Thermal degradation of poly(3-hydroxyalkanoates): preparation of well-defined oligomers

The thermal degradation of the biodegradable bacterial polyesters poly(3-hydroxybutyrate), PHB, poly(3-hydroxyvalerate), PHV, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), 0-21 mol % of hydroxyvalerate, was studied. At moderately low temperatures (170-200 degrees C), the main product is a well-defined oligomer, especially a 500-10,000 g/mol macromolecule, which contains one unsaturated end group, predominantly a trans-alkenyl end group, as well as a carboxylic end group. The process was studied regarding the effect of the copolymer composition and reaction time at 190 degrees C. During the first few hours of reaction, the thermal degradation of PHB and PHV followed a kinetic model of random scission, but eventually auto-acceleration of the pyrolysis was detected, probably due to the influence of the crotonate end groups of the oligomers formed. Ten-time scale-up experiments on a Brabender instrument were successfully undertaken.     

New biodegradable thermogelling copolymers having very low gelation concentrations

New biodegradable multiblock amphiphilic and thermosensitive poly(ether ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] (PHB), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) blocks were synthesized, and their aqueous solutions were found to undergo a reversible sol-gel transition upon temperature change at very low copolymer concentrations. The multiblock poly(ether ester urethane)s were synthesized from diols of PHB, PEG, and PPG using 1,6-hexamethylene diisocyanate as a coupling reagent. The chemical structures and molecular characteristics of the copolymers were studied by GPC, 1H NMR, 13C NMR, and FTIR. The thermal stability of the poly(PEG/PPG/PHB urethane)s was studied by thermogravimetry analysis (TGA), and the PHB contents were calculated based on the thermal degradation profile. The results were in good agreement with those obtained from the 1H NMR measurements. The poly(PEG/PPG/ PHB urethane)s presented better thermal stability than the PHB precursors. The water soluble poly(ether ester urethane)s had very low critical micellization concentration (CMC). Aqueous solutions of the new poly(ether ester urethane)s underwent a sol-gel-sol transition as the temperature increased from 4 to 80 degrees C, and showed a very low critical gelation concentration (CGC) ranging from 2 to 5 wt %. As a result of its multiblock architecture, a novel associated micelle packing model can be proposed for the sol-gel transition for the copolymer gels of this system. The new material is thought to be a promising candidate for injectable drug systems that can be formulated at low temperatures and forms a gel depot in situ upon subcutaneous injection.     

A themogravimetric analysis for poly(3-hydroxybutyrate) quantification

Summary A simple and quick thermogravimetric analysis method has been suggested for poly(3-hydroxybutyrate) [PHB] quantification. During the analysis, a rapid thermal degradation of PHB occurred in the range of 250 to 320 °C. From the gravimetric change during the thermal degradation, we could quantify PHB contents of various samples. Due to the simultaneous thermal degradation of cellular materials, the PHB contents were estimated slightly higher than those by gas chromatographic analysis. We have proposed a way to compensate for such errors using a linear correlation to allow accurate determination of PHB contents.     

Chemical recycling of polyhydroxyalkanoates as a method towards sustainable development

Chemical recycling of bio-based polymers polyhydroxyalkanoates (PHAs) by thermal degradation was investigated from the viewpoint of biorefinery. The thermal degradation resulted in successful transformation of PHAs into vinyl monomers using alkali earth compound (AEC) catalysts. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s (PHBVs) were smoothly and selectively depolymerized into crotonic (CA) and 2-pentenoic (2-PA) acids at lower degradation temperatures in the presence of CaO and Mg(OH)₂ as catalysts. Obtained CA from 3-hydroxybutyrate sequences in PHBV was copolymerized with acrylic acid to produce useful water-soluble copolymers, poly(crotonic acid-co-acrylic acid) that have high glass-transition temperatures. The copolymerization of CA derived from PHA pyrolysis is an example of cascade utilization of PHAs, which meets the idea of sustainable development.     

