Ralstonia Pickettii T1 Research Articles

Miscibility and enzymatic degradability of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)-based polyester blends by PHB depolymerase and lipase

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) containing 5 and 17 mol% 3-hydroxyhexanoate has been blended with atactic poly(3-hydroxybutyrate) (PHB), polycaprolactone (PCL), and polybutylene succinate (PBS) at different ratios by the solvent-casting method. PHBH-5%/atactic PHB and PHBH-17%/atactic PHB showed a single glass-transition temperature (Tg), indicating miscibility in the amorphous state. Immiscibility was observed in the PHBH-5%/PCL and PHBH-17%/PCL blends based on the non-transitioning Tg of the PHBH component, presence of two distinct Tg values for some of the blend films, and individual crystallization manner. The thermal properties and spherulitic morphologies of the PHBH-5%/PBS blend films suggested partial miscibility. The enzymatic degradability of the three types of blends by Ralstonia pickettii T1 PHB depolymerase and Burkholderia cepacia lipase was investigated. Depending on the enzyme–substrate specificity, miscibility, and phase structure of the blend, the variation of the enzymatic degradation rate with the blend ratio was different among the three types of blends. The weight loss rate of PHBH-5%/atactic PHB was higher than that of PHBH-17%/atactic PHB when degraded by PHB depolymerase. The PCL component accelerated the weight loss rate of PHBH-17%/PCL but decreased that of PHBH-5%/PCL. Lipase barely eroded PHBH-5%/PBS, but the weight loss rate increased as the PCL content increased. From quantitative analysis of the water-soluble products liberated during degradation, the degradation manners of the blends by PHB depolymerase and lipase are discussed.

Cell Surface Display of Poly(3-hydroxybutyrate) Depolymerase and its Application.

We have expressed extracellular poly(3-hydroxybutyrate) (PHB) depolymerase of Ralstonia pickettii T1 on the Escherichia coli surface using Pseudomonas OprF protein as a fusion partner by C-terminal deletion-fusion strategy. Surface display of depolymerase was confirmed by flow cytometry, immunofluorescence microscopy and whole cell hydrolase activity. For the application, depolymerase was used as an immobilized catalyst of enantioselective hydrolysis reaction for the first time. After 48 h, (R)-methyl mandelate was completely hydrolyzed, and (S)-mandelic acid was produced with over 99% enantiomeric excess. Our findings suggest that surface displayed depolymerase on E. coli can be used as an enantioselective biocatalyst.

Effects of mutation at position 285 of Ralstonia pickettii T1 poly[(R)-3-hydroxybutyrate] depolymerase on its activities

Asn at position 285 (N285) in the catalytic domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 most likely participates in the cleavage of ester bonds as revealed by our previous evolutionary engineering study using the error-prone polymerase chain reaction (PCR) method. To exhaustively examine the effects of mutations at that position, we conducted site-directed saturation mutagenesis at that position and the resultant mutant enzymes (N285X) were evaluated in p-nitrophenyl ester (pNPCn) hydrolysis and PHB degradation. Kinetic studies demonstrated that the PHB-degrading activities of N285X were reciprocally related to their pNPCn-hydrolyzing activities, with the exception of N285A and N285G, and that His residue could functionally substitute for Asn285 on PHB degradation.

Transcriptional repression of the poly(3-hydroxybutyrate) depolymerase in Ralstonia pickettii T1 by a tetR-like gene

Ralstonia pickettii T1 secretes a poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZ) and a 3-hydroxybutyrate (3HB)-oligomer hydrolase, and extracellularly degrades PHB to produce 3HB. However, it is not clear how the expression of phaZ is regulated. In this study, the mechanism by which phaZ expression is controlled in R. pickettii T1 was examined using a mutant made by the random insertion of a transposon, Tn5. The mutant produced a larger amount of PhaZ than the wild type in nutrient broth or a minimal salt (SM) medium supplemented with succinate. However, there was essentially no difference in the activity or amount of PhaZ in the culture supernatant between the wild type and mutant when the two were grown on 3HB. The gene disrupted by the insertion of Tn5 (epdR) was cloned and its nucleotide sequence was determined. In a BLAST search, epdR showed a high degree of similarity to genes for TetR transcriptional regulators of several bacteria. The introduction of epdR into the wild type and mutant grown on the three media described above decreased the amount of PhaZ. These results indicated EpdR to be involved in the repression of phaZ in R. pickettii T1. A quantitative RT-PCR analysis indicated that mRNA levels corresponded with the activity detected and the amounts of PhaZ in the wild type and mutant. Furthermore, the amount of epdR transcript was inversely proportional to the amount of phaZ transcript. In addition, the existence of a positive element acting on phaZ expression was suggested, because in the mutant lacking EpdR, the amount of phaZ transcript varied in cells grown in SM-3HB, SM-succinate or nutrient broth. Based on the above results, a model for the regulation of PhaZ expression in R. pickettii T1 is proposed.

