Retardation Effect of Enzymatic Degradation of Microbial Polyesters at the Surface by Blending with Polystyrene

The enzymatic surface degradation behavior of the blend films of polystyrene (PS) with poly[((R)‐3‐hydroxybutyrate)‐co‐((L )‐3‐hydroxyvalerate)] (P(3HB‐co‐3HV)) or poly((R)‐3‐hydroxybutyrate (P(3HB)) were investigated using atomic force microscopy (AFM). It was found that the blends of PS with P(3HB‐co‐3HV) or P(3HB) are immiscible in both the amorphous and melt states. The degradation of both P(3HB‐co‐HV) and P(3HB) was significantly retarded at the initial stages of enzymatic attack by hydrophobic PS (up to 20 wt.‐%).     

Adsorption effects of poly(hydroxybutyric acid) depolymerase on chain-folding surface of polyester single crystals revealed by mutant enzyme and frictional force microscopy

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/μm² 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.     

Enzymatic biodegradation of chemosynthetic atactic P(3HB) enhanced by an amorphous non‐biodegradable polymer: blend of atactic P(3HB) with PMMA

Communication: The phase structure and biodegradability were investigated for a blend of chemosynthetic atactic poly((R,S)‐3‐hydroxybutyrate), a‐P(3HB), and poly(methyl methacrylate), PMMA. The thermal analysis indicated that amorphous a‐P(3HB)/PMMA blends with 20 wt.‐% and 40 wt.‐% PMMA shows sophisticated phase behavior and is partially miscible. The depolymerase of natural poly((R)‐3‐hydroxybutyrate) purified from Alcaligenes faecalis T1 did not degrade chemosynthesized a‐P(3HB) at all in the pure state, but it degraded a‐P(3HB) in some a‐P(3HB)/PMMA blends. The results suggested that enzymatic degradation of a‐P(3HB) can be enhanced by an amorphous non‐biodegradable polymer.     

Degradation of commercially important polyhydroxyalkanoates in tropical mangrove ecosystem

This paper presents the degradation trends of selected polyhydroxyalkanoate (PHA) films in a tropical mangrove environment. The biodegradability of homopolymer poly(3-hydroxybutyrate) [P(3HB)] and its co-polymers, poly(3-hydroxybutyrate-co-5 mol% 3-hydroxyvalerate) [P(3HB-co-5 mol% 3HV)] and poly(3-hydroxybutyrate-co-5 mol% 3-hydroxyhexanoate) [P(3HB-co-5 mol% 3HHx)], was investigated along with P(3HB) films containing 38 wt% titanium dioxide (TiO₂) [P(3HB)-38 wt% TiO₂]. The degradation of these formulations was monitored for 8 weeks at three different zones in an intermediate mangrove compartment along Sungai Pinang, adjacent to a famous fishing village on south of Penang Island. The degradation rate was observed both on the surface and in the sediment and was expressed in percentage of weight loss. The microbial enumeration done using sediment from the different zones indicated similar colony-forming unit (CFU) counts even though differences were noticed in the degradation profile of the various films in the respective zones. The results obtained revealed that co-polymers disintegrated at similar or higher rate than the homopolymer, P(3HB). However, the incorporation of TiO₂ into PHB films caused the degradation rate of P(3HB)-38 wt% TiO₂ composite film to be far slower than all the other PHA films. The overall rate of degradation of all PHA films placed on the sediment surface was slower than those buried in the sediment. Microscopic analyses showed that the surface morphology of P(3HB-co-5 mol% 3HHx) was more porous compared to P(3HB) and P(3HB-co-5 mol% 3HV) films, which may be an important factor for its rapid degradation.     

Characterization and enzymatic degradation of microbial copolyester P(3HB-co-3HV)s produced by metabolic reaction model-based system

The two types of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s [P(3HB-co-3HV)s] were produced by Paracoccus denitrificans ATCC 17741 using two different feeding methods. The produced P(3HB-co-3HV)s were fractionated and the copolymer sequence distributions were analyzed by ¹H and ¹³C NMR spectroscopy. It was found that the P(3HB-co-3HV) samples produced by conventional feeding method were statistically random copolymers. The sequence distributions of P(3HB-co-3HV) samples produced by optimization method were different from random P(3HB-co-3HV)s. The thermal properties and melting behaviors were analyzed by differential scanning calorimetry (DSC). These results demonstrated that P(3HB-co-3HV) samples produced by optimization method are close in nature to P(3HB-co-3HV)s rich in long-sequence of block 3HB units, but less in 3HV random regions. The enzymatic degradation profile of P(3HB-co-3HV) films was investigated in the presence of 3-hydroxybutyrate depolymerase from Pseudomonase lemoignei. The degradation process was observed by monitoring the time-dependent change in the weight loss of copolymer films. The surface erosion of copolymer films was qualitatively monitored by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The highest degradation rate of 2.6% per day was observed for random P(3HB-co-38%3HV) produced by conventional method. In comparison, the hydrolysis degradation rates of random P(3HB-co-3HV)s were about one time faster than those of P(3HB-co-3HV)s produced by optimization method.     

