Isodimorphism in bacterial poly(β-hydroxybutyrate-co-β-hydroxyvalerate)

Bluhm, Terry L.; Hamer, Gordon K.; Marchessault, R. H.; Fyfe, C. A.; Veregin, Richard P. N.

doi:10.1021/ma00165a035

Cited by 410 publications

(325 citation statements)

References 3 publications

(4 reference statements)

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“…However, despite this problem, GPC chromatography of the resulting product reveals that ∼50% of the radioactivity is associated with a large molecular mass polymer (Figure 9). The size of the polymer is similar to that of a control in which [ 3 H]- 2 Previous studies with R. eutropha identified a peptide containing C319, but methods of analysis precluded us from establishing that the cysteine was actually modified. HB-CoA is incubated with synthase and analyzed under identical conditions.…”

Section: Pha Synthase Expression and Purificationmentioning

confidence: 64%

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

et al. 1998

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Polyhydroxyalkanoate synthase (PHA) from Chromatium Vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a ∼1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His) 6 -tagged construct. The PhaC-(His) 6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His) 6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His) 6 to PhaC-(His) 6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ( Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [ 3 H]-sT while the other two contained, in addition to the [ 3 H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.Polyhydroxyalkanoates (PHAs 1 ) are polyoxoesters with properties that range from elastomers to thermoplastics (1-4). They are produced by a wide range of bacteria when they are placed in an environment of nutrient limitation (5). Copolymers of polyhydroxybutyrate (PHB) and polyhydroxyvalerate in the correct ratio have properties similar to the petrochemically based polypropylenes, the major component of bulk plastics (6). In 1997, the US market for thermoplastics was on the order of 40 million tons per year (7). PHAs have recently received much attention because they are biodegradable and can be generated from biorenewable sources: bacteria and plants (8). The major focus of many investigators is to make their production economically competitive with the polypropylenes. To achieve this goal, the requirements for the polymerization process need to be established. This paper focuses on the PHA synthase from Chromatium Vinosum which catalyzes the formation of PHB from 3-hydroxybutyryl-CoA (HB-CoA). Evidence is presented that two cysteines and covalent catalysis play an important role in the initiation and elongation of the polymerization process.PHA synthases from 20 organisms have now been identified. They have been divided into three classes based on their substrate specificity and subunit composition (9). The class I synthases, with the Ralstonia eutropha synthase as a prototype, are composed of a single polypeptide (∼65 k...

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Section: Pha Synthase Expression and Purificationmentioning

confidence: 64%

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

et al. 1998

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“…Depending on the HV distribution in polymer chains, polymer samples containing 0-37% of HV crystallize in the PHB lattice. It was previously noted by Marchessault, 13 Lauzier 14 and, recently by Menezes, 15 a partial exclusion of HV units from the PHB crystal lattice, increasing the contribution of HV to the amorphous phases. In particular, the higher changes observed for 14 and 22% HV contents can be explained by the additional contribution due to the inhomogeneous distribution of the HV sequence in the amorphous and crystalline phases.…”

Section: Bulk Propertiesmentioning

confidence: 86%

“…10 In the crystalline phase, it can be observed from the d-spacing measurements by X diffraction that, up to 29 mol % of the HV content sequence, the HV units are accommodated in the PHB crystal lattice (isomorphism) by small lateral expansion of the unit cell but the crystal packing (internal morphology) is disturbed by inclusion of these HV units. 6,[8][9][10][11] Additionally, NMR evidence indicates that from 20-40% HV total contents in the copolymer structure, some segments with relative higher HV units, are partially excluded from the PHB lattice. 12 From the polyhydroxyalkanoates data shown in Table 1, it can be inferred that the glass transition temperature (Tg), measured for copolymers with 14 and 22 mole % HV contents, were lower than the predicted by the Fox's equation (1/Tg = W PHB /Tg PHB + W HV /Tg HV, where W is the content of the polymer).…”

