traversing the surface of the parallel arrays are the protein machinery that mediates structure, synthesis, and depolymerization.…”

Section: Discussionmentioning

confidence: 60%

Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy

Dennis¹,

Liebig²,

Holley³

et al. 2003

FEMS Microbiology Letters

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Atomic force microscopy analysis of polyhydroxyalkanoate (PHA) inclusions isolated from sonicated Ralstonia eutropha cells revealed that they exhibit two types of surface structure and shape; rough and ovoid, or smooth and spherical. Smooth inclusions possessed linear surface structures that were in parallel arrays with 7-nm spacing. Occasionally, cracks or fissures could be seen on the surface of the rough inclusions, which allowed a measurement of approximately 4 nm for the thickness of the boundary layer. When the rough inclusions were imaged at higher resolution, globular structures, 35 nm in diameter, having a central pore could be seen. These globular structures were connected by a network of 4-nm-wide linear structures. When the inclusions were treated with sodium lauryl sulfate, the boundary layer of the inclusion deteriorated in a manner that would be consistent with a lipid envelope. When the boundary layer was largely gone, 35-nm globular disks could be imaged laying on the surface of the filter beside the inclusions. These data have facilitated the development of a preliminary model for PHA inclusion structure that is more advanced than previous models.

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Section: Discussionmentioning

confidence: 60%

Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy

Dennis¹,

Liebig²,

Holley³

et al. 2003

FEMS Microbiology Letters

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“…Murase et al30,31 studied the enzymatic degradation behavior of P[( R )‐3HB] single crystals fixed on highly ordered pyrolytic graphite (HOPG), and reported that PHB single crystals were preferentially degraded through the formation of some cracks and crevices along the crystallographic a ‐ and b ‐axes, respectively. Lee et al29 have reported on the melting behavior and enzymatic degradation of P[( R )‐3HB] single crystals.…”

Section: Resultsmentioning

confidence: 99%

“…To study the enzymatic degradation of the crystalline region of PHA, a number of studies using single crystals of P[( R )‐3HB] and its copolymers have been performed 22–31. It has been reported that the enzymatic degradation by the extracellular PHB depolymerase takes place at the edges and the ends of single crystals to yield a splintered morphology along the crystallographic a ‐axis rather than at their chain‐folding surface.…”

Section: Introductionmentioning

confidence: 99%

“…They have suggested that regions with less regularity in the molecular chains are sensitive to heat, and that such disordered regions are preferentially degraded by PHB depolymerase. In addition, Murase et al30,31 have proposed that straight degradation pathways exist in P[( R )‐3HB] single crystals, in which the slits and crevices are formed along the a ‐ and b ‐axes during the enzymatic degradation process.…”

Section: Introductionmentioning

confidence: 99%

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

Kikkawa

Hirota

Numata

et al. 2004

Macromolecular Bioscience

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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.

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“…The enzymatic degradation and adsorption on P(3HB) single crystals by extracellular PHB depolymerases has been reported by Hocking et al,14 Nobes et al,15 Iwata et al16,17 and Murase et al18,19 The partially degraded crystalline lamellae were splintered along the long axes of the crystals, despite the fact that there was no decrease in molecular weight. From these observations, an edge‐attack model for the degradation of P(3HB) single crystals was proposed 15.…”

Section: Introductionmentioning

confidence: 78%

Surface structures of poly[(R)-3-hydroxybutyrate] and its copolymer single crystals before and after enzymatic degradation with an extracellular PHB depolymerase

Iwata

Shiromo

Doi

2002

Macromol. Chem. Phys.

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Lamellar single crystals of poly[(R)‐3‐hydroxybutyrate] (P(3HB)) with three types of molecular weights and four kinds of P(3HB) copolymers with different secondary monomer units were grown from dilute solutions of chloroform and ethanol under isothermal crystallization conditions. The surface morphologies of all the single crystals, before and after enzymatic degradation by an extracellular PHB depolymerase purified from Alcaligenes faecalis T1, were investigated by the coating of these crystals with polyethylene and observation by transmission electron microscopy. All the single crystals have a lath‐shaped morphology with a crystallographic a axis along the long axis of the crystals. The surface regularity of P(3HB) single crystals was dependant on molecular weight. The crystal surfaces of the copolymer single crystals were disordered to a degree which depended on the copolymer composition, and tightly chain‐packed crystalline regions in copolymer single crystals were reduced by the introduction of secondary monomer units. The surface regularity of the copolymer single crystals corresponded to the thickness of the crystalline core regions measured by small‐angle X‐ray scattering. Furthermore, it was revealed that chain‐folding regions of copolymer single crystals were embedded under the crystal surfaces which consisted of molecular chains excluded from the crystalline core. It was directly confirmed by the polyethylene‐coating method that enzymatic degradation does not progress along the chain‐folded regions of the crystal surface.

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Atomic Force Microscopy Investigation of Poly[(R)-3-hydroxybutyrate] Lamellar Single Crystals: Relationship between Molecular Weight and Enzymatic Degradation Behavior

Cited by 28 publications

References 14 publications

Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy

Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy

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

Surface structures of poly[(R)-3-hydroxybutyrate] and its copolymer single crystals before and after enzymatic degradation with an extracellular PHB depolymerase

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