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

Enzymatic degradation behavior of a lamella of single crystals of poly(R)‐3‐hydroxybutyrate (P(3HB)) with an extracellular polyhydroxybutyrate (PHB) depolymerase purified from Alcaligenes faecalis T1 has been investigated by atomic force microscopy (AFM) in order to obtain further information for the chain packing state of P(3HB) in a lamellar single crystal. Two kinds of P(3HB) single crystals with different molecular weights, denoted respectively as H‐ and L‐P(3HB) for high and low molecular weights, respectively, were prepared. The enzymatic treatment was conducted for P(3HB) single crystals adsorbed on a surface of highly ordered pyrolytic graphite. The enzymatic degradation of both P(3HB) single crystals generates several crevices crosswise across the crystal at an early stage. Subsequently, the enzymatic degradation yields numbers of cracks lengthwise along the crystal. In addition to these common features, the interval between cracks crosswise across a lamella in H‐P(3HB) single crystal is longer than that in L‐P(3HB) single crystal, and each crack has V‐shaped and rectangular shaped morphology for H‐ and L‐P(3HB) single crystals, respectively. Based on these results, it is concluded that a lamella of P(3HB) single crystal has straight degradation pathways, that may correspond to a switchboard region, along the long axis of the crystal, independent of molecular weight of P(3HB) samples, and that a H‐P(3HB) single crystal has broader degradation pathways with longer intervals crosswise across the crystal than a L‐P(3HB) single crystal.     

Origin of the double melting peaks of poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) with a high HV content as revealed by in situ synchrotron WAXD/SAXS analyses

We here reported the dual melting behaviors with a large temperature difference more than 50 °C without discernible recrystallization endothermic peak in isomorphous poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (P(HB‐co‐HV)) with a high HV content of 36.2 mol %, and the structure evolution upon heating was monitored by in situ synchrotron wide‐angle X‐ray diffraction/small‐angle X‐ray scattering (WAXD/SAXS) to unveil the essence of such double endothermic phenomena. It illustrated that the thinner lamellae with the larger unit cell and the thicker crystals having the smaller unit cell were melted around the first low and second high melting ranges, respectively. By analyzing in situ WAXD/SAXS data, and then coupling the features of melting behavior, the evolution of the parameters of both crystal unit cell and lamellar crystals, we proposed that the thinner unstable lamellae possess a uniform structure with HV units total inclusion, and the thicker stable lamellae reflect the sandwich structure with HV units partial inclusion. It further affirmed that the thicker sandwich and thinner uniform lamellae formed during the cooling and subsequent isothermal crystallization processes, respectively. These findings fully verify that it is the change of structure of lamellae rather than the melting/recrystallization that is responsible for double melting peaks of isomorphous P(HB‐co‐36.2%HV), and enhance our understanding upon multiple endothermic behaviors of polymers. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019 , 57, 1453–1461     

Enhancement of Stress Tolerance in the Polyhydroxyalkanoate Producers without Mobilization of the Accumulated Granules

Poly(3-hydroxybutyrate) [P(3HB)], a polymer belonging to the polyhydroxyalkanoate (PHA) family, is accumulated by numerous bacteria as carbon and energy storage material. The mobilization of accumulated P(3HB) is associated with increased stress and starvation tolerance. However, the potential function of accumulated copolymer such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] remained unknown. In this study, Delftia acidovorans DS 17 was used to evaluate the contributions of P(3HB) and P(3HB-co-3HV) granules during simulated exogenous carbon deprivation on cell survival by transferring cells with PHAs to carbon-free mineral salt medium supplemented with 1 % (w/v) nitrogen source. By mobilizing the intracellular P(3HB) and P(3HB-co-3HV) at 11 and 40 mol% 3HV compositions, the cells survived starvation. Surprisingly, D. acidovorans containing P(3HB-co-94 mol% 3HV) also survived although the mobilization was not as effective. Similarly, recombinant Escherichia coli pGEM-T::phbCAB _Cn (harboring the PHA biosynthesis genes of Cupriavidus necator) containing P(3HB) granules had a higher viable cell counts compared to those without P(3HB) granules but without any P(3HB) mobilization when exposed to oxidative stress by photoactivated titanium dioxide. This study provided strong evidence that enhancement of stress tolerance in PHA producers can be achieved without mobilization of the previously accumulated granules. Instead, PHA biosynthesis may improve bacterial survival via multiple mechanisms.     

