Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Abstract Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of d (−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of d (−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing d (−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M.

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

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.     

Effects of mutations in the substrate-binding domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 on PHB degradation

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

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase

(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by β-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g?L^?1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g?L^?1, with a productivity of 0.22 g?L^?1 h^?1. The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of ¹³C carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.     

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

[This corrects the article on p. 3236 in vol. 59.].     

Structure and enzymatic degradation of poly(3-hydroxybutyrate) copolymer single crystals with an extracellular PHB depolymerase from Alcaligenes faecalis T1.

Lamellar single crystals of four random copolymers of (R)-3-hydroxybutyrate with different hydroxyalkanoates: poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyvalerate) (P(3HB-co-8%-3HV)), poly(3-hydroxybutyrate-co-10 mol%-4-hydroxybutyrate) (P(3HB-co-10%-4HB)), poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyhexanoate) (P(3HB-co-8%-3HH)) and poly(3-hydroxybutyrate-co-10 mol%-6-hydroxyhexanoate) (P(3HB-co-10%-6HH)), were grown from dilute solutions of chloroform and ethanol. All single crystals have lath-shaped morphology and the second monomer units seem to be excluded from the P(3HB) crystal, on the basis of the electron diffraction diagrams. The enzymatic degradation of P(3HB-co-8%-3HH) and P(3HB-co-10%-6HH) single crystals was investigated with an extracellular PHB depolymerase from Alcaligenes faecalis T1. Adsorption of an extracellular PHB depolymerase, examined using an immuno-gold labelling technique, demonstrated a homogeneous distribution of enzyme molecules with a low concentration on the crystal surfaces. Enzymatic degradation of single crystals progressed from the edges and ends of crystals to yield narrow cracks along their long axes and the small crystal fragments. Lamellar thicknesses of single crystals and molecular weights of copolymer chains remained unchanged during the enzymatic hydrolysis. The above results support the hypothesis that the hydrophobic adsorption of the enzyme contributes to increase the mobility of molecular chains of single crystals and generate the disordered chain-packing regions. The active-site of PHB depolymerase takes place preferentially at the disordered chain-packing regions of crystal edges and ends with endo-exo enzymatic hydrolysis behaviour, termed processive degradation.     

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)     

Requirement for the enzymes acetoacetyl coenzyme A synthetase and poly-3-hydroxybutyrate (PHB) synthase for growth of Sinorhizobium meliloti on PHB cycle intermediates

We have identified two Sinorhizobium meliloti chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. One locus was identified as the gene acsA, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the acsA nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown acsA::Tn5 mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the S. meliloti poly-3-hydroxybutyrate cycle. The second locus was identified as phbC, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits.     

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.     

Structure of native poly(3-hydroxybutyrate) granules characterized by X-ray diffraction

The structure of native poly(3-hydroxybutyrate) (PHB) granules of Alcaligenes eutrophus was characterized in wet cells or wet granules by analysis of X-ray diffraction. The PHB granules in intact cells were completely amorphous, but became crystalline after treatment with alkali or sodium hypochlorite. The native PHB granules were isolated from the cells by treatment with enzymes and sonic oscillation. The isolated PHB granules remained amorphous in suspension. The PHB granules were crystallized by various treatments with aqueous acetone, alkaline solution (of either NaOH or sodium hypochlorite), and lipase in an aqueous environment. These results suggest that crystallization of PHB molecules is started by the removal of a lipid component from native granules by various treatments.     

Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases.

Native poly(hydroxybutyrate) (PHB) granules, purified PHB and artificial amorphous PHB granules were examined as putative substrates for hydrolysis by the intracellular depolymerase system of Rhodospirillum rubrum and the extracellular depolymerase of Pseudomonas lemoignei. The R. rubrum depolymerizing system requires pretreatment of granules with a heat stable 'activator' fraction; the activator can be replaced by mild trypsin treatment. Artificial granules were prepared with a cationic detergent, cetyltrimethylammonium bromide (CTAB) and an anionic detergent, (sodium cholate). Cholate and CTAB PHB granules were hydrolyzed by both enzyme systems; however, some differences were noted. Cholate granules were hydrolyzed in the absence of the R. rubrum activator fraction. Activator was required for the hydrolysis of CTAB granules but could be replaced by heparin in the extracellular depolymerase system but not in the intracellular depolymerase system. A Triton X-114 extract of native PHB granules inhibited the hydrolysis of trypsin-activated granules by the intracellular depolymerase. The inhibition was reversed by the activator fraction. Detergent extracts of granules activated with the R. rubrum activator were unable to inhibit the hydrolysis of trypsin-activated granules. These data suggest that the activator acts to modify an inhibitor present on native granules.     