聚羟基烷酸酯 (PHA) 改性研究进展

本文简述了生物制造聚羟基烷酸酯(PHA),包括聚3-羟基丁酸酯(PHB)、聚(3-羟基丁酸酯-3-羟基戊酸酯)(PHBV)、聚(3-羟基丁酸酯-4-羟基丁酸酯)(P3/4HB)、聚(3-羟基丁酸酯-3-羟基己酸酯)(PHBH)的产业化现状,综述了针对PHA材料热稳定性差、加工窗口较窄等缺点而进行的一些改性研究。选用适当方法对PHA进行改性,可使其性能得到优化,应用领域得到拓展。     

Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis

Purification of poly(3-hydroxybutyrate) depolymerase (EC 3.1.1.75) from Paucimonas lemoignei is complicated because the bacterium produces several isoenzymes which are difficult to separate from each other. The phaZ5 gene of P. lemoignei encoding extracellular poly(3-hydroxybutyrate) depolymerase A was functionally expressed from the constitutive P43 promoter of pWB980 in a multiple protease-negative mutant of Bacillus subtilis (strain WB800) and secreted to the culture medium. The depolymerase (apparent M(r), 42 kDa; 1.9 mg purified protein per liter culture) was purified from cell-free culture fluid to homogenity by applying only one chromatography step in comparison to at least two necessary steps if poly(3-hydroxybutyrate) depolymerases are purified from P. lemoignei. The recombinant depolymerase lacked any carbohydrate content in contrast to the glycosylated depolymerase of the wild-type. Glycosylation was not essential for activity but enhanced the thermal stability of the enzyme at high temperature. Overexpression of poly(3-hydroxybutyrate) depolymerase in B. subtilis is more efficient than in Escherichia coli.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9.

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

THROMBIN IMMOBILIZATION TO METHACRYLIC ACID GRAFTED POLY(3-HYDROXYBUTYRATE) AND ITS IN VITRO APPLICATION

Poly(3-hydroxybutyrate) is nontoxic and biodegradable, with good biocompatibility and potential support for long-term implants. For this reason, it is a good support for enzyme immobilization. Enzyme immobilization could not be done directly because poly(3-hydroxybutyrate) has no functional groups. Therefore, modification should be done for enzyme immobilization. In this study, methacrylic acid was graft polymerized to poly(3-hydroxybutyrate) and thrombin was immobilized to polymethacrylic acid grafted poly(3-hydroxybutyrate). In fact, graft polymerization of methacrylic acid to poly(3-hydroxybutyrate) and thrombin immobilization was a model study. Biomolecule immobilized poly(3-hydroxybutyrate) could be used as an implant. Thrombin was selected as a biomolecule for this model study and it was immobilized to methacrylic acid grafted poly(3-hydroxybutyrate). Then the developed product was used to stop bleeding.     

Synthesis, properties, and biological activity of poly[di(sodium carboxylatoethylphenoxy)phosphazene

A new water-soluble polyphosphazene polyelectrolyte containing carboxylate functionalities, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) was synthesized via reaction of macromolecular substitution. The polymer was characterized using (1)H, (31)P NMR, and gel permeation chromatography with multiangle laser light scattering detection. PCEP was shown to undergo hydrolytic degradation in aqueous solutions, as indicated by the decrease in the molecular weight and the release of side groups. A series of incompletely substituted copolymers of PCEP containing varying amounts of residual chlorine atoms was also prepared. The rate of degradation for such copolymers increased with the rise in the content of chlorine atoms. In vivo studies demonstrated high potency of PCEP as a vaccine immunoadjuvant. The new polyphosphazene was also shown to be capable of forming microspheres in aqueous solutions via reactions of ionic complexation with physiologically occurring amines, such as spermine.     

