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We developed molded plastic samples with microscale textured features, and investigated the relationship between the tactile sensory response and the physical properties of the surfaces. Samples with various pitch features, and features with various height protrusions were prepared. Changes in the tactile sensory perception of the surfaces were correlated with the magnitude of the fluctuation in the coefficients of friction that occurred as a fingerprint moved across the surface, caused by the intrusion of the features of the textured surface into the grooves of the fingerprint. This occurred when the pitch of the features on the textured surface approached that of the pattern of the fingerprint; the height of the features was not significant. A change in the tactile perception of the surfaces occurred when the pitch of the features at the surface was approximately 100 μm.

To investigate the conversion of lignin derivatives to biopolyesters, 11 polyhydroxyalkanoate (PHA)-accumulating strains were cultured on mineral salt media containing each of the 18 lignin derivatives and hydroxybenzoic acids, including intermediates derived from the metabolism of lignin derivatives in bacteria. Most of the strains grew poorly in media containing lignin derivatives such as p-coumaric acid, caffeic acid, ferulic acid, and sinapinic acid. One of the strains, Pseudomonas putida Gpo1, grew in the presence of p-coumaric acid, ferulic acid, vanillic acid, 4-hydroxybenzoic acid (4-HBA), and 3,4-dihydroxybenzoic acid (3,4-DHBA). Pseudomonas putida JCM 13063 also grew in the presence of 4-HBA, 3,4-DHBA, and vanillic acid. Another strain, Ralstonia eutropha H16, synthesized PHA from 4-HBA, 2,5-DHBA, and 3,4-DHBA. On the basis of the data obtained from these experiments, we suggest that the conversion of lignin derivatives into intermediates such as 4-HBA, 2,5-DHBA, 3,4-DHBA, and vanillic acid repr...

Because cyanobacteria directly harvest CO2 and light energy, their carbon metabolism is important for both basic and applied sciences. Here, we show that overexpression of the sigma factor sigE in Synechocystis sp. PCC 6803 widely changes sugar catabolism and increases production of the biodegradable polyester polyhydroxybutyrate (PHB) during nitrogen starvation. sigE overexpression elevates the levels of proteins implicated in glycogen catabolism, the oxidative pentose phosphate pathway, and polyhydroxyalkanoate biosynthesis. PHB accumulation is enhanced by sigE overexpression under nitrogen-limited conditions, yet the molecular weights of PHBs synthesized by the parental glucose-tolerant and sigE overexpression strain are similar. Although gene expression induced by nitrogen starvation is changed and other metabolites (such as GDP-mannose and citrate) accumulate under sigE overexpression, genetic engineering of this sigma factor altered the metabolic pathway from glycogen to PHB during nitrogen starvation.

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaC Cs ) with specificities for short-chain-length (SCL) [( R )-3-hydroxybutyrate (3HB) and ( R )-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [( R )-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC Cs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C 4 and C 5 ) or MCL (C 6 ) substrates substantiates the fundamental classification of PHA synthases.

The purpose of this study was to develop a plastic molding with a distinguishing tactile character for various industrial applications. Grooved textures with pitches and depths ranging from several micrometers to several hundred micrometers were fabricated on plastic molding surfaces by first micromachining aluminum alloy molds, and then utilizing a vacuum hot-press procedure. The effect of texture shape on the human tactile sense was investigated by sensory evaluation and multivariate statistical analysis. The sensory evaluation revealed that tactile recognition depends on the pitch of the texture, and not on the height; an obvious change was observed at a pitch of around 100 μm. Force measurements revealed that this change in tactile response was caused by the coefficient of friction and force fluctuations.

This is the first report on synthesis of poly(hydroxyalkanoate)s (PHAs) by various deep-sea bacteria (4 types of Colwellia spp., 11 types of Moritella spp., and 18 types of Shewanella spp.) from glucose, fructose, gluconate, or from one of the several plant oils as the sole source of carbon. The overall results provide important and basic information regarding the production of PHAs by deep-sea bacteria and on the diversity of PHA synthase enzymes in nature.

This study describes the biosynthesis of polyhydroxyalkanoate (PHA) containing 5-hydroxyvalerate (5HV) units. Monomer units like 5HV, with variation in length of the backbone segment of the repeat unit, are known to more greatly impact mechanical properties of polymers. Introduction of 5HV units was also aimed at enhancing biodegradability of the material, as it had been found previously that a homopolymer of 5HV could be degraded by lipase. The produced poly[( R )-3-hydroxybutyrate- co -3-hydroxypropionate- co -5-hydroxyvalerate]s [P(3HB- co -3HP- co -5HV)s] with 5HV content ranging from 1 to 32 mol% by the choice of strain, type and concentration of carbon substrate, exhibited low melting temperature ( T m ), reduced crystallinity and elastomeric behavior. Enzymatic degradation test confirmed the role of 5HV unit in enabling lipase-mediated degradation of P(3HB- co -3HP- co -5HV)s, as poly[( R )-3-hydroxybutyrate] and poly[( R )-3-hydroxypropionate] films were not hydrolyzed by porcine pancreas lipase. Additionally, viability assay of human mesenchymal stem cells revealed that the P(3HB- co -3HP- co -5HV) films were less cytotoxic and supported cell proliferation, highlighting the potential of P(3HB- co -3HP- co -5HV)s as biomaterials.