Properties and enzymatic degradation of poly(acrylic acid) grafted polyhydroxyalkanoate films by plasma-initiated polymerization

Using plasma-initiated polymerization, poly(acrylic acid) (PAA) was grafted onto the film surfaces of polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate] (P(3HB)), and two kinds of poly[(R)-3-hydroxybutyrate-co-4-hydroxybutyrate] (P(3HB-co-4HB)) with 4.7 mol% or 9.4 mol% of 4HB content. The graft ratio of PAA on the film surface increased drastically with acrylic acid concentration over 8%. Attenuated total reflection Fourier transform infrared spectroscopy demonstrated that the PAA grafted on the film surface was at least 70 nm thick. Mechanical and surface properties of PHA films grafted by PAA (PAA–PHA) were analyzed by tensile test, contact angle test, and scanning electron microscopy. While mechanical properties before and after plasma treatment remained unchanged, surface properties changed from hydrophobic to hydrophilic. Enzymatic degradation of PAA–PHA films was performed using an extracellular PHB depolymerase purified from Ralstonia pickettii T1. The rate of enzymatic degradation was inhibited by PAA graft. However, the rate of enzymatic degradability was accelerated by exposing ungrafted polymer to the enzyme solution, when a PAA-grafted film was divided into several pieces.

Mechanical properties, structure analysis and enzymatic degradation of uniaxially cold-drawn films of poly[( R)-3-hydroxybutyrate- co-4-hydroxybutyrate

Poly[( R)-3-hydroxybutyrate- co-4-hydroxybutyrate] (P(3HB- co-4HB)) films were prepared by uniaxial cold-drawing from an amorphous preform at a temperature below, but close to the glass transition temperature. Molecular and highly-ordered structures and physical properties of cold-drawn films were investigated by tensile testing, wide-angle X-ray diffraction and small-angle X-ray scattering. Enzymatic degradation of P(3HB- co-4HB) films was performed using an extracellular polyhydroxybutyrate depolymerase purified from Ralstonia pickettii T1. Tensile strength, elongation to break and Young’s modulus of P(3HB- co-4HB) with cold-drawn ratio 1200% reached 290 MPa, 58% and 2.8 GPa, respectively. X-ray fibre diagrams of cold-drawn P(3HB- co-4HB) films showed a strong reflection on the equatorial line, indicating a planar zigzag conformation ( β-form) together with 2 1 helix conformation ( α-form). The β-form seems to contribute to the high tensile strength, and a new mechanism of generation of the β-form is proposed. The enzymatic degradation rate increased with increasing draw ratio, and increased greatly with increasing 4HB content.

Characterization of a novel 3-hydroxybutyrate dehydrogenase from Ralstonia pickettii T1