Enzymatic hydrolysis of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4

Enzymatic degradability has been investigated for a series of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s (P(3HB-co-3HP)s) with 3-hydroxypropionate (3HP) unit contents from 11 to 86 mol % as well as poly(3-hydroxybutyrate) (P(3HB)) and chemosynthesized poly(3-hydroxypropionate) (P(3HP)). The behavior of degradation by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pikettii T1 and Acidovorax Sp. TP4, defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were compared in relation to the thermal properties and crystalline structures of the PHA samples elucidated by differential scanning calorimetry and wide-angle X-ray diffraction. The degradation products were characterized by high-performance liquid chromatography and one- (1D) and two-dimension (2D) (1)H NMR spectroscopy. It was found that the PHA depolymerase of Acidovorax Sp. TP4 showed degradation behavior different from that shown by depolymerase of R. pikettii T1. PHA depolymerase from Acidovorax Sp. TP4 degraded the P(3HB-co-3HP) films with lower crystallinity in higher rates than those with higher crystallinity, no matter what kinds of crystalline structures they formed. In contrast, PHA depolymerase from R. pikettii T1 degraded P(3HB-co-3HP) films forming P(3HB) crystalline structure in higher rates than those forming P(3HP)s. The increase in amorphous nature of the P(3HB-co-3HP) films with P(3HB)-homopolymer-like crystalline structure increases and then decreases the rate of degradation by depolymerase from R. pikettii T1. The 3-hydroxybutyrate (3HB) monomer was produced as a major product by the hydrolysis of P(3HB) film by PHA depolymerase from Acidovorax Sp. TP4. The P(3HB-co-3HP) films could be degraded into 3HB and 3-hydroxypropionate (3HP) monomer at last, indicating that the catalytic domain of the enzyme recognized at least two monomeric units as substrates. While the PHA depolymerase from R. pikettii T1 hydrolyzed P(3HB) film into 3HB dimer as a major product, and the catalytic domain recognized at least three monomeric units. The degradation behavior of P(3HB-co-3HP) films by the PHA depolymerase of Acidovorax Sp. TP4 could be distinguished from that by the depolymerase of R. pikettii T1.     

Hydrolytic Degradation of Poly(3-Hydroxybutyrate) and Its Copolymer with 3-Hydroxyvalerate of Different Molecular Weights in vitro

The hydrolytic degradation of polymer films of poly(3-hydroxybutyrate) of different molecular weights and its copolymers with 3-hydroxyvalerate (9 mol % 3-hydroxyvalerate in the poly(3-hydroxybutyrate) chain) of different molecular weights was studied in model conditions in vitro. The changes in the physicochemical properties of the polymers were investigated using different analytical techniques: viscometry, differential scanning calorimetry, gravimetrical method, and water contact angle measurement for polymers. The data showed that in a period of 6 months the weight of polymer films decreased insignificantly. The molecular weight of the samples was reduced significantly; the largest decline (up to 80% of the initial molecular weight of the polymer) was observed in the high-molecular-weight poly(3-hydroxybutyrate). The surface of all investigated polymers became more hydrophilic. In this work, we focus on a mathematical model that can be used for the analysis of the kinetics of hydrolytic degradation of poly(3-hydroxyaklannoate)s by noncatalytic and autocatalytic hydrolysis mechanisms. It was also shown that the degree of crystallinity of some polymers changes differently during degradation in vitro. Thus, the studied polymers can be used to develop biodegradable medical devices such that they can perform their functions for a long period of time.     