Poly(hydroxyalkanoate)s (PHAs) have emerged as a family of aliphatic polyesters produced by a number of bacteria. PHAs have attracted much attention as biodegradable thermoplastics to solve the waste disposal challenge. Currently, from the viewpoint of effective utilization of natural resources, the PHAs are recognized as bio-based polymeric materials produced from renewable biomass resources. In this article, we have attempted to give an overview of research and development on biosynthesis, structure and properties, and biodegradability of PHAs, and to relate prospects of the field. In addition, the present situation regarding industrial production of PHA materials is presented.

Polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae FA440 (PhaC(Ac), BAA21815) is one of the most valuable PHA synthase, because of its function to synthesize a practical bioplastic, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. However, biochemical activity and active intermediates of PhaC(Ac) have not been clarified until now. In the present study, a gene of PhaC(Ac) was cloned and overexpressed by a cell-free protein expression system. Both the polymerization activity and oligomerization behavior of the purified PhaC(Ac) were characterized in order to clarify the active intermediates of PhaC(Ac) based on the hydrodynamic diameters and specific activities of PhaC(Ac). The influences of a substrate, (R)-3-hydroxybutyryl-CoA (3HB-CoA), on the oligomerization of PhaC(Ac) (7.5 μM) were also investigated, and then the Hill coefficient (n = 2.6 ± 0.4) and the microscopic dissociation constant (K(m) = 77 ± 5 μM) were determined. Based on the results, the active intermediate of PhaC(Ac) was concluded to be the dimeric PhaC(Ac) containing 3HB-CoA as an activator for its dimerization. This information is critical for revealing the relationships between its dimerization and function in PHA synthesis.

Like most of the materials used by humans, polymeric materials are proposed in the literature and occasionally exploited clinically, as such, as devices or as part of devices, by surgeons, dentists, and pharmacists to treat traumata and diseases. Applications have in common the fact that polymers function in contact with animal and human cells, tissues, and/or organs. More recently, people have realized that polymers that are used as plastics in packaging, as colloidal suspension in paints, and under many other forms in the environment, are also in contact with living systems and raise problems related to sustainability, delivery of chemicals or pollutants, and elimination of wastes. These problems are basically comparable to those found in therapy. Last but not least, biotechnology and renewable resources are regarded as attractive sources of polymers. In all cases, water, ions, biopolymers, cells, and tissues are involved. Polymer scientists, therapists, biologists, and ecologists should thus use the same terminology to reflect similar properties, phenomena, and mechanisms. Of particular interest is the domain of the so-called “degradable or biodegradable polymers” that are aimed at providing materials with specific time-limited applications in medicine and in the environment where the respect of living systems, the elimination, and/or the bio-recycling are mandatory, at least ideally.

Marine bacteria have recently attracted attention as potentially useful candidates for the production of practical materials from marine ecosystems, including the oceanic carbon dioxide cycle. The advantages of using marine bacteria for the biosynthesis of poly(hydroxyalkanoate) (PHA), one of the eco-friendly bioplastics, include avoiding contamination with bacteria that lack salt-water resistance, ability to use filtered seawater as a culture medium, and the potential for extracellular production of PHA, all of which would contribute to large-scale industrial production of PHA. A novel marine bacterium, Vibrio sp. strain KN01, was isolated and characterized in PHA productivity using various carbon sources under aerobic and aerobic-anaerobic marine conditions. The PHA contents of all the samples under the aerobic-anaerobic condition, especially when using soybean oil as the sole carbon source, were enhanced by limiting the amount of dissolved oxygen. The PHA accumulated using soybean oil as a sole carbon source under the aerobic-anaerobic condition contained 14% 3-hydroxypropionate (3HP) and 3% 5-hydroxyvalerate (5HV) units in addition to (R)-3-hydroxybutyrate (3HB) units and had a molecular weight of 42 × 10³ g/mol. The present result indicates that the activity of the beta-oxidation pathway under the aerobic-anaerobic condition is reduced due to a reduction in the amount of dissolved oxygen. These findings have potential for use in controlling the biosynthesis of long main-chain PHA by regulating the activity of the beta-oxidation pathway, which also could be regulated by varying the dissolved oxygen concentration.

This is the first report on dual synthesis of poly[(R)-3-hydroxybutyric acid] (PHB) and protein polymer by recombinant microorganisms for use in the fabrication of a biofunctional PHB-based material hybridized with functional proteins. PHB and recombinant silk protein, a model protein developed in this study, were synthesized by a recombinant Escherichia coli system, and then films of PHB hybridized with the silk protein (PHB/Silk) were prepared. The presence of the silk protein in the amorphous phase of the PHB/Silk film was demonstrated based on wide-angle X-ray diffraction and differential scanning calorimetry measurements, which also revealed the enhanced tensile strength and elongation at break of the PHB/Silk film. The cell adhesion of human mesenchymal stem cells (hMSCs) for the PHB/Silk film increased because of the addition of silk molecules to the film. This might have been because the silk molecules existed in the amorphous phase at the surface of the PHB/Silk films, and were exposed to hMSCs. The overall results illustrate the potential of this dual synthesis system as a versatile and useful platform method for preparation of biofunctional PHB-based materials, especially for biomedical applications.

