Display of functionally active PHB depolymerase on Escherichia coli cell surface

The display of PHB depolymerase (PhaZ(RpiT1) ) from R. pickettii T1 on the surface of E. coli JM109 cells is realized using OprI of P. aeruginosa as the anchoring motif. The fusion protein is stably expressed and its surface localization is verified by immunofluorescence microscopy. The displayed PhaZ(RpiT1) retains its cleaving ability for soluble substrates as well as its ability to adsorb to the PHB surface, and also remains catalycically active in the degradation of insoluble polyester materials, in spite of the possible suppression of the enzyme movement on the polymer surface. The results demonstrate that PhaZ(RpiT1) -displaying E. coli shows potential for use as a whole-cell biocatalyst for the production of (R)-3-hydroxybutyrate monomers from insoluble PHB materials.     

Tyrosine 105 of Paucimonas lemoignei PHB depolymerase PhaZ7 is essential for polymer binding

The 3-dimensional structure of the Paucimonas lemoignei poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 has significant similarity to Bacillus subtilis lipase LipA but differs from the latter by the presence of an additional domain. Analysis of this lid-like domain revealed the presence of many hydrophobic amino acid residues including Tyr₁₀₅. In this study we constructed His-tag fusions of PhaZ7 for simplified purification and investigated the effect of amino acid exchange of eight tyrosine codons of the lid-like domain. Exchanges of Tyr₁₀₃, Tyr₁₇₂, Tyr₁₇₃, Tyr₂₀₃ or Tyr₂₀₄ to alanine or serine had no phenotype but muteins with substitution of Tyr₁₈₉, Tyr₁₉₀ and Tyr₁₀₅ to alanine showed a lag phase of the in vitro PHB depolymerase reaction. Replacement of Tyr₁₀₅ by glutamate further increased the lag phase. Binding assays of the purified PHB depolymerase proteins with the natural substrate, native PHB granules, revealed a significantly reduced binding ability of the Tyr₁₀₅Glu mutant compared to the wild type protein and confirmed that Tyr₁₀₅ is involved in interaction with the polymeric substrate.     

Silver sulfide/poly(3-hydroxybutyrate) nanocomposites: Thermal stability and kinetic analysis of thermal degradation

The non-isothermal degradation of poly(3-hydroxybutyrate) (PHB) and silver sulfide/poly(3-hydroxybutyrate) (Ag₂S/PHB) nanocomposites was investigated using thermogravimetric (TG) analysis. In the composite materials, Ag₂S caused the degradation of PHB at a lower temperature as opposed to that of neat PHB. Moreover, an increase Ag₂S loading in the PHB decreased the onset temperature (T_onset) of thermal degradation, whereas it was raised upon augmenting the heating rate. From Kissinger plots, the observed trend of the degradation activation energy, E_d, was attributed to polymer-particle surface interactions and the agglomeration of Ag₂S. The thermal degradation rate constant, k, was linearly related to the Ag₂S loading in PHB. Thus, the Ag₂S nanoparticles effectively catalyzed the thermal degradation of PHB in the Ag₂S/PHB nanocomposites. Differential scanning calorimetry (DSC) data also supported the catalytic property of Ag₂S.     

A convenient route to PHB macromonomers via anionically controlled moderate‐temperature degradation of PHB

The degradation of poly(3‐hydroxybutyrate)s in homo‐ and heterogeneous mixtures with selected salts of organic and mineral acids was investigated. Nonvolatile degradation products, of processes conducted at moderate temperatures (150–170 °C), were analyzed using ¹H NMR. Analysis of results revealed a significant decrease in poly([R]‐3‐hydroxybutyrate) (PHB) thermal stability in the presence of acetic acid and carbonic acid salts of alkali metals (Cs, K, Li) as well as a less substantial effect with respect to bivalent metal (Ca, Mg, Zn) salts. This significant decrease in PHB thermal stability in the presence of salts of weak Bronsted‐Lowry acids can be explained in terms of an anionic degradation reaction proceeding via an E1cB mechanism. Furthermore, continuous poly(3‐hydroxybutyrate) controlled degradation was developed by a moderate‐temperature process using carbonic acid salts as “initiators” of anionic degradation. Foamed PHB macromonomers, bearing one crotonate terminal group, were obtainable via a reactive extrusion process. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Thermal Degradation of Poly(ε-caprolactone), Poly(L-lactic acid) and their Blends with Poly(3-hydroxy-butyrate) Studied by TGA/FT-IR Spectroscopy

Summary: The thermal degradation behavior of poly(ε-caprolactone) (PCL) and poly(L-lactic acid) (PLA) have been studied in different environment. It was found that these polymers undergo completely different degradation processes in nitrogen and oxygen atmosphere. In oxygen environment PCL and PLA mainly decompose to CO₂, CO, water and short-chain acids. In nitrogen atmosphere PCL releases 5-hexenioc acid, CO₂, CO and ε-caprolactone, whereas PLA decomposes to acetaldehyde, CO₂, CO and lactide. The polymer blends of poly(3-hydroxybutyrate) (PHB) with PCL and PLA decompose similar to the individual homopolymers with crotonic acid as the initial decomposition product of PHB.     