Study of enzymatic degradation of microbial copolyesters consisting of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates

Enzymatic degradation of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) (PHBA) biopolyester consisting of 3-hydroxybutyrate (HB) and 15 mol% medium-chain-length 3-hydroxyalkanoates (HA) was studied using a polyhydroxyalkanoates (PHA) depolymerase produced by Ralstonia pickettii T1. It was found that PHBA films did not lose their weight after 25 h of depolymerase treatment. In contrast, three commercially available PHAs including poly-3-hydroxybutyrate (PHB), poly(3-hydroxybutyrate-19 mol% 3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-19 mol% 3-hydroxyhexanoate) (PHBHHx) lost 75%, 94% and 39% of their original weights. Slow degradation of PHBA was also confirmed by the absence of HA monomers, dimers or trimers as degradation products in their depolymerase solution compared with abundance of degradation products released by the other three PHAs under the same condition. Surface erosion of PHBA was only observed after 48 h of enzymatic treatment compared with those of PHB, PHBV and PHBHHx which already had obvious surface changes after 7.5 h of same treatment. Although the crystallinities of PHB, PHBV, PHBHHx and PHBA were in the order PHB > PHBV > PHBHHx > PHBA valued at 55.8%, 47.8%, 45.9% and 40.9%, respectively, the order of degradability was PHBV > PHB > PHBHHx > PHBA. It can be proposed that PHA enzymatic degradation using this depolymerase was structure related: longer side-chain PHA including PHBHHx and PHBA was less favorable for the depolymerase degradation, longer the side chain, less the biodegradation.     

Biosynthesis of (R)-3-hydroxyalkanoic acids by metabolically engineered Escherichia coli

An efficient system for the production of (R)-hydroxyalkanoicacids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced.     

Influence of feeding strategies of mixed microbial cultures on the chemical composition and microstructure of copolyesters P(3HB‐co‐3HV) analyzed by NMR and statistical analysis

NMR spectroscopy was applied for quantitative and qualitative characterization of the chemical composition and microstructure of a series of poly(3‐hydroxybutyrate‐co‐3‐hydoxyvalerate) copolymers, P(3HB‐co‐3HV), synthesized by mixed microbial cultures at several different feeding strategies. The monomer sequence distribution of the bacterially synthesized P(3HB‐co‐3HV) was defined by analysis of their high‐resolution 1D ¹³C NMR and 2D ¹H/¹³C HSQC and ¹H/¹³C HMBC NMR spectra. The results were verified by employment of statistical methods and suggest a block copolymer microstructure of the P(3HB‐co‐3HV) copolymers studied. Definitive distinction between block copolymers or a mixture of random copolymers could not be achieved. NMR spectral analysis indicates that the chemical composition and microstructure of the copolymers can be tuned by choosing a correct feeding strategy. Copyright © 2009 John Wiley & Sons, Ltd.     

Characteristic Interactions between Poly(hydroxybutyrate) Depolymerase and Poly[(R)‐3‐hydroxybutyrate] Film Studied by a Quartz Crystal Microbalance

Enzymatic degradation of poly[(R)‐3‐hydroxybutyrate] (P(3HB)) film by the poly(hydroxybutyrate) (PHB) depolymerase from Ralstonia picketti T1 was studied in 0.01 M phosphate buffer solution (pH 7.4) at 37 °C by using a quartz crystal microbalance (QCM) technique. Enzymatic degradation of P(3HB) film was quantitatively followed by QCM as a positive frequency shift. While, the amount of depolymerases adsorbed on the film could be evaluated as a negative frequency shift by using a mutant enzyme which had no hydrolytic activity in a catalytic site. The degradation rate increased with enzyme concentration to reach a maximum value at 1.0 μg · mL^?1, and then the rate decreased at higher enzyme concentration. This enzyme concentration dependence could be quantitatively explained in terms of a change of coverage of the film surface by the adsorbed enzyme. When the wild‐type enzyme solution in a QCM cell was replaced with the mutant enzyme solution in the middle of the reaction, the degradation rate was reduced markedly, indicating that the wild‐type enzyme adsorbed on the P(3HB) surface is easily substituted by the mutant enzyme in the solution. On the other hand, replacement of the wild‐type enzyme solution with other proteins or buffer solutions did not affect the degradation rate at all, suggesting that the adsorbed enzyme was not desorbed from the film surface. Thus, the adsorbed PHB depolymerase is released from the P(3HB) surface only by interaction with the same depolymerase in solution.