Thin films of poly(L-lactide) (PLLA)/atactic poly(3-hydroxybutyrate) (ataPHB) blends with different miscibility were prepared and characterized by using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). The DSC analysis suggested that the blend thin films exhibited different phase structures, such as miscible, partially miscible, and immiscible depending on the blending ratio as well as molecular weight of ataPHB component. The different miscibility was further confirmed by the surface morphological observation by AFM. Both the immiscible and partially miscible blends of PLLA/ataPHB revealed the formation of phase-separated morphology of PLLA and ataPHB components, whereas the homogeneous surface morphology was observed for the miscible blend. On the basis of the changes in the depth profile from the surface level of the thin films, the enzymatic degradation rates of the PLLA and ataPHB domains were determined in the presence of either PHB depolymerase or proteinase K, respectively. The erosion rate of PLLA/ataPHB blends was strongly dependent on the blend composition and the degree of dispersion of the two components. The enzymatic degradation behaviors were discussed in terms of phase structure, molecular mobility, and retardation effect of the components in the blends.

Polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp 61-3 (PhaC1(Ps)) is able to synthesize P(3HB-co-3HA), consisting of a 3HB unit and medium-chain-length 3HA units of 6-12 carbon atoms. Expression vectors encoding 76 PhaC1(Ps) mutants with an amino acid replacement at position 130, 325, 477 or 481 were individually introduced into Ralstonia eutropha. The mutant enzyme genes were evaluated in terms of their abilities to synthesize P(3HB-co-3HA) using soybean oil as a carbon source. 20 mutants showed significantly high accumulation levels of PHA exceeding 30 wt.-% and as high as 57 wt.-%. It was found that hydrophobic amino acids at the positions are more likely to enhance accumulation of PHA in R. eutropha.

Poly(hydroxyalkanoate)s (PHAs) have attracted much attention as environmentally compatible polymeric materials that can be produced from renewable carbon resources. Biodegradation of PHA materials occurs by the function of extracellular PHA depolymerase secreted from microorganisms. Thus, elucidation of the enzymatic degradation mechanism for PHA materials is important to design PHA materials with desirable properties and controlled biodegradability. The solid PHA polymer is a water-insoluble substrate but PHA depolymerases are soluble in water. Therefore, the enzymatic degradation of PHA materials is a heterogeneous reaction on the material’s surface. Two distinct processes are involved during the degradation, namely, adsorption of the enzyme on the surface of PHA material and the subsequent hydrolysis of polymer chains. Atomic force microscopy (AFM) is a powerful tool that has been used for the quantitative analysis of PHA crystal degradation. AFM enables the characterization of the crystal surface nanostructure in a buffer solution. By using in-situ (real-time) AFM observations, we recently succeeded in observing the degradation processes of PHA crystals. Subsequently, we were also able to investigate the degradation rates of PHA crystals using the same technique. In this review, we have attempted to give an overview concerning the direct visualization of the adsorption, as well as the hydrolysis reactions of PHA depolymerases at the nanometer scale. In addition, we present other analytical techniques besides AFM as a complimentary approach to analyze the effect of enzyme adsorption on PHA crystals.Key words: poly(hydroxyalkanoate) (PHA), enzymatic degradation, lamellar crystal, PHA depolymerase.

Effect of metal compounds on the thermal degradation behaviors of poly(3-hydroxybutyric acid) (P(3HB)), poly(4-hydroxybutyric acid) (P(4HB)), and poly(ɛ-caprolactone) (PCL) was investigated by means of thermogravimetric and pyrolysis-gas chromatograph mass spectrometric analyses. Na and Ca compounds accelerated a random chain scission of P(3HB) molecules resulting in a decrease of thermal degradation temperature, whereas the contribution of Zn, Sn, Al compounds to the thermal degradation of P(3HB) was very small. In contrast to P(3HB), Zn, Sn and Al compounds induced the thermal degradation of PCL at lower temperature range by catalyzing the selective unzipping depolymerization from ω-hydroxyl chain end. Transesterification reaction of PCL molecules could be facilitated by the presence of Ca compound, while the gravimetric change was detected at almost identical temperature region regardless of the content of Ca compound. According to the lactonizing characteristic of monomer unit, the thermal degradation of P(4HB) progressed by the cyclic rupture via unzipping reaction from the ω-hydroxyl chain end or/and random intramolecular transesterification at the main chain with a release of γ-butyrolactone as volatile product. Each of metal compounds used in this study was effective to catalyze the cyclic rupture of P(4HB) molecules, and the degradation rate was accelerated by the presence of metal compounds.