We previously reported that the activities of two 3-hydroxybutyrate dehydrogenases (BDH1 and BDH2) were greatly influenced by culture conditions when Ralstonia pickettii T1, a strain growing on extracellular poly-3-hydroxybutyrate (PHB), was grown on different carbon sources such as 3HB and succinate. In this study, knockout mutants of bdh1 or bdh2 were constructed and characterized under different culture conditions. In addition, a novel BDH (BDH3) was found in bdh2 mutants, and bdh3 was cloned. Apparent kinetic parameters for the substrates of BDH3 indicated that the enzyme is suitable for the oxidation reaction of 3-hydroxybutyrate (3HB) to acetoacetate. In Western blotting, it was clear that BDH3 is produced only in cells grown on 3HB or PHB as a carbon source, while BDH1 and BDH2 are produced in cells grown on various carbon sources such as sugars, amino acids, organic acids, 3HB, and PHB. Both the bdh1 and bdh2 mutants lagged behind the wild type in growth rates when the cells were cultured with 3HB, citrate, succinate, or nutrient broth. A test of sensitivity to diamide as an oxidative stress revealed that the lack of BDH1 or BDH2 caused a decline in the capacity to neutralize the stress. These results suggested that BDH1 and BDH2 are needed to regulate the cytoplasmic redox state as well as to utilize 3HB, while BDH3 is specialized to utilize 3HB. The expression of bdh3 may be coordinately regulated with a gene encoding putative 3HB permease.

Secretion pathway for the poly(3-hydroxybutyrate) depolymerase in Ralstonia pickettii T1

The extracellular poly(3-hydroxybutyrate) depolymerase from Ralstonia pickettii T1 has been purified, its function and character investigated in detail, and its gene cloned and sequenced. However, the mechanism by which this enzyme is secreted has not been elucidated. A mutant unable to degrade poly(3-hydroxybutyrate), N17, was obtained with the random insertion of a mini-transposon, Tn5. Western analysis using antiserum against the poly(3-hydroxybutyrate) depolymerase of Ralstonia pickettii T1, revealed that N17 accumulated the poly(3-hydroxybutyrate) depolymerase in the periplasm and cytoplasm, and did not secrete the enzyme into the external medium. It was also found that 3-hydroxybutyrate-oligomer hydrolase was secreted but inactive. The disrupted gene in N17, depO, was analyzed by Southern hybridization and its nucleotide sequence was determined. One complete open reading frame was found in the cloned 2.3-kbp DNA fragment. From a BLAST search, this gene product was found to be homologous to PulO of Ralstonia eutropha JMP134 (60% identity) and XcpA of Pseudomonas aeruginosa (60% identity). These proteins are prepilin peptidase/N-metyltransferases, a component of the Type II secretion pathway. DepO also had the four cysteines highly conserved in most prepilin peptidases at the same positions. The transcript of depO was examined by Northern hybridization using depO as a probe. In the total RNA of Ralstonia pickettii T1 in the early stationary phase, a band at 2.6-kb was detected, suggesting depO to be a functional gene. In this study, it was found that poly(3-hydroxybutyrate) depolymerase was secreted by the Type II pathway.

Crystalline-Structure-Dependent Enzymatic Degradation of Polymorphic Poly(3-hydroxypropionate)

The crystalline structure dependence of enzymatic degradation behavior was investigated for the polymorphic poly(3-hydroxypropionate) (P3HP), which has a basic backbone chemical structure of bacterial poly(3-hydroxyalkanoate)s (P3HAs). The P3HP films consisting of the beta-, gamma-, and/or delta-form crystal were cast or melt-crystallized as reported previously (Macromolecules 2005, 38, 6455; Macromolecules 2006, 39, 194-203) by controlling the molecular weight, crystallization temperature, and/or temperature of the melt. Their thermal properties, crystalline structures, morphologies, and (13)C solid spin-lattice relaxation dynamics were characterized by the differential scanning calorimetry, the wide-angle X-ray diffraction, the small-angle X-ray scattering (SAXS), and the (13)C solid-state NMR spectra (SNMR), respectively. Both the crystallinities and the lamellar thicknesses of P3HP films were found to decrease roughly in the order of beta-form > (or approximately) gamma-form > delta-form. From previous work, which indicates that the P3HA enzymatic degradation depends only on the degree of crystallinity and the lamellar thickness, their enzymatic degradation rates are then expected to increase in the order of beta-form < (or approximately) gamma-form < delta-form. Unexpectedly, their experimental P3HP enzymatic degradation rates in the presence of P3HA depolymerase isolated from Ralstonia pickettii T1 increase in the reverse order, i.e., delta-form < gamma-form < beta-form. The weight loss rate of the delta-form film is almost 1 order of magnitude smaller than that of the fastest degraded beta-form film. It is then strongly indicated that the crystalline structure plays a strikingly decisive role in the enzymatic degradation of P3HP. In particular, only when the conformation of crystalline chain accords with that of the bacterial poly(3-hydroxybutyrate) (P3HB) sample, i.e., the 2 1 helix conformation, is the P3HP sample degraded as slow as the P3HB sample. The inherent reason responsible for the unique P3HP enzymatic degradation behavior has been further clarified by comparing the molecular interaction and dynamics of polymorphic P3HP crystals.

