A highly elastic absorbable monofilament suture fabricated from poly(3-hydroxybutyrate-co-4-hydroxybutyrate).

To address the growing demand for more elastic sutures free from unwanted knot loosening, we fabricated an absorbable monofilament suture from poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and subjected it to physical property characterization and performance evaluation (in vitro and in vivo degradability tests and a porcine abdominal wall suture test). As this flexible, highly stretchable, and difficult-to-untie suture exhibited additional advantages of small knot size and medium to long-term bioabsorbability, it was concluded to be a safe alternative to existing monofilament sutures, with far-reaching potential applications.

Marine biodegradation of poly[(R)-3-hydroxybutyrate-co-4-hydroxybutyrate] elastic fibers in seawater: dependence of decomposition rate on highly ordered structure.

Here, we report the marine degradability of polymers with highly ordered structures in natural environmental water using microbial degradation and biochemical oxygen demand (BOD) tests. Three types of elastic fibers (non-porous as-spun, non-porous drawn, and porous drawn) with different highly ordered structures were prepared using poly[(R)-3-hydroxybutyrate-co-16 mol%-4-hydroxybutyrate] [P(3HB-co-16 mol%-4HB)], a well-known polyhydroxyalkanoate. Scanning electron microscopy (SEM) images indicated that microorganisms attached to the fiber surface within several days of testing and degraded the fiber without causing physical disintegration. The results of BOD tests revealed that more than 80% of P(3HB-co-16 mol%-4HB) was degraded by microorganisms in the ocean. The plastisphere was composed of a wide variety of microorganisms, and the microorganisms accumulated on the fiber surfaces differed from those in the biofilms. The microbial degradation rate increased as the degree of molecular orientation and porosity of the fiber increased: as-spun fiber < non-porous drawn fiber < porous drawn fiber. The drawing process induced significant changes in the highly ordered structure of the fiber, such as molecular orientation and porosity, without affecting the crystallinity. The results of SEM observations and X-ray measurements indicated that drawing the fibers oriented the amorphous chains, which promoted enzymatic degradation by microorganisms.

Mechanism of Elastic Properties of Biodegradable Poly[(R)-3-Hydroxybutyrate-co-4-hydroxybutyrate] Films Revealed by Synchrotron Radiation.

Reversible elastic films of biobased and biodegradable poly[(R)-3-hydroxybutyrate-co-4-hydroxybutyrate] [P(3HB-co-4HB)] were prepared by uniaxial drawing procedures. Mechanical properties and highly ordered film structures were investigated by tensile testing and both static-state and in situ wide-angle X-ray diffraction and small-angle X-ray scattering with synchrotron radiation during stretching and relaxing. Despite the crystalline nature of the polymers, the elongation at break of these films was greater than 1500%. Reversible elasticity was achieved after the first 10 times of uniaxial stretching. X-ray measurement results indicated that on stretching, ß-form molecular chains with a planar zigzag conformation were introduced from molecular chains with random coils in the amorphous regions between α-form lamellar crystals. Notably, the orientation of the α-form lamellar crystals increased after relaxation of the molecular chains with a planar zigzag conformation (ß-form) between the lamellar crystals (α-form). Reversible elastic properties were regenerated by a planar zigzag conformation between the lamellar crystals, the extension of molecular chains in lamellar crystals by the rotation of molecular conformation, and changes in the degree of orientation of the lamellar crystals.

Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): Functional mapping of amino acid residues responsible for DNA binding.

PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PHA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).

Expression profiles of polyhydroxyalkanoate synthesis-related genes in Paracoccus denitrificans.

A facultative methylotrophic bacterium, Paracoccus denitrificans can synthesize polyhydroxyalkanoate acids (PHA) from various alcohols. Recently, six genes, phaA, B, C, P, R, and Z, related to PHA synthesis have been cloned and characterized. PHA synthesis and the expression of phaA, B, C, P, R, and Z in P. denitrificans were examined at the transcriptional and translational levels under both nitrogen-sufficient and nitrogen-deficient conditions. The results showed that PHA synthesis is not regulated at the mRNA or protein level in phaA, B, and C. We also observed the condensation of acetyl-coenzyme A (acetyl-CoA) in the cells by high-performance liquid chromatography (HPLC). The results suggest that the amount of acetyl-CoA would regulate PHA synthesis. Finally, we discuss a possible regulation mechanism for PHA synthesis in P. denitrificans.

A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA.

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.