Poly(L-lactide) (PLLA) with various alkyl ester chain end groups were synthesized by ring-opening polymerization of L-lactide in the presence of zinc alkoxide as a catalyst. The structural effect of chain end groups on the rate of enzymatic and nonenzymatic degradations for amorphous films of PLLA were investigated at 37 degrees C in a Tris-HCl buffer solution (pH 8.6) with proteinase K and at 60 degrees C in a phosphate buffer solution (pH 7.4), respectively. The rate of enzymatic degradation for PLLA films was dependent on the carbon numbers of alkyl ester chain end groups, and the rates of PLLA samples with dodecyl (C12), tridecyl (C13), and tetracocyl (C14) ester end groups were much lower than those of the other samples. The surface morphologies of PLLA films after enzymatic degradation were characterized by scanning electron microscopy. After the enzymatic degradation, non-end-capped PLLA, PLLA with methyl (C1) and hexyl (C6) ester chain ends, were degraded homogeneously by proteinase K and the film surface was very smooth. In contrast, the PLLA with alkyl ester chain ends of carbon numbers over 12 were degraded heterogeneously by the enzyme, and the sponge-like network structure was formed on the film surface. These results indicated that the long alkyl ester groups at the chain ends of PLLA molecules aggregated in the amorphous films and the erosion rate was depressed due to the coverage of the aggregated terminal groups on the film surface. For the nonenzymatic degradation, the molecular weight of non-end-capped PLLA was remarkably decreased with progress of degradation. In contrast, the molecular weight of the end-capped PLLA gradually reduced at the initial stage of degradation and then the rate of degradation was accelerated. The decreases of molecular weight of PLLA by autocatalyzed degradation were retarded by the capping of carboxyl chain ends.

A short-chain poly3-hydroxybutyrate including four comonomers, originating from a complex with calcium polyphosphate, was isolated from commercial baker's yeast cells (Saccharomyces cerevisiae) and characterized as the second complexed poly(3-hydroxyalkanoate) (cPHA) in eukaryotes. The number-average molecular weight of 4982.5 Da with a polydispersity index of 1.11 was much lower than that of beet cPHA previously isolated. End-group analysis suggested that at least 60% of the molecules form the cyclic structures. Here, the organism-dependent structural diversity of cPHAs was completely established. It was also found that a change of culture medium influences the molecular weight but not the polydispersity of baker's yeast cPHA.

This study investigates the biosynthesis and characterization of P(3HB- co -3HV- co -3HHx) terpolymer from mixtures of palm kernel oil and 3HV-precursors by using recombinant Cupriavidus necator PHB − 4/pBBREE32d13. Sodium valerate and propionate have been evaluated for the generation of 3HV monomers. The feeding time of these precursors was a crucial factor that significantly affected the 3HV molar fractions, which ranged from 2 to 60 mol%. Sodium valerate was generally the better precursor in initiating the accumulation of 3HV monomers while maintaining high cell dry weight (7.9 g/L) and good PHA accumulation (79 wt%). However, the 3HHx molar fractions in the terpolymers at 72 h were consistent at about 2–7 mol%. P(3HB- co -3HV) copolymers have been known to exhibit approximately the same degree of crystallinity throughout a wide range of 3HV composition. Interestingly, in this study, terpolymers containing 58 mol% 3HB, 39 mol% 3HV and 3 mol% 3HHx showed elastomeric behavior. This study demonstrates the suitability of palm kernel oil as the main carbon source and both sodium propionate and sodium valerate as 3HV-precursors for the synthesis of novel compositions of P(3HB- co -3HV- co -3HHx) terpolymers with interesting properties.

Aeromonas caviae polyhydroxyalkanoate synthase (PhaC(Ac)) is an important biocatalyst for the synthesis of practically useful two-component polyhydroxyalkanoate copolymer, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. In a previous study, two PhaC(Ac) mutants that have a single amino acid substitution of either asparagine 149 by serine (N149S) or aspartate 171 by glycine (D171G) were isolated as higher active enzymes by means of evolutionary engineering. In this study, the synergistic effects of N149S and D171G double mutation (NSDG) in PhaC(Ac) on polyhydroxyalkanoate biosynthesis were investigated in recombinant Ralstonia eutropha. The PhaC(Ac) NSDG mutant showed enhanced incorporation of longer 3-hydroxyalkanoate (3HA) units into the polyhydroxyalkanoate copolymer from octanoate (3HA fraction: 18.5 mol%) and soybean oil (5.4 mol%) as a carbon source. Besides, the NSDG mutant synthesized P(3HB) homopolymer with a very high molecular weight (M(w)=368 x 10(4)) when fructose was used as a carbon source. Thus, a combination of the beneficial mutations synergistically altered enzymatic properties, leading to synthesis of a polyhydroxyalkanoate copolymer with enhanced 3HA fraction and increased molecular weight.

Atomic force microscopy (AFM) was used for modifying the surface structures of poly(e-caprolactone) (PCL) thin film. Oriented growth of PCL crystals at a desired area of the film surface was induced by scanning with a strong, normal load. PCL crystals were first grown edge-on from the induction line and then their orientation changed to flat-on at a lamellar length. The effects of molecular weight, crystallization temperature, scanning rate, and normal load on the AFM-tip-induced crystallization were examined. The growth kinetics of lamellar crystals in the AFM-tip-induced crystallization was the same as that in spherulitic crystallization. It was found that the formation of precursors strongly depends on the applied tip load and is facilitated when the applied load is higher than a threshold.