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.

Adsorption and Hydrolysis Reactions of Poly(hydroxybutyric acid) Depolymerases Secreted from Ralstonia pickettii T1 and Penicillium funiculosum onto Poly[(R)-3-hydroxybutyric acid

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.

Mechanical properties and enzymatic degradation of poly[( R)-3-hydroxybutyrate] fibers stretched after isothermal crystallization near Tg

Poly[( R)-3-hydroxybutyrate] (P(3HB)) fibers with high tensile strength were prepared by stretching the fibers after isothermal crystallization near the glass transition temperature. Two samples with different molecular weights ( M w = 0.7 × 10 6 and 4.3 × 10 6) were used to investigate the effect on tensile strength. Increasing the time for isothermal crystallization of P(3HB) fibers resulted in a decrease in the maximum draw ratio. But, the tensile strength of P(3HB) fibers increased remarkably when the isothermal crystallization time was prolonged to more than 24 h. The tensile strength of low-molecular-weight drawn fibers was higher than that of high-molecular-weight fibers. Therefore, it can be concluded that this procedure does not increase the tensile strength of the high-molecular-weight drawn fibers. This is because, in this drawing method, small crystal nuclei grow initially during the isothermal crystallization process. Then, the molecular chains between the small crystal nuclei that acted as the entanglement points are oriented by stretching. In the case of the high-molecular-weight fibers, because the molecular length between the entanglement points of the small crystal nuclei is too long, the molecular chains are not sufficiently oriented by the stretching process. However, in the case of the low-molecular-weight fibers, the molecular length is suitable for generating the extended chains. Based on the result of X-ray analysis of P(3HB) fibers stretched after isothermal crystallization, fibers have the oriented α-form crystal with 2 1 helix conformation and β-form with planar zigzag conformation. The enzymatic degradation of the stretched P(3HB) fibers was performed by using an extracellular PHB depolymerase purified from Ralstonia pickettii T1. The enzymatic erosion rate of β-form was faster than that of α-form in the P(3HB) fibers stretched after isothermal crystallization.

Microbeam X-ray Diffraction and Enzymatic Degradation of Poly[(R)-3-hydroxybutyrate] Fibers with Two Kinds of Molecular Conformations

A new core−sheath structure constructed by two kinds of molecular conformations2/1 helix (α-form) and planar zigzag (β-form) conformationsin biodegradable poly[(R)-3-hydroxybutyrate] (P(3HB)) fiber was revealed by 0.5 μm microbeam X-ray diffraction in synchrotron radiation. P(3HB) fiber processed by a method combining cold-drawing and two-step-drawing has both α-form and β-form molecular chains in the core region, while the sheath region consists of only α-form chains. The crystallinity and orientation of α-form crystals in the core region were higher than those in the sheath region. Microbeam X-ray diffraction during two-step-drawing of cold-drawn fiber at room temperature revealed the generation mechanism of this interesting core−sheath structure at the highly ordered structural level. The β-form in core region was generated from amorphous chains between α-form lamellar crystals by the strong stretching of two-step-drawing. However, in the sheath region, since the α-form lamellar crystals rotated along the two-step-drawing direction, molecular chains between lamellar crystals could not elongate sufficiently. The enzymatic degradation of P(3HB) fibers was performed with an extracellular poly(hydroxybutyrate) depolymerase purified from Ralstonia pickettii T1. The intensity of a reflection derived from the β-form decreased faster than that of the α-form in X-ray fiber diagram, despite the β-form existing in the core region. The enzymatic degradation progressed from (1) the amorphous chains between α-form lamellar crystals in the sheath region, (2) the β-form molecular chains in the core region, and (3) the α-form lamellar crystals in the whole fiber. The result that the rate of enzymatic erosion of the β-form was faster than that of the α-form indicates that the rate of enzymatic degradation can be controlled by the molecular conformation, despite the same chemical structure.