Time courses of frequency changes (ΔF) or weight changes (Δw) observed during enzymatic degradation of P(3HB) film by PHB depolymerase from R. picketti T1 at 37 °C.     

Purification and Properties of an Extracellular Polyhydroxybutyrate Depolymerase from Pseudomonas mendocina DSWY0601

An extracellular polyhydroxybutyrate(PHB) depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was approximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20―50 ℃ and sensitive to pH value within a pH range of 8.0―9.5. PHB depolymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate(P3/4HB) and poly-3-hydroxybutyrate-co-3- hydroxyvalerate(PHBV) but did not degrade poly(lactic acid)(PLA), poly(butylene succinate)(PBS) or poly- (caprolactone)(PCL). PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride(PMSF), H₂O₂ and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer(3HB) by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis of phaZ_pm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA(dPHA_SCL, PHA=polyhydroxyalkanoate) depolymerase containing catalytic domain, linker and substrate-binding domain.     

Crystalline morphology and thermal properties for random copolyesters of (R)‐3‐hydroxybutyric acid with different hydroxyalkanoic groups

The solid‐state structures and thermal properties of melt‐crystallized films of random copolymers of (R)‐3‐hydroxybutyric acid (3HB) with different hydroxyalkanoic acids such as (R)‐3‐hydroxypentanoic acid (3HV), (R)‐3‐hydroxyhexanoic acid (3HH), medium‐chain‐length (R)‐3‐hydroxyalkanoic acids (mcl‐3HA; C8‐C12), 4‐hydroxybutyric acid (4HB), and 6‐hydroxyhexanoic acid (6HH) were characterized by means of small‐angle X‐ray scattering, differential scanning calorimetry, and optical microscopy. The randomly distributed second monomer units except for 3HV in copolyesters act as defects of P(3HB) crystal and are excluded from the P(3HB) crystalline lamellae. The lamellar thickness of copolymers decreased with an increase in either the main‐chain or the side‐chain carbon numbers of second monomer units. In addition, the growth rate of spherulites decreased with an increase in the carbon numbers of second monomer units for copolymers with an identical comonomer composition. These results indicate that the steric bulkiness of second monomer unit affects on the crystallization of 3HB segments in random copolyesters.     

Surface modification and characterization of SEBS films obtained by in situ and ex situ oxidization with potassium permanganate

The surface morphologies and compositions of the asymmetric films of polystyrene‐b‐poly(ethylene‐co‐butylene)‐b‐polystyrene (SEBS) prepared by in situ and ex situ oxidization with the KMnO₄ aqueous solution and KMnO₄/H₂SO₄ mixed solution were investigated by using scanning electron microscope, atomic force microscope (AFM), X‐ray photoelectron spectroscopy (XPS) and attenuated total reflectance infrared spectroscopy (ATR‐FTIR). The effect of the oxidization reagents on morphological changes and the influence of in situ and ex situ preparation methods on surface compositions were discussed. Different from the in situ oxidation by degrading the copolymers to form a gradient film, the ex situ oxidation preferentially degraded the uppermost layer of the film. Although both the KMnO₄ oxidation and the KMnO₄/H₂SO₄ oxidation gave hierarchical structures, distinctive differences were found that large ridges and smaller granules were fabricated in the former film and the latter produced large and deep ravines and fine sponge‐like morphologies. Additionally, the oxygen concentration and the oxo‐species implanted by these oxidation treatments were characterized and evaluated to provide a quantitative comparison. Oxygen, as well as manganese was found to be implanted in the surface layer of the oxidized film, forming predominantly O? C and O? C?O groups, as well as a small fraction of O? H and Mn? O compounds. Changes in contact angle of water on these films and total surface oxygen content are related but not directly. The hystereses of water contact angle at a value of 119 ± 3° due primarily to surface roughness and at a value of 63 ± 3° due primarily to chemical heterogeneity are led by different oxidation degrees and oxidation methods. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2010     

Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer from wild-type Comamonas sp. EB172

Poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer was produced by Comamonas sp. EB172 using single and mixture of carbon sources. Poly(3-hydroxyvalerate) P(3HV) incorporation in the copolymer was obtained when propionic and valeric acid was used as precursors. Incorporation of 3HV fractions in the copolymer varied from 45 to 86 mol% when initial pH of the medium was regulated. In fed-batch cultivation, organic acids derived from anaerobically treated palm oil mill effluent (POME) were shown to be suitable carbon sources for polyhydroxyalkanoate (PHA) production by Comamonas sp. EB172. Number average molecular weight (M_n) produced by the strain was in the range of 153-412 kDa with polydispersity index (M_w/M_n) in the range of 2.2-2.6, respectively. Incorporation of higher 3HV units improved the thermal stability of P(3HB-co-3HV) copolymer. Thus the newly isolated bacterium Comamonas sp. EB172 is a suitable candidate for PHA production using POME as renewable and alternative cheap raw materials.     

Preparation and Characterization of Polyhydroxyalkanoates Macroporous Scaffold Through Enzyme-Mediated Modifications

Polyhydroxyalkanoates (PHAs) are hydrophobic biodegradable thermoplastics that have received considerable attention in biomedical applications due to their biocompatibility, mechanical properties, and biodegradability. In this study, the degradation rate was regulated by optimizing the interaction of parameters that influence the enzymatic degradation of P(3HB) film using response surface methodology (RSM). The RSM model was experimentally validated yielding a maximum 21 % weight loss, which represents onefold increment in percentage weight loss in comparison with the conventional method. By using the optimized condition, the enzymatic degradation by an extracellular PHA depolymerase from Acidovorax sp. DP5 was studied at 37 °C and pH 9.0 on different types of PHA films with various monomer compositions. Surface modification of scaffold was employed using enzymatic technique to create highly porous scaffold with a large surface to volume ratio, which makes them attractive as potential tissue scaffold in biomedical field. Scanning electron microscopy revealed that the surface of salt-leached films was more porous compared with the solvent-cast films, and hence, increased the degradation rate of salt-leached films. Apparently, enzymatic degradation behaviors of PHA films were determined by several factors such as monomer composition, crystallinity, molecular weight, porosity, and roughness of the surface. The hydrophilicity and water uptake of degraded salt-leached film of P(3HB-co-70%4HB) were enhanced by incorporating chitosan or alginate. Salt-leached technique followed by partial enzymatic degradation would enhance the cell attachment and suitable for biomedical as a scaffold.     

Mechanical Properties of Uniaxially Cold-Drawn Films of Poly[(R)-3-hydroxybutyrate] and Its Copolymers

Uniaxially oriented films of poly[(R)-3-hydroxybutyrate] (P(3HB)) and two kind of copolymers, poly[(R)-3-hydroxybutyrate-co-8%-[R]-3-hydroxyvalerate] (P(3HB-co-8%-3HV)), and poly[(R)-3-hydroxybutyrate-co-[R]-5%-3-hydroxyhexanoate] (P(3HB-co-5%-3HH)), were prepared by cold-drawing from amorphous preforms at temperatures near to the respective glass transition temperatures. Melt-quenched films in a rubber state could be stretched reproducibly to a draw ratio of 500%∼1800%, and subsequent annealing under tension led to improvement of the tensile strength and Young's modulus. Two-step drawing resulted in further improvement of the mechanical properties. The mechanical properties remained unchanged after storing for 6 months at room temperature, suggesting that high orientation and crystallinity suppress the secondary crystallization.     

Adsorption of biopolyester depolymerase on silicon wafer and poly[(R)-3-hydroxybutyric acid] single crystal revealed by real-time AFM

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.     

TH‐11, a Streptomyces sp. Strain that Degrades Poly(3‐Hydroxybutyrate) and Poly(Ethylene Succinate)

TH‐11, a bacterial strain with strong depolymerase activity that breaks down aliphatic esters such as poly(3‐hydroxybutyrate) (PHB) and poly(ethylene succinate) (PES) was isolated from a soil sample collected from the sediment of Tou‐Chain River, Taiwan, R.O.C. It was phenotypically and genetically characterized to be a Streptomyces strain. The degradation of PHB and PES were tested both using emulsified polymers in solid agar and thin polymer films in liquid culture media. The degradations were measured by clear‐zone formation on solid agar plates, or direct weight measurements and electromicroscope inspection of the incubated polymer films in the liquid culture. The depolymerase activities can be detected in the cell‐free preparation of the culture medium, and can be enhanced by gelatin.