In this article the effects of the number of molecular branches (chain ends) and the stereochemistry of poly(lactide)s (PLAs) on the enzymatic degradation and alkaline hydrolysis are studied. Various linear and branched PLAs were synthesized using lipase PS (Pseudomonas fluorescens)-catalyzed ring-opening polymerization (ROP) of lactide monomers having different stereochemistries (L-lactide, D-lactide, and D,L-lactide). Five different alcohols were used as initiators for the ROP, and the monomer-to-initiator molar feed ratio was varied from 10 to 100 and 1000 for each branch in the polymer architecture. The properties of branched PLAs that would affect the enzymatic and alkaline degradations, i.e., the glass transition temperature, the melting temperature, the melting enthalpy, and the advancing contact angle, were determined. The PLA films were degraded using proteinase K or 1.0 M NaOH solution, and the weight loss and changes in the number average molecular weight (Mn) of the polymer were studied during 12 h of degradation. The results suggest that an increase in the number of molecular branches of branched PLAs enhances its enzymatic degradability and alkali hydrolyzability. Moreover, the change in Mn of the branched poly(L-lactide) (PLLA) by alkaline hydrolysis indicated that the decrease in Mn was in the first place dependent on the number of molecular branches and thereafter on the length of the molecular branch of branched PLA. The branched PLLA, poly(D-lactide) (PDLA), and poly(D,L-lactide) (PDLLA) differed in weight loss and change in Mn of the PLA segment during the enzymatic degradation. It is suggested that the branched PDLLA was degraded preferentially by proteinase K.

A cDNA encoding Penicillium funiculosum P(3HB) depolymerase (PhaZPfu) was cloned from a cDNA library. This cDNA contained a 1,020-bp open reading frame (ORF) that encoded 339 amino acids. Edman degradation of PhaZPfu indicated that 20 amino acids from the N terminus function as a signal peptide. Homology analysis revealed that PhaZPfu lacks linker and substrate-binding domains, both of which are observed in bacterial P(3HB) depolymerases. This may account for a weak binding affinity of PhaZPfu to the P(3HB) surface.

The annealing behavior of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (P(3HB-co-3HV)) crystal was studied by synchrotron radiation small-angle X-ray scattering (SAXS) and wide-angle X-ray ...

We investigated the changes in morphology of medium and high molecular weight polyhydroxybutyrate blends (MMWT/HMWT PHB) during shearing, using various in-situ rheological methods. On single shearing, fibres were formed only at the highest MMWT/HMWT ratio of (97/3). This was attributed to a critical orientation molecular weight. Fibre formation took place with all compositions and there was a rapid initial shish formation on multiple shearing. This is evidence for a partial shish based on the Janeschitz–Kriegl model.

Reaction processes of poly[(R)-3-hydroxybutyric acid] (P(3HB)) with two types of poly(hydroxybutyric acid) (PHB) depolymerases secreted from Ralstonia pickettii T1 and Penicillium funiculosum were characterized by means of atomic force microscopy (AFM) and quartz crystal microbalance (QCM). The PHB depolymerase from R. pickettii T1 consists of catalytic, linker, and substrate-binding domains, whereas the one from P. funiculosum lacks a substrate-binding domain. We succeeded in observing the adsorption of single molecules of the PHB depolymerase from R. pickettii T1 onto P(3HB) single crystals and the degradation of the single crystals in a phosphate buffer solution at 37 degrees C by real-time AFM. On the contrary, the enzyme molecule from P. funiculosum was hardly observed at the surface of P(3HB) single crystals by real-time AFM, even though the enzymatic degradation of the single crystals was surely progressed. On the basis of the AFM observations in air of the P(3HB) single crystals after the enzymatic treatments, however, not only the PHB depolymerase from R. pickettii T1 but also that from P. funiculosum adsorbed onto the surface of P(3HB) crystals, and both concentrations of the enzymes on the surface were nearly identical. This means both enzymes were adsorbed onto the surface of P(3HB) single crystals. Moreover, QCM measurements clarified quantitatively the differences in detachment behavior between two types of PHB depolymerases, namely the enzyme from R. pickettii T1 was hardly detached but the enzyme from P. funiculosum was released easily from the surface of P(3HB) crystals under an aqueous condition.

In this study, a new metabolic pathway for the synthesis of poly[(R)-3-hydroxybutyrate] [P(3HB)] was constructed in a recombinant Escherichia coli strain that utilized forward and reverse reactions catalyzed by two substrate-specific enoyl-CoA hydratases, R-hydratase (PhaJ) and S-hydratase (FadB), to epimerize (S)-3HB-CoA to (R)-3HB-CoA via a crotonyl-CoA intermediate. The R-hydratase gene (phaJ(Ac)) from Aeromonas caviae was coexpressed with the PHA synthase gene (phaC(Re)) and 3-ketothiolase gene (phaA(Re)) from Ralstonia eutropha in fadR mutant E. coli strains (CAG18497 and LS5218), which had constitutive levels of the beta-oxidation multienzyme FadB(Ec). When grown on glucose as the sole carbon source, the cells accumulated P(3HB) up to an amount 6.5 wt% of the dry cell weight, whereas the control cells without phaJ(Ac) or fadR mutation accumulated significantly smaller amounts of P(3HB). These results suggest that PhaJ(Ac) and FadB(Ec) played an important role in supplying monomers for P(3HB) synthesis in the pathway. Furthermore, by using this pathway, a P(3HB)-concentration-dependent fluorescent staining screening technique was developed to rapidly identify cells that possess active R-hydratase.