Characterization of two 3-hydroxybutyrate dehydrogenases in poly(3-hydroxybutyrate)-degradable bacterium, Ralstonia pickettii T1

Two D-(-)-3-hydroxybutyrate (3HB) dehydrogenases, BDH1 and BDH2, were isolated and purified from a poly(3-hydroxybutyrate) (PHB)-degradable bacterium, Ralstonia pickettii T1. BDH1 activity increased in R. pickettii T1 cells grown on several organic acids as a carbon source but not on 3HB, whereas BDH2 activity markedly increased in the same cells grown on 3HB or PHB. To examine their biochemical properties, bdh1 and bdh2 were cloned and overexpressed in Escherichia coli, and their purified products were characterized. The kinetic parameters indicate that BDH1 is more suitable for converting acetoacetate to 3HB than BDH2, whereas BDH2 is more efficient for the reverse reaction than BDH1. Thus, R. pickettii T1 contains two BDHs with different biochemical properties and physiological roles: BDH1 for cell growth on organic acids other than 3HB and BDH2 for cell growth on 3HB or PHB.

Enzymatic Hydrolysis of Thioester Linkages in Bacterial Poly(3-hydroxybutyrate-co-3-mercaptopropionate)s by Poly(3-hydroxybutyrate) Depolymerase Isolated from Ralstonia pickettii T1

Enzymatic Hydrolysis of Thioester Linkages in Bacterial Poly(3-hydroxybutyrate- co -3-mercaptopropionate)s by Poly(3-hydroxybutyrate) Depolymerase Isolated from Ralstonia pickettii T1

Dynamic Adsorption Behavior of Poly(3-hydroxybutyrate) Depolymerase onto Polyester Surface Investigated by QCM and AFM

Time-dependent adsorption behavior of poly(3-hydroxybutyrate) (PHB) depolymerase from Ralstonia pickettiiT1 on a polyester surface was studied by complementary techniques of quarts crystal microbalance (QCM) and atomic force microscopy (AFM). Amorphous poly(l-lactide) (PLLA) thin films were used as adsorption substrates. Effects of enzyme concentration on adsorption onto the PLLA surface were determined time-dependently by QCM. Adsorption of PHB depolymerase took place immediately after replacement of the buffer solutions with the enzyme solutions in the cell, followed by a gradual increase in the amount over 30 min. The amount of PHB depolymerase molecules adsorbed on the surface of amorphous PLLA thin films increased with an increase in the enzyme concentration. Time-dependent AFM observation of enzyme molecules was performed during the adsorption of PHB depolymerase. The phase response of the AFM signal revealed that the nature of the PLLA surface around the PHB depolymerase molecule was changed due to the adsorption function of the enzyme and that PHB depolymerase adsorbed onto the PLLA surface as a monolayer at a lower enzyme concentration. The number of PHB depolymerase molecules on the PLLA surface depended on the enzyme concentration and adsorption time. In addition, the height of the adsorbed enzyme was found to increase with time when the PLLA surface was crowded with the enzymes. In the case of higher enzyme concentrations, multilayered PHB depolymerases were observed on the PLLA thin film. These QCM and AFM results indicate that two-step adsorption of PHB depolymerase occurs on the amorphous PLLA thin film. First, adsorption of PHB depolymerase molecules takes place through the characteristic interaction between the binding domain of PHB depolymerase and the free surface of an amorphous PLLA thin film. As the adsorption proceeded, the surface region of the thin film was almost covered with the enzyme, which was accompanied by morphological changes. Second, the hydrophobic interactions among the enzymes in the adlayer and the solution become more dominant to stack as a second layer.

Direct observation of poly(3-hydroxybutyrate) depolymerase adsorbed on polyester thin film by atomic force microscopy.