Kinetics and mechanism of heterogeneous hydrolysis of poly[(R)-3-hydroxybutyrate] film by PHA depolymerases

The kinetics and mechanism of enzymatic degradation on the surface of poly[(R)-3-hydroxybutyrate] (P[(R)-3HB]) film have been studied using three types of extracellular poly(hydroxyalkanoate) (PHA) depolymerases from Alcaligenes faecalis, Pseudomonas pickettii and Comamonas testosteroni. The monomer and dimer of 3-hydroxybutyric acid were produced during the course of the enzymatic degradation of P[(R)-3HB] film, and the rate of production was determined by monitoring the increase in absorbance at 210 nm on a spectrophotometer. The rate of enzymatic degradation increased to a maximum value with the concentration of PHA depolymerase, followed by a gradual decrease. The kinetic data were accounted for in terms of a heterogeneous enzymatic reaction, involving enzymatic degradation on the surface of P[(R)-3HB] film via two steps of adsorption and hydrolysis by a PHA depolymerase with binding and catalytic domains. The kinetic results suggest that the properties of the catalytic domains are very similar among the three PHA depolymerases, but that those of the binding domains are strongly dependent on the type of depolymerase.     

Poly(ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] and poly(ethylene glycol) as candidate biomaterials: characterization and mechanical property study

Poly(ester urethane)s with poly[(R)-3-hydroxybutyrate] (PHB) as the hard and hydrophobic segment and poly(ethylene glycol) (PEG) as the soft and hydrophilic segment were synthesized from telechelic hydroxylated PHB (PHB-diol) and PEG using 1,6-hexamethylene diisocyanate as a nontoxic coupling reagent. Their chemical structures and molecular characteristics were studied by gel permeation chromatography, 1H NMR, and Fourier transform infrared spectroscopy. Results of differential scanning calorimetry and X-ray diffraction indicated that the PHB segment and PEG segment in the poly(ester urethane)s formed separate crystalline phases with lower crystallinity and a lower melting point than those of their corresponding precursors, except no PHB crystalline phase was observed in those with a relatively low PHB fraction. Thermogravimetric analysis showed that the poly(ester urethane)s had better thermal stability than their precursors. The segment compositions were calculated from the two-step thermal decomposition profiles, which were in good agreement with those obtained from 1H NMR. Water contact angle measurement and water swelling analysis revealed that both surface hydrophilicity and bulk hydrophilicity of the poly(ester urethane)s were enhanced by incorporating the PEG segment into PHB polymer chains. The mechanical properties of the poly(ester urethane)s were also assessed by tensile strength measurement. It was found that the poly(ester urethane)s were ductile, while natural source PHB is brittle. Young's modulus and the stress at break increased with increasing PHB segment length or PEG segment length, whereas the strain at break increased with increasing PEG segment length or decreasing PHB segment length.     

Isolation of poly(3-hydroxybutyrate) (PHB)-degrading microorganisms and characterization of PHB-depolymerase from Arthrobacter sp. strain W6.

Microbial degraders of poly(3-hydroxybutyrate) (PHB) were isolated from soil. Arthrobacter sp. strain W6 used not only PHB as a carbon source, but also PHAs such as poly(3-hydroxybutyrate-co-[5%]3-hydroxyvalerate), poly(3-hydroxybutyrate-co-[14%]3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-[22%]3-hydroxyvalerate). PHB-depolymerase was purified to homogeneity from the culture broth of Arthrobacter sp. strain W6 by a procedure involving DEAE- and butyl-Toyopearl column chromatographies. The Mr of the enzyme was estimated to be about 47,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 8.5 and 50 degrees C, and was inhibited by phenylmethylsulfonyl fluoride, Hg2+, Ag+, and Pb2+.     

New poly(3-hydroxybutyrate)-based systems for controlled release of dipyridamole and indomethacin

New poly(3-hydroxybutyrate)-based systems for controlled release of anti-inflammatory and anti-thrombogenic drugs have been studied. The release occurs via two mechanisms (diffusion and degradation) operating simultaneously. Dipyridamole and indomethacin diffusion processes determine the rate of the release at the early stages of the contact of the system with the environment (the first 6–8 h). The coefficient of the release diffusion of a drug depends on its nature, the thickness of the poly(3-hydroxybutyrate) films containing the drug, the concentrations of dipyridamole and indomethacin, and the molecular weight of the poly(3-hydroxybutyrate). The results obtained are critical for developing systems of release of diverse drugs, thus, enabling the attainment of the requisite physiological effects on tissues and organs of humans.     