Adsorption effects of poly(hydroxybutyric acid) (PHB) depolymerase from Ralstonia pickettii T1 on various polymer single crystals were studied using a catalytically inactive mutant of PHB depolymerase by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), and frictional force microscopy (FFM). Six types of polymer single crystals, poly[( R )-3-hydroxybutyric acid] (P(3HB)), poly[( R )-3-hydroxybutyric acid- co -6 mol% ( R )-3-hydroxyvaleric acid] (P(3HB- co -6 mol% 3HV)), poly[( R )-3-hydroxybutyric acid- co -8 mol% ( R )-3-hydroxyhexanoic acid] (P(3HB- co -8 mol% 3HH)), poly( l -lactic acid) (PLLA), poly( d -lactic acid) (PDLA), and polyethylene (PE), were prepared to examine the influence of an ester bond and stereoregularity of a polymer on the enzymatic adsorption. The numbers of PHB depolymerase enzymes adsorbed on P(3HB) and P(3HB- co -6 mol% 3HV) single crystals were determined as 171 and 183 enzymes/μm 2 by AFM, respectively. AFM observation revealed that the concentration of PHB depolymerase enzymes adsorbed onto PLLA and PDLA single crystals is much higher compared to those on a P(3HB) single crystal, whereas the concentration of enzyme adsorbed onto PE and P(3HB- co -8 mol% 3HH) single crystals is much less. In addition, the single crystals of each polymer were characterized by TEM and FFM before and after enzymatic treatment by mutant for 1 h at 37 °C. The surface properties of P(3HB), P(3HB- co -6 mol% 3HV), and P(3HB- co -8 mol% 3HH) single crystals were changed by the enzymatic adsorption, whereas the internal structures were not affected. On the basis of these results, the properties of the binding domain of PHB depolymerase to polymer chain-folding surfaces have been discussed.

Four novel series of periodic copolymers composed of ester and amide units with the same carbon numbers were synthesized by two-step polycondensation reactions using adipate, linear aliphatic diols, and diamines with various chain lengths, ranging from 3 to 6 methylene groups. Effects of the comonomeric and sequential structures on the physical properties and crystalline structures were investigated for the obtained copolymer series by means of differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD), and tensile testing. The thermal properties of obtained periodic copolymer series were found to be strongly dependent on both comonomeric and sequential structures, and the values showed remarkable differences between neighboring methylene numbers in the comonomer units. Such thermal behavior was strongly correlated to the wide-angle X-ray diffraction patterns, with some copolymers displaying patterns similar to homopolyesters, while other copolymers had diffraction patterns that were ver...

Poly[( R )-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ RpiT1 ) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZ RpiT1 was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZ RpiT1 . Genetic analysis of the isolated mutants with lowered activity showed that Ser, Tyr, Val, Ala, and Leu residues in the SBD were replaced by other residues at high frequency. Some of the mutant enzymes, which contained the residues replaced at high frequency, were applied to assays of dPHB degradation and adsorption, revealing that those residues are essential for full activity of both dPHB degradation and adsorption. These results suggested that PhaZ RpiT1 adsorbs on the surface of dPHB not only via hydrogen bonds between hydroxyl groups of Ser in the enzyme and carbonyl groups in the PHB polymer but also via hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer. The L441H enzyme, which displayed lower dPHB degradation and adsorption abilities, was purified and applied to a dPHB degradation assay to compare it with the wild-type enzyme. The kinetic analysis of the dPHB degradation suggested that lowering the affinity of the SBD towards dPHB causes a decrease in the dPHB degradation rate without the loss of its hydrolytic activity for the polymer chain.

Thermal degradation behavior of poly(4-hydroxybutyric acid) (P(4HB)) was investigated by thermogravimetric and pyrolysis–gas chromatography mass spectrometric analyses under both isothermal and non-isothermal conditions. Based on the thermogravimetric analysis, it was found that two distinct processes occurred at temperatures below and above 350 °C during the non-isothermal degradation of P(4HB) samples depending on both the molecular weight and the heating rate. From 1 H NMR analysis of the residual P(4HB) molecules after isothermal degradations at different temperatures, it was confirmed that the ω-hydroxyl chain-end was remained unchanged in the residual P(4HB) molecules at temperatures below 300 °C, while the ω-chain-end of P(4HB) molecules was converted to 3-butenoyl units at temperatures above 300 °C. In contrast, the majority of the volatile products evolved during thermal degradation of P(4HB) was γ-butyrolactone regardless of the degradation temperature. From these results, it is concluded that during the thermal degradation of P(4HB), the selective formation of γ-butyrolactone via unzipping reaction from the ω-hydroxyl chain-end predominantly occurs at temperatures below 300 °C. At temperatures above 300 °C, both the cis -elimination reaction of 4HB unit and the formation of cyclic macromolecules of P(4HB) via intramolecular transesterification take place in addition to unzipping reaction from the ω-hydroxyl chain-end. Finally, the primary reaction of thermal degradation of P(4HB) at temperatures above 350 °C progresses by the cyclic rupture via intramolecular transesterification of P(4HB) molecules with a release of γ-butyrolactone as volatile product. Moreover, we carried out the thermal degradation tests for copolymer of 93 mol% of 4HB with 7 mol% of 3-hydroxybutyric acid (3HB) to examine the effect of 3HB units on thermal stability of P(4HB).