Biosynthesis of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16 from jatropha oil as carbon source

Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer that can be synthesized through bacterial fermentation. In this study, Cupriavidus necator H16 is used to synthesize PHB by using Jatropha oil as its sole carbon source. Different variables mainly jatropha oil and urea concentrations, and agitation rate were investigated to determine the optimum condition for microbial fermentation in batch culture. Based on the results, the highest cell dry weight and PHB concentrations of 20.1 and 15.5 g/L, respectively, were obtained when 20 g/L of jatropha oil was used. Ethanol was used as external stress factor and the addition of 1.5 % ethanol at 38 h had a positive effect with a high PHB yield of 0.987 g PHB/g jatropha oil. The kinetic studies for cell growth rate and PHB production were conducted and the data were fitted with Logistic and Leudeking–Piret models. The rate constants were evaluated and the theoretical values were in accordance with the experimental data obtained.     

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.     

The role of phasins in the morphogenesis of poly(3-hydroxybutyrate) granules

Recent developments in the understanding of the structure of polyhydroxyalkanoate, PHA, granules in bacteria are documented in the literature and point to the role of structural proteins, phasins, in granule formation and stabilization. We have previously conceived a computer program which successfully simulates granule formation in vitro, in the absence of phasins. Now we are extending the computer model to a more complex system, including phasins, to quantify their anticipated effect on the granule properties. The simulation enabled us to propose real experiments to test the validity of the model and provide a framework for a better understanding of PHA granule formation in vivo.     

Effect of poly(3-hydroxybutyrate) (PHB) content on the starvation-survival of bacteria in natural waters

Abstract The effect of poly(3-hydroxybutyrate) (PHB) content on the survival of wild-type strains and PHB negative mutants of Bacillus megaterium and Alcaligenes eutrophus in natural waters was studied. The survival strategy of B. megaterium was dominated by the development of resistant forms, but the number of the wild-type vegetative cells was higher than that of PHB mutant strain. In some environmental conditions the mutant spores needed a heat shock for germination, a fact that suggests, for the first time, that PHB plays a role in this phenomenon. Survival of A. eutrophus wild-type strain in all experiments was higher compared to the PHB mutant, and differences were significant. In raw river water, survival of both species was lower than in sterile river water.     

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.     

Change of surface structure of poly(3-hydroxybutyrate) film upon enzymatic hydrolysis by PHB depolymerase

The change in the surface structure of poly[(R)-3-hydroxybutyrate] [PHB] films upon the enzymatic hydrolysis was analyzed by attenuated total reflection infrared [ATR/IR] spectrometry. As enzymes, PHB depolymerases isolated from Ralstonia pickettii T1 and Pseudomonas stutzeri were used. By curve decomposition of the carbonyl stretching band of ATR/IR spectra, the change in the surface crystallinity of PHB films by exposure to buffer containing 0, 1, and 4 microg of PHB depolymerases was estimated. It has been widely believed that the enzymatic hydrolysis first occurs in the amorphous phase, followed by the degradation in the crystalline phase, and extracellular PHB depolymerase can degrade only polymer chains in the surface layer of the film. Therefore, the surface crystallinity had been expected to increase upon the enzymatic degradation. However, the results were contrary to this expectation. The surface crystallinity was decreased by the enzymatic attack. Because ATR/IR spectrometry is sensitive to a small change in molecular structure of the sample surface, the decrease in the crystallinity shown by ATR/IR experiments probably does not indicate the complete loss of regularity of the crystalline phase. Because the chains at crystalline surface are more mobile than those inside the crystals, the C=O band for crystalline surface may appear at a position similar to those of the amorphous or interfacial phase in ATR/IR spectra of PHB. Only the chains inside the crystals may contribute to the C=O band of the crystalline phase. Thus, we rather suppose that the decrease in the crystalline peak of the ATR/IR spectra reflects the change in chain mobility or the increase of crystalline surface area by cracking of lamellas at the surface layers of PHB films or both.