Purification and Properties of an Extracellular Polyhydroxybutyrate Depolymerase from Pseudomonas mendocina DSWY0601

An extracellular polyhydroxybutyrate(PHB) depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was approximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20―50 ℃ and sensitive to pH value within a pH range of 8.0―9.5. PHB depolymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate(P3/4HB) and poly-3-hydroxybutyrate-co-3- hydroxyvalerate(PHBV) but did not degrade poly(lactic acid)(PLA), poly(butylene succinate)(PBS) or poly- (caprolactone)(PCL). PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride(PMSF), H₂O₂ and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer(3HB) by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis of phaZ_pm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA(dPHA_SCL, PHA=polyhydroxyalkanoate) depolymerase containing catalytic domain, linker and substrate-binding domain.     

PHB (poly‐β‐hydroxybutyrate) and its enzymatic degradation

Our daily life needs depend on plastics, as they are cheap and durable, so they become the most commonly used synthetic chemical products. But from an environmentalist's point of view, a major concern related to these plastics is their non‐biodegradable nature. Driven by growing demand to search for sustainable solutions to dispose off generating huge volume of synthetic plastic wastes, shifted the mind of researcher towards the use of biodegradable plastics which can be completely disposed‐off by microbial enzymatic degradation. These biodegradable plastics or “bioplastics” are also synthesized by microbes under certain stressed environmental conditions out of which poly(R‐3‐hydroxybutyrate) (PHB) is the most ubiquitous and best known representatives of polyhydroxyalkanoate family. The PHB is most intensively used for the innovative biomedical applications owing to suitable combination of biocompatibility, transport characteristics, and mechanical properties. These challenging aspects of PHB can be used for designing of novel medical devices, in tissue engineering, and for systematic sustained drug delivery. Lots of research reports on PHB degrading enzymes and their producing microorganisms including biochemical aspects are available but in scattered form. So this review highlighted all the relevant information of PHB and PHB‐degrading enzymes starting with basic classification, synthesis, mechanism, and applications that are environment friendly and are of public interest.     

Purification,Characterization and Kinetic Studies of a Novel Poly(β) Hydroxybutyrate (PHB) Depolymerase PhaZ<Subscript><Emphasis Type="BoldItalic">Pen</Emphasis></Subscript> from <Emphasis Type="Italic">Penicillium citrinum</Emphasis> S2

A fungal isolate, identified as Penicillium citrinum S2, produced ≈1 U/mL of PHB depolymerase by 72 h when grown in BHM containing 0.2%, w/v PHB, pH 6.0 at 30 °C. Partial purification of an extracellular poly(-β-)hydroxybutyrate (PHB) depolymerase PhaZ _Pen from P. citrinum S2 by two steps using ammonium sulphate (80% saturation) and affinity chromatography using concanavalin A yielded 16.18-fold purity and 21.53% recovery of protein. The enzyme was composed of three polypeptide chains of 66, 43 and 20 kDa, respectively, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All the three bands stained positive for glycoprotein by PAS staining. Optimum enzyme activity was detected at pH 6.0 and 50 °C. The enzyme was stable between pH 4.0 and 7.0 at 50 °C, 2 h. β-hydroxybutyrate monomer was detected as the major end product of PHB hydrolysis. The enzyme also showed distinct behaviour towards different inhibitors tested, which suggests the role of serine, serine residue, carboxyl group, tyrosine and sulfhydryl groups in its active site.     

Effect of partial crosslinking on morphology and properties of the poly(β-hydroxybutyrate)/poly(d,l-lactic acid) blends

Poly(β-hydroxybutyrate) (PHB) is a bio-based and biodegradable aliphatic polyester, however its application is limited by some disadvantages such as high price, brittleness, poor processability and low melt-strength due to serious thermal degradation. Partial crosslinking initiated by dicumyl peroxide (DCP) was applied in this work to improve the performance of poly(β-hydroxybutyrate)/poly(d,l-lactic acid) (PHB/PDLLA) blends. The partial crosslinking of the blends and its effect on the properties, morphology, rheology and thermal behavior of the blends were investigated. The tensile strength and impact toughness of the PHB were increased by incorporation of the PDLLA, which were improved further after the partial crosslinking because of an increased compatibility between the PHB and the PDLLA phases. The rheological study revealed that the storage modulus (G′) and complex viscosity (η*) of the blends were increased after addition of the DCP. On the other hand, the crystallization of PHB in the blends was restricted to a certain extent by the formation of partially crosslinked network while its crystal form was not modified.     