In MMWT/UHMWT PHB blends (99.5/0.5), (99/1), (98/2) and (97/3) we demonstrate that by applying our new shear technique, “semi continuous shear flow”, copious fibre formation is guaranteed, irrespective of the ultra high molecular weight composition. The shishes formed via this technique are extremely stable being able to maintain their stability for at least 5–10 min. When this technique was applied to MMWT/UHMWT PHB blends of compositions (95/5), (90/10) and (85/15) disorientated fibres were observed in the flow direction. On increasing the UHMWT component, distinct unordered and intertwining of fibres resulted and with severe shearing orientation occurred, however shish formation was limited. We propose the optimum conditions for shish formation and the critical molecular weight necessary for entanglement.

In this paper, the photosynthetic production of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymers is reported. The wild-type and highly active doubly mutated PHA synthase 1 (S325T/Q481K, abbreviated ST/QK) genes from Pseudomonas sp. 61-3 were introduced into Arabidopsis thaliana. Peroxisome targeting signal 1 (PTS1) was used to target PHA synthases into the peroxisome to synthesize PHA from the intermediates of the β-oxidation pathway. The transgenic Arabidopsis produced PHA copolymers consisting of 40–57 mol% 3-hydroxybutyrate, 21–49 mol% 3-hydroxyvalerate, 8–18 mol% 3-hydroxyhexanoate, and 2–8 mol% 3-hydroxyoctanoate. The maximum PHA contents were 220μ g/g cell dry weight (cdw) in leaves, and 36μ g/g cdw in stems, respectively. The expression of the ST/QK mutated PHA synthase in leaves gene did not lead to significant difference in PHA content and monomer composition of PHAs, compared to the wild-type PHA synthase gene, suggesting that the supply of monomers may be a rate-determining step of PHA biosynthesis in the peroxisome. However, in stems, there were significant differences dependent on whether the wild-type or ST/QK mutated PHA synthase was expressed. These results suggest that tissue-specific monomer availability is important in determining the final mol% composition of PHA copolymers produced by the peroxisome in plants.

Surface properties and enzymatic degradation of poly( l -lactide) (PLLA) end-capped with hydrophobic dodecyl and dodecanoyl groups were investigated by means of advancing contact angle ( θ a ) measurement, quartz crystal microbalance (QCM) and atomic force microscopy (AFM). The θ a values of end-capped PLLA films were larger than those of non-end-capped PLLA films, suggesting that the hydrophobic dodecyl and dodecanoyl groups were segregated on the film surface. The weight changes of end-capped PLLA thin films during enzymatic degradation in the presence of proteinase K were monitored by using a QCM technique. The relatively fast weight loss of PLLA film occurred during first few hours of degradation, followed by a decrease in the erosion rate. The erosion rate of PLLA films at the initial stage of degradation was dependent on the chain-end structure of PLLA molecules, and the value decreased with an increase in the amount of hydrophobic functional groups. The surface morphologies of PLLA thin films before and after degradation were characterized by AFM. After the enzymatic degradation, the surface of non-end-capped PLLA films was blemished homogeneously. In contrast, the end-capped PLLA thin films were degraded heterogeneously by the enzyme, and many hollows were formed on the film surface. From these results, it has been concluded that the introduction of hydrophobic functional groups at the chain-ends of PLLA molecules depressed the erosion rate at the initial stage of enzymatic degradation.

Thermal degradation behaviours of poly(3-hydroxybutyric acid) (P(3HB); bacterial poly[( R )-3-hydroxybutyric acid] and synthetic poly[( R , S )-3-hydroxybutyric acid] samples, were examined under both isothermal and non-isothermal conditions. The inverse of number-average degree of polymerisation for all P(3HB) samples decreased linearly with degradation time during the initial stage of isothermal degradation at a temperature ranging from 170–190 °C. In addition, crotonyl unit was detected in the residual polymer samples as main ω-chain-end. These results indicate that the dominant thermal degradation reaction for P(3HB) is a random chain scission via cis -elimination reaction of P(3HB) molecules. It was found that the presence of either Ca or Mg ions enhances the depolymerisation of P(3HB) molecules, while that Zn ions hardly catalyse the reaction. As a result, a shift of thermogravimetric curves toward the lower temperature regions was observed for the P(3HB) samples containing high amounts of Ca and Mg compounds.