Section: Bulk Propertiesmentioning

confidence: 99%

“…The minimum of the melting point (≅80 o C, pseudoeutectic point) of the copolymers is observed for a 30% mole fraction of HV. 5,6 The accurate determination of the thermal properties of polymers and particularly of PHAs is of fundamental importance in many processes and engineering projects aiming at the expansion of the uses of these biodegradable materials. In the present work, the effect of HV contents in the P(3HB-co-3HV) on the thermal and electronic properties was examined.…”

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confidence: 99%

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Structural impact of hydroxyvalerate in polyhydroxyalkanoates (PHAscl) dense film monitored by XPS and photothermal methods

Silva¹,

Vargas²,

Poley³

et al. 2005

J. Braz. Chem. Soc.

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Filmes densos de poli(3-hidrobutirato) e poli(3-hidroxibutirato-co-3-hidroxivalerato) com espessuras de 90 µm foram obtidos utilizando o método "casting" de deposição e caracterizados por espectroscopia de fotoelétrons, calorimetria diferencial e métodos fotoacústicos. As frações molares de hidroxivalerato utilizadas foram de 8, 14 e 22 % molar. Seguindo uma mesma tendência, a capacidade térmica específica e a temperatura de transição vítrea diminuem com o aumento da concentração de hidroxivalerato.Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) polymer dense films with thickness of 90 µm were obtained by the casting method and characterized using X-ray photoelectron spectroscopy, differential scanning calorimetric and photo-thermal methods. The HV molar fractions in the copolymer structure were 8, 14 and 22 mol %. The measured specific thermal capacity and the glass transition temperature of the P3(HB-co-x%HV) decrease for increasing hydroxyvalerate content following a similar trend. Keywords: polymers, photoacoustics, XPS, thermal properties IntroductionIn the last years, science and industry have given special attention to the development of biodegradable polymer materials as consequence of today ecological problems caused by most of the chemical and petrochemical synthesized polymers disposed in the environment. 1 Chemically and petrochemically synthesized polymers are generally resistant to enzymatic attack. Although some of these synthetic polymers can be microbiologically degraded, the process of degradation is slow in contrast to the biodegradability of the polymers of biological origin. 2 The biodegradability is not the only characteristic that makes the polymers of biological origin attractive, also their biosynthesis from renewable carbon sources, based on agricultural products, allows a sustainable closed process for the production and use of such polymers.One important family of the biologically produced thermoplastic polyesters is formed by the polyhydroxyalkanoaes (PHAs). Important members of this family are the P3HB and P(3HB-co-3HV)copolymers synthesized and intracellularly stored by numerous prokaryotes by well know fermentation process. The stereoregularity of P3HB makes it a highly crystalline material having melting point of 177 o C, close to that of polypropylene among others similarities, although the biopolymer is stiffer and more brittle. 3,4 The P(3HB-co-3HV) chains also show crystalline conformations and the co-monomer distribution is statistically random. The properties of the copolymers of 3HB and 3 HV changes depending on their HV contents. The minimum of the melting point (≅80 o C, pseudoeutectic point) of the copolymers is observed for a 30% mole fraction of HV. 5,6 The accurate determination of the thermal properties of polymers and particularly of PHAs is of fundamental importance in many processes and engineering projects aiming at the expansion of the uses of these biodegradable materials. In the present work, the effect of HV contents in the P(3HB-co-...

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“…For instance, polyhydroxyalkanoates (PHA) produced by bacteria from biodegradable aliphatic homo or copolyesters with over 150 unlike kinds composed of dissimilar monomers (Steinbüchel and Lütke-Eversloh, 2003). Poly(3-hydroxy butyrate) (PHB) and its copolymers with 3-hydroxyvalerate (PHBHV), 3-hydroxyoctanoate (PHBHO), and 3-hydroxyhexanoate (PHBHHx) units are among the largely employed PHAs (Bluhm et al, 1986;Doi et al, 1988;Noda et al, 2004). The high crystallinity of PHB homopolymer results in a hard and brittle material, inconvenient to blend with PLA.…”

Section: Pla Modificationsmentioning

confidence: 99%