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerases adsorbed on poly(L-lactide) (PLLA) thin film were directly observed by atomic force microscopy (AFM). A PLLA thin film of 100 nm thickness was prepared on a silicon wafer by spin-cast method. The PLLA thin film was treated at 220 degrees C and quenched to room temperature, resulting in the formation of a completely amorphous film with a smooth surface. Then, the PHB depolymerases from Pseudomonas stutzeri YM1006 and Ralstonia pickettii T1 were dispersed on the amorphous PLLA thin film. Direct AFM observation has revealed that the PHB depolymerases bind in an elliptic shape on the surface of the PLLA thin film and that a small ridge is created around each enzyme molecule. After removal of the enzymes with 40% ethanol aqueous solution, small hollows were found on the PLLA thin film. These results suggest that a PHB depolymerase interacts with polyester molecules during their adsorption to make a hollow on the substrate surface.

Roles of poly(3-hydroxybutyrate) depolymerase and 3HB-oligomer hydrolase in bacterial PHB metabolism.

Many poly-3-hydroxybutyrate (PHB)-degrading enzymes have been studied. But biological roles of 3HB-oligomer hydrolases (3HBOHs) and how PHB depolymerases (PHBDPs) and 3HBOHs cooperate in PHB metabolism are not fully elucidated. In this study, several PHBDPs and 3HBOHs from three types of bacteria were purified, and their substrate specificity, kinetic properties, and degradation products were investigated. From the results, PHBDP and 3HBOH seemed to play a role in PHB metabolism in three types of bacteria, as follows: (A) In Ralstonia pickettii T1, an extracellular PHBDP degrades extracellular PHB to various-sized 3HB-oligomers, which an extracellular 3HBOH hydrolyzes to 3HB-monomers. (B) In Acidovorax sp. SA1, an extracellular PHBDP hydrolyzes extracellular PHB to small 3HB-oligomers (dimer and trimer), which an intracellular 3HBOH efficiently degrades to 3HB in the cell. (C) In Ralstonia eutropha H16, an intracellular 3HBOH helps in the degradation of intracellular PHB inclusions by PHBDP.

Enzymatic degradation behavior of comonomer compositionally fractionated bacterial poly(3-hydroxybutyrate -co-3-hydroxyvalerate)s by poly(3-hydroxyalkanoate) depolymerases isolated from Ralstonia pickettii T1 and Acidovorax sp. TP4

The enzymatic hydrolysis behavior of compositionally well-fractionated bacterial poly(3-hydroxybutyrate- co-3-hydroxyvalerate) [P(3HB- co-3HV)] with 3HV-unit content from 8 to 98 mol% and bacterial poly(3-hydroxybutyrate) [P(3HB)] has been investigated in the presence of poly(3-hydroxyalkanoate) depolymerases isolated from Ralstonia pickettii T1 and Acidovorax sp. TP4. The water-soluble degradation products were characterized by HPLC and 1H NMR spectroscopy. P(3HB- co-3HV)s with the whole range of 3HV-unit content can be degraded by R. pickettii T1 depolymerase, while only P(3HB- co-3HV)s with 3HV-unit content less than 80 mol% can be degraded by Acidovorax sp. TP4 depolymerase. It was found that the enzymatic degradation behavior of fractionated P(3HB- co-3HV)s was affected not only by the comonomer unit composition and its distribution but also by the solid-state structure of P(3HB- co-3HV)s and further by the bacterial source of depolymerases. Based on the results, the mechanism of enzymatic degradation was discussed.

Mechanical properties and enzymatic degradation of poly([ R]-3-hydroxybutyrate- co-[ R]-3-hydroxyhexanoate) uniaxially cold-drawn films

Uniaxially oriented films of poly([ R]-3-hydroxybutyrate- co-[ R]-3-hydroxyhexanoate) (P(3HB- co-3HH)) with 5 and 12 mol% 3HH comonomer were prepared by cold-drawing from an amorphous preform 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%. Subsequent annealing under tension led to improvement of the tensile strength and Young's modulus. Two step drawing of P(3HB- co-5%-3HH) films resulted in further improvement of the mechanical properties. The X-ray fiber diagrams showed increasing orientation and crystallinity of the films with draw ratio. The mechanical properties remained unchanged after storing for several months at room temperature, suggesting that high orientation and crystallinity suppress the secondary crystallization. Cold-drawn P(3HB- co-3HH) films were enzymatically degraded with an extracellular PHB depolymerase purified from Ralstonia pickettii T1. The enzymatic degradation was investigated by means of weight-loss profiles and scanning electron microscopy.