Novel aliphatic poly(ester−amide)s with a periodic sequential structure consisting of ester and amide groups were synthesized by two-step polycondensation reactions using adipate, butane-1,4-diamine, and linear diols with different chain lengths, ranging from 3 to 6 methylene groups. Effects of the monomeric structure and sequential length of ester units on the thermal properties and crystalline structure of the obtained periodic copolymers were determined by means of differential scanning calorimetry (DSC) and wide-angle X-ray diffraction (WAXD). On the basis of DSC measurements, the melting temperatures of the periodic copolymers were higher than those of homopolyesters consisting of the same ester units and tended to increase with an increase in amide content. The copolymers from adipate, butane-1,4-diol, and butane-1,4-diamine had especially high thermal stability compared with the poly(tetramethylene adipate) homopolyester, and the melting temperatures detected were above 200 °C. The X-ray diffractio...

Biodegradable fibers of poly[(R)-3-hydroxybutyrate-co-8%-(R)-3-hydroxyvalerate] (P(3HB-co-8%-3HV)) produced from wild-type bacteria were prepared by melt-spinning, followed in an amorphous state by quenching near the glass transition temperature (Tg). High tensile strength fibers of over 1 GPa were prepared by one-step drawing with small crystal nuclei grown by isothermal crystallization near the Tg. This new drawing technique is a very attractive method for obtaining strong fibers from low-molecular-weight polyesters produced by wild-type bacteria. The highly ordered structure of strong P(3HB-co-8%-3HV) fibers was investigated by tensile measurement, scanning electron microscopy, and wide- and small-angle X-ray scatterings (WAXD and SAXS) with synchrotron radiation (SPring-8, Japan). The WAXD patterns of strong fibers showed sharp reflections corresponding to highly oriented α-form (21 helix conformation) and β-form (planar zigzag conformation). The weak reflections along the meridian in the SAXS pattern...

Poly[(R)-3-hydroxybutyrate] (P(3HB)) single crystals were grown isothermally at different crystallization temperatures (Tcs) in a dilute solution. Structural changes of the sedimented single-crystal mats on heating were followed by real-time synchrotron small-angle X-ray scattering (SAXS) and wide-angle X-ray diffraction (WAXD) techniques, and the dependence of the annealing behavior on Tc was examined. The two-dimensional (2D) SAXS pattern clearly demonstrated that the P(3HB) single crystal exhibits a discontinuous increase in the lamellar thickness at an annealing temperature. The onset of the discontinuous thickening depended on Tc or the initial lamellar thickness. The discontinuous thickening occurred at higher temperature as the initial lamellar thickness increased. In the transition region where the discontinuous thickening progressed, the new scattering peak intensity increased while the original one decreased. On the other hand, the crystallinity, estimated from the 2D WAXD patterns, first decrea...

The adsorption behavior of PHB depolymerase from R. pickettii T1 on a silicon wafer and on P(3HB) single crystals has been studied by real-time and AFM in air and a buffer solution. First, the morphology of PHB depolymerase adsorbed on a silicon wafer was characterized to show that one molecule of PHB depolymerase has dimensions of 2.2 +/- 0.7 nm height and 16 +/- 5 nm width. The observation of PHB depolymerase adsorbed on a P(3HB) single crystal indicated that the dimensions of enzyme on the crystalline surface in air were 1.2 +/- 0.5 nm high and 28 +/- 7 nm wide, while enzyme molecules with dimensions of 2.1 +/- 0.6 nm height and 16 +/- 7 nm width were detected in a buffer solution. Comparison of the dimensions of PHB depolymerase in air with those in a buffer solution showed that the enzyme was squashed in air, but not in a buffer solution. In addition, the influence of enzymatic adsorption on the molecular state of the P(3HB) crystalline surface was investigated. The AFM images of P(3HB) single crystals after enzymatic adsorption and washing with ethanol indicated that the adhesion of PHB depolymerase changed the molecular state and generated holes on the crystalline surface.

Phase structures and enzymatic degradation of poly(l-lactide) (PLLA)/atactic poly(3-hydroxybutyrate) (ata-PHB) blends with different compositions were characterized by using atomic force microscopy (AFM). Differential scanning calorimetry (DSC) thermograms of PLLA/ata-PHB blends with different compositions showed two glass transition temperatures, indicating that the PLLA/ata-PHB blends are immiscible in the melt. Surface morphologies of the thin films for PLLA/ata-PHB blends were determined by AFM. Phase separated morphology was recognized from the AFM topography and phase images. The domain size of the components was dependent on the blend ratio. Enzymatic degradation of the PLLA/ata-PHB blends was performed by using both PHB depolymerase and proteinase K. Either PLLA or ata-PHB domains were eroded depending on the kinds of enzyme. Surface morphologies after enzymatic degradation have revealed the phase structure along the depth direction. Enzymatic adsorption of PHB depolymerase was examined on the surface of PLLA/ata-PHB blends. The enzyme molecules were found on both domains of the binary blends. The larger number of enzyme molecules was found on the PLLA domains relative to those on the ata-PHB domains, suggesting the higher affinity of the enzyme against PLLA domain.