Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in soils.

The microbial degradation of tensile test pieces made of poly(3-hydroxybutyrate) [P(3HB)] or a copolymer of 90% 3-hydroxybutyric acid and 10% 3-hydroxyvaleric acid was studied in soils incubated at a constant temperature of 15, 28, or 40 degrees C for up to 200 days. In addition, hydrolytic degradation in sterile buffer at temperatures ranging from 4 to 55 degrees C was monitored for 98 days. Degradation was measured through loss of weight (surface erosion), molecular weight, and mechanical strength. While no weight loss was recorded in sterile buffer, samples incubated in soils were degraded at an erosion rate of 0.03 to 0.64% weight loss per day, depending on the polymer, the soil, and the incubation temperature. The erosion rate was enhanced by incubation at higher temperatures, and in most cases the copolymer lost weight at a higher rate than the homopolymer. The molecular weights of samples incubated at 40 degrees C in soils and those incubated at 40 degrees C in sterile buffer decreased at similar rates, while the molecular weights of samples incubated at lower temperatures remained almost unaffected, indicating that molecular weight decrease is due to simple hydrolysis and not to the action of biodegrading microorganisms. The degradation resulted in loss of mechanical properties. From the samples used in the biodegradation studies, 295 dominant microbial strains capable of degrading P (3HB) and the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer in vitro were isolated and identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro biocompatibility of electrospun poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fiber mats

In the present contribution, the potential for use of the ultrafine electrospun fiber mats of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as scaffolding materials for skin and nerve regeneration was evaluated in vitro using mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) as reference cell lines. Comparison was made with PHB and PHBV films that were prepared by solution-casting technique. Indirect cytotoxicity assessment of the as-spun PHB and PHBV fiber mats with mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) indicated that the materials were acceptable to both types of cells. The attachment of L929 on all of the fibrous scaffolds was significantly better than that on both the film scaffolds and tissue-culture polystyrene plate (TCPS), while RT4-D6P2T appeared to attach on the flat surfaces of TCPS and the film scaffolds much better than on the rough surfaces of the fibrous scaffolds. For L929, all of the fibrous scaffolds were superior in supporting the cell proliferation to the film counterparts, but inferior to TCPS at days 3 and 5, while, for RT4-D6P2T, the rough surfaces of the fibrous scaffolds appeared to be very poor in supporting the cell proliferation when comparing with the smooth surfaces of TCPS and the film scaffolds. Scanning electron microscopy was also used to observe the behavior of both types of cells that were cultured on both the fibrous and the film scaffolds and glass substrate for 24 h.     

Anaerobic biodegradation of aliphatic polyesters: poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) and poly(epsilon-caprolactone)

Poly(3-hydroxybutyrate-co-3-hydroxyoctanoate), PHBO, represents a class of PHA copolymers that contain both short-chain-length and medium-chain-length repeat units. Radiolabeled and cold PHBO, containing 90 mol % 3-hydroxybutyrate and 10 mol % 3-hydroxyoctanoate were chemically synthesized using a new difunctional alkoxyzinc initiator. (14)C-PHBO was incubated with samples of anaerobic digester sludge, septage, freshwater sediment, and marine sediment under conditions resembling those in situ. In addition, it was incubated in laboratory-scale landfill reactors. (14)C-PCL (poly-epsilon-caprolactone) was incubated with anaerobic digester sludge and in landfill reactors. Biodegradation was determined by measuring generation of (14)CO(2) and (14)CH(4) resulting from mineralization of the radiolabeled polymers. PHBO was extensively mineralized in digester sludge, septage sediments, and the landfill reactors, with half-lives less than 30 days. PCL was not significantly mineralized in digester sludge over 122 days. In the landfill reactors, PCL mineralization was slow and was preceded by a long lag period (>200 days), suggesting that PCL mineralization is limited by its rate of hydrolysis. The results indicate that PHBO is practically biodegradable in the major anaerobic habitats that it may enter. In contrast, anaerobic biodegradation of PCL is less ubiquitous and much slower.     

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T₁ has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(−)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(−)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.     

Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate)-based biopolymer systems

Biodegradable biopolymers attract much attention in biology and medicine due to its wide application. The present review considers a biodegradable and biocompatible polymer of bacterial origin, poly(3-hydroxybutyrate), which has wide perspectives in medicine and pharmaceutics. It highlights basic properties of biopolymer (biodegradability and biocompatibility) and also biopolymer systems: various materials, devices and compositions based on the biopolymer. Application of poly(3-hydroxybutyrate)-based biopolymer systems in medicine as surgical implants, in bioengineering as cell culture scaffolds, and in pharmacy as novel drug dosage forms and drug systems are also considered.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Viscoelastic relaxations and thermal properties of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