Cyclohexane carboxylate and benzoate formation from crotonate in Syntrophus aciditrophicus

The anaerobic, syntrophic bacterium Syntrophus aciditrophicus grown in pure culture produced 1.4 +/- 0.24 mol of acetate and 0.16 +/- 0.02 mol of cyclohexane carboxylate per mole of crotonate metabolized. [U-13C]crotonate was metabolized to [1,2-(13)C]acetate and [1,2,3,4,5,7-(13)C]cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [13C]sodium bicarbonate formed [6-(13)C]cyclohexane carboxylate. Trimethylsilyl (TMS) derivatives of cyclohexane carboxylate, cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates by comparison of retention times and mass spectral profiles to authentic standards. With [U-(13)C]crotonate, the m/z-15 ion of TMS-derivatized glutarate, 3-hydroxybutyrate, and acetoacetate each increased by +4 mass units, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +6 mass units. With [13C]sodium bicarbonate and unlabeled crotonate, the m/z-15 ion of TMS derivatives of glutarate, pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +1 mass unit, suggesting that carboxylation occurred after the synthesis of a four-carbon intermediate. With [1,2-(13)C]acetate and unlabeled crotonate, the m/z-15 ion of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, respectively, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4, and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO2 addition, rather than the use of the intact four-carbon skeleton of crotonate.     

Biodegradable films of partly branched poly(l-lactide)-co-poly(epsilon-caprolactone) copolymer: modulation of phase morphology, plasticization properties and thermal depolymerization

We report on the modulation of phase morphology, plasticization properties, and thermal stability of films of partly branched poly(l-lactide)-co-poly(epsilon-caprolactone) copolymer (PLLA-co-PCL) with additions of low molecular weight compounds, namely, triethyl citrate ester, diethyl phthalate, diepoxy polyether (poly(propylene glycol) diglycidyl ether), and with epoxidized soybean oil (ESO). The PLLA-co-PCL/polyether films showed significant stability against thermal depolymerization, high film flexibility, and good plasticizing properties, probably due to cross-linking and chain branching formation between diepoxy groups with both the end carboxyl and hydroxyl groups of the PLLA copolymer (initially present or generated during the degradation process) to produce primary ester and ether bonds, respectively. Diethyl phthalate and triethyl citrate ester were found to be efficient plasticizers for PLLA copolymer in terms of glass transition and mechanical properties, but the more water-soluble plasticizer triethyl citrate induced a dramatic loss in the molecular weight of the copolymer. Although ESO cannot play the role of a plasticizer, it substantially stabilizes and retards thermal depolymerization of the PLLA copolymer matrix, possibly because of a reaction between epoxy groups with the end carboxyl and hydroxyl groups of the PLLA copolymer. The presence of ESO in PLLA-co-PCL/ESO/triethyl citrate blends enhanced the compatibility and miscibility of the plasticizer with the PLLA copolymer matrix, considerably improved the mechanical properties (elongation at break), and substantially stabilized the copolymer against thermal depolymerization. It seems likely that the epoxy groups interact not only with the end hydroxyl and carboxyl group of the copolymer but as well with the hydroxyl group of triethyl citrate plasticizer to produce a new ether bond (C-O-C) as the cross-linking unit. On the other hand, for PLLA-co-PCL/ESO/polyether blends, (80/10/10) epoxidized oil distorts the compactness of the blend by diminishing the proposed entanglements between carboxyl, hydroxyl, and diepoxy groups of polyether and reduces the high elongation properties otherwise observed in the PLLA-co-PCL/polyether films. The multicomponent approach toward modulating poly(l-lactide)-co-poly(epsilon-caprolactone) copolymer films using epoxy compounds and plasticizers and the insight into the nature of various PLLA matrixes presented here offer advantages to a broad engineering of PLLA copolymer films having desirable physical properties and multiphase behavior for efficient uses in future technical applications.