3-Hydroxybutyrate-3-hydroxyvalerate (3HB-3HV) as well as 3-hydroxybutyrate-4-hydroxybutyrate (3HB-4HB) copolyesters have been investigated by differential scanning calorimetry, thermogravimetric analysis and dynamic mechanical spectroscopy, over a wide range of compositions (0-95 mol% 3HV; 0-82 mol% 4HB). Both series of isolated copolyesters are partially crystalline at all compositions. Quenched samples show a glass transition that decreases linearly with increasing co-monomer molar fraction, more markedly when the co-monomer is 4HB. Above Tg, all copolyesters, rich in 3HB units, show a cold crystallization phenomenon followed by melting, while at the other end crystallization on heating is observed only in 3HB-3HV copolymers. The viscoelastic spectrum, strongly affected by thermal history, shows two relaxation regions: the glass transition, whose location depends on copolymer type and composition, and a secondary dispersion region at low temperatures (-130/-80 degrees C). The latter results from a water-related relaxation analogous to that of P(3HB) and, in 3HB-4HB copolymers, from another overlapping absorption peak centered at -130 degrees C, attributed to local motion of the methylene groups in the linear 4HB units.     

Gamma-ray crosslinking of poly(3-hydroxyoctanoate-co-undecenoate)

The gamma-ray crosslinking of films made of poly(3-hydroxyoctanoate) containing undecenoate moieties (up to 33 mole%) were studied. X-ray diffraction, thermal analysis, dynamic mechanical analysis (DMA), solid state nuclear magnetic resonance (NMR) spectroscopy and degree of crosslinking (swelling analysis) as a function of irradiation dose were evaluated for treatments in air or in N(2) atmosphere. After uncrosslinked material was isolated by CHCl(3) extraction, solid state NMR data suggested that only a small percentage of the double bonds took part in the formation of irradiation crosslinks. Crosslinking in N(2) was more efficient than in air and a 20 kGy dose was sufficient for optimal crosslinking. The X-ray diffraction patterns of the polymer films were unaffected by moderate irradiation. The use of sodium hypochlorite to isolate poly(3-hydroxyoctanoate-co-undecenoate) samples resulted in partial chlorination of the double bonds and considerable depolymerization.     

Study of the interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)Pt(II) complex with poly(I), poly(C) and poly(I) x poly(C)

The interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)platinum(II) (Ptpyrr) with the polynucleotides poly(I), poly(C) and poly(I) x poly(C) acids was studied by circular dichroism, molecular fluorescence and (1)H NMR spectroscopies. Multivariate Curve Resolution, a factor analysis method, was applied for the analysis and interpretation of spectroscopic data obtained in mole ratio and kinetics studies. This procedure allows the determination of the number of different interaction complexes present during the experiments and the resolution of both concentration profiles and pure spectra for all of them. Two different interaction complexes were observed at the experimental conditions studied. The first one, at low Ptpyrr:polynucleotide ratio (r(Ptpyrr:poly)) values, corresponds to the interaction of Ptpyrr with hypoxanthine bases in the poly(I) moiety. This interaction leads to the destabilization and dissociation of the double-stranded conformation. The second complex was observed at higher r(Ptpyrr:poly) values and corresponds to the interaction of Ptpyrr to cytosine bases in poly(C) moiety. The formation of both complexes showed that the interaction of Ptpyrr with hypoxanthine bases occurred at the first stages of the reaction and with cytosine bases at longer reaction times. The results obtained show the utility of the Multivariate Curve Resolution approach for the analysis of data obtained by monitoring spectroscopically the interaction equilibria of platinum compounds with nucleic acids.     

FT-IR imaging spectroscopy of phase separation in blends of poly(3-hydroxybutyrate) with poly(L-lactic acid) and poly(epsilon-caprolactone)

The detection of phase separation and identification of miscibility in biopolymer blends is an important aspect for the improvement of their physical properties. In this article, the phase separation in blends of poly(3-hydroxybutyrate) (PHB) with poly(L-lactic acid) (PLA) and poly(epsilon-caprolactone) (PCL), respectively, has been studied as a function of the blend composition by FT-IR imaging spectroscopy. For both polymer blend systems, a miscibility gap has been found around the 50:50% (w/w) composition of the two components. Furthermore, the separating phases have been identified as blends of the two polymer components and their compositions could be determined from calibrations based on the spectra of the blends in the compositional range of miscibility. The data derived from FT-IR spectroscopic imaging were corroborated by additional DSC analyses and mechanical stress-strain measurements of polymer blend films, which exhibited a characteristic fracture behavior as a function of PHB composition.     

Oligomerization of activated derivatives of 3''-amino-3''-deoxyguanosine on poly(C) and poly(dC) templates.

3'-amino-3'-deoxyuridine reacts with the nucleoside 5'-phosphorimidazolides in aqueous solution to give dinucleoside phosphoramidates. The reactions are one to two orders of magnitude faster than the corresponding reactions of uridine. In the presence of poly(C) or poly(dC) it is known that guanosine-5'-phosphorimidazolide does not condense efficiently or regiospecifically. However, the introduction of a methyl group at the 2-position of the imidazole ring leads to efficient synthesis of long 3'-5'-linked oligomers. The corresponding imidazole derivatives of 3'-amino-3'-deoxyguanosine-5'-phosphate both condense on these templates to give virtually identical families of products. Our results suggest that the intrinsically greater nucleophilicity of the amine groups will permit a much wider range of efficient template-directed syntheses with 3'-amino-3'-deoxynucleoside derivatives than with the corresponding derivatives of the parent nucleosides.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

Preparation and hydrolytic degradation of semi-interpenetrating networks of poly(3-hydroxyundecenoate) and poly(lactide-co-glycolide)

Semi-interpenetrating networks (semi-IPNs), where poly(lactide-co-glycolide) (PLGA) molecules were entrapped in the crosslinked matrices of poly(3-hydroxyundecenoate) (PHU), were prepared by irradiating homogeneous solutions of PHU and PLGA in chloroform with UV light. Attenuated total reflectance infrared spectroscopy showed that the PLGA chains were entrapped in PHU networks. The semi-IPNs showed enhanced mechanical strength as the PLGA content increased. The semi-IPNs were incubated at 37 °C in a 0.01N NaOH solution, and the extent of hydrolytic degradation was investigated by monitoring changes in various parameters such as water uptake, pH, mass, and morphology. Hydrolysis of semi-IPNs were significantly affected by the presence of PLGA. A semi-IPN prepared from a 9:1 (by weight) mixture of PHU and PLGA lost 25% of its original weight in 12 weeks while a PHU sample containing no PLGA lost only 5% of its weight during the same period under identical conditions. The hydrolysis was most likely accelerated when the pH of the medium was lowered by the hydrolyzed products of PLGA, 2-hydroxyalkanoic acids. These results showed that hydrolysis of PHA could be enhanced by incorporating a second component that lowered the pH of the hydrolysis system.     

Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Large scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by Aeromonas hydrophila 4AK4 was examined in a 20,000 l fermentor. Cells were first grown using glucose as a carbon source, and polyhydroxyalkanoate (PHA) biosynthesis was triggered by the addition of lauric acid under conditions of limited nitrogen or phosphorus. When cells first grown in a medium containing 50 g glucose l(-1) were further cultivated after the addition of 50 g lauric acid l(-1) under phosphorus limitation, a final cell concentration, PHA concentration and PHA content of 50 g l(-1), 25 g l(-1), and 50 wt%, respectively, were obtained in 46 h, equivalent to PHA productivity of 0.54 g l(-1)t h(-1). The copolymer produced was found to be a random copolymer, and the 3HHx fraction was 11 mol%.     

Carboxylate-induced degradation of poly(3-hydroxybutyrate)s

This communication shows that thermal degradation of poly(3-hydroxybutyrate)s (PHBs) is induced by carboxylate groups via a newly proposed E1cB mechanism. In PHBs with end groups in the form of carboxylic acid salts with Na+, K+, and Bu4N+ counterions, the proposed mechanism explains the dependence of thermal stability on the size of the counterion. The degradation via intermolecular alpha-deprotonation by carboxylate is suggested to be the main PHB decomposition pathway at moderate temperatures. The results of the present study show the ability to control the degradation and stability of poly(3-hydroxybutyrate)s as well as of their blends via chemical structure and concentration of the carboxylate polymer end groups.     

Ribosome-inactivating proteins depurinate poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase and have transforming activity for 3T3 fibroblasts

It has been known that ribosome-inactivating proteins (RIPs) from plants damage ribosomes by removing adenine from a precise position of rRNA. Subsequently it was observed that all tested RIPs depurinate DNA, and some of them also non-ribosomal RNAs and poly(A), hence the denomination of adenine polynucleotide glycosylases was proposed. We report now that ricin, saporin-L2, saporin-S6, gelonin and momordin depurinate also poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase (auto modified enzyme), an enzyme involved in DNA repair. We observed also that all RIPs but gelonin induce transformation of fibroblasts, possibly as a consequence of damage to DNA and of the altered DNA repair system.     

Acid-tolerant poly(3-hydroxybutyrate) hydrolases from moulds

Abstract We have isolated some mould strains that can grow under acid conditions with poly(3-hydroxybutyrate) (PHB) as sole carbon source, and secrete PHB hydrolases active at pH values at least down to 3. An improved assay method for such enzymes using a pH stat has been developed, and used to determine the dependence of reaction rate on enzyme and polymer concentrations. The implications of these kinetic properties of the PHB hydrolase for its mode of action are discussed.     

Synthesis and characterization of poly(LysAla3)

The synthesis and characterization of poly(LysAla₃) are described. The polytetrapeptide is a model for short sequences found in proelastin, and is presumably involved in desmosine or isodesmosine cross-link formation in the native protein. Poly(LysAla₃) is found to possess a mixture of conformations in aqueous solution dependent on molecular weight and pH. Low-molecular-weight (ca. 3000) material appears to be a mixture of random and extended helix at neutral pH. However, as the molecular weight is increased an increasing amount of α-helix is observed rising to >50% for mol wt = 21,000. The α-helical chain segments are thermally stable, melting to a mixture of extended and random forms at T_m = 25°C. High pH (10.5) promotes further α-helix formation but at pH >11.0 the polypeptide becomes insoluble. The inference is that short chain segments of the peptide in elastin are unlikely to be α-helical in the equilibrium state but may fluctuate through such a conformation.