Biosynthesis of Poly-(3-hydroxybutyrate) under the Control of an Anaerobically Induced Promoter by Recombinant Escherichia coli from Sucrose

Poly-(3-hydroxybutyrate) (PHB) is a polyester with biodegradable and biocompatible characteristics and has many potential applications. To reduce the raw material costs and microbial energy consumption during PHB production, cheaper carbon sources such as sucrose were evaluated for the synthesis of PHB under anaerobic conditions. In this study, metabolic network analysis was conducted to construct an optimized pathway for PHB production using sucrose as the sole carbon source and to guide the gene knockout to reduce the generation of mixed acid byproducts. The plasmid pMCS-sacC was constructed to utilize sucrose as a sole carbon source, and the cascaded promoter P_3nirB was used to enhance PHB synthesis under anaerobic conditions. The mixed acid fermentation pathway was knocked out in Escherichia coli S17-1 to reduce the synthesis of byproducts. As a result, PHB yield was improved to 80% in 6.21 g/L cell dry weight by the resulted recombinant Escherichia coli in a 5 L bed fermentation, using sucrose as the sole carbon source under anaerobic conditions. As a result, the production costs of PHB will be significantly reduced.     

改性PHB/PET液晶共聚酯的合成

精选八种具有不同结构特征的第三单体,以熔融缩聚法合成了八种改性PHB/PET共聚酯。发现含香草酸第三单体的PHB/PET/VA三元共聚酯具有较快共聚反应速率、较低熔融温度、典型热致液晶行为和良好成纤性。香草酸是最适合于PHB/PET体系共聚改性的第三单体。     

Compatibility and fungal degradation of poly[(R)-3-hydroxybutyrate]/aliphatic copolyester blend

Poly[(R)-3-hydroxybutyrate] (PHB) was blended with an aliphatic copolyester, which was synthesized by the esterification of adipic acid, ethylene glycol, and lactic acid. The blend showed a single T_g, which varied systematically but convexly upwards with the composition. The growth rate of PHB spherulites, the crystallization temperature, and the equilibrium melting temperature of the blend were decreased as the amount of the copolyester was increased. Therefore, the blend system was determined to be compatible. However, the degree of crystallinity, and the enthalpies of crystallization and fusion of PHB in the blend remained almost constant, regardless of the compositional change, although the crystallization rate was decreased upon blending. No chemical change such as transesterification was observed as a result of the blending, yet there was a slight change in the crystalline morphology of PHB. The rate of fungal degradation was lowered with an increase in the copolyester content of the blend. © 1996 John Wiley & Sons, Inc.     

甲烷氧化细菌生物催化合成聚-β-羟基丁酸酯的分子量调控

在甲烷氧化细菌Methylosinus trichosporium IMV3011细胞内生物催化合成聚-β-羟基丁酸酯(PHB)的过程中,对影响聚合物分子量的各种因素进行了研究.发现碳源、培养基组分NH4+,NO3-,HPO24-,Mg2+,某些导向PHB合成的关键中间产物以及PHB的提取方法均会对PHB的分子量产生影响.同时,通过对胞内PHB合成酶系中关键作用酶的活性变化进行研究,发现β-酮硫解酶催化着控制进入PHB循环入口的关键反应,而PHB分子量的变化则主要取决于PHB合成酶和PHB降解酶的协同作用.     

The bifunctionality of poly[(R)-3-hydroxybutyrate] in self-reinforced composite materials

The poly[(R)-3-hydroxybutyrate] (PHB) is a highly crystalline, biosourced polymer. The advantages of the PHB are its biodegradability and biocompatibility; however, the brittleness caused by its high crystallinity decreases the application ability of the PHB in comparison with the polyolefins. Excellent results were observed for the reactive extrusion of PHB in the presence of peroxides in many investigations of the modifications of PHB mechanical properties. The disadvantage must be considered to be the thermal degradation of PHB during extended extrusion and its limitation in natural composite preparation. The peroxides are highly reactive with natural fillers, and this causes a decrease of the filler's mechanical properties. Consequently, the reactive extrusion may be a useful tool for the production of additives only. The results we present of this investigation is based on a different material preparation strategy. The two-stage method incorporated additives preparation via reactive extrusion of PHB and the blending of the obtained product with neat PHB. Theself-reinforced composite material obtained in this way revealed significantly higher values of stress and strain compared to neat PHB. The thermal degradation of the PHB matrix was retarded and total crystallinity of the composite was decreased. The materials were characterized using DSC, SEM and SEC techniques. The samples were also investigated employing tensile and impact strength tests.     

Correlation between traditional techniques and TD-NMR to determine the morphology of PHB/PCL blends

Blends of two different biodegradable polyesters, poly(3-hydroxybutyrate) (PHB) and low molecular weight polycaprolactone (PCL), were obtained through solution casting and their miscibility and crystallinity were studied. The materials were characterized by wide angle X-ray diffraction, differential scanning calorimetry (DSC) and time-domain nuclear magnetic resonance (TD-NMR). Blends with PCL concentrations higher than 60% (w/w) were not obtained due the inability of low molecular weight PCL to form films by this method. The DSC technique revealed that the films were not miscible since there were no changes in the PHB glass transition temperature (T_g) after the PCL addition. However, the TD-NMR technique showed some partial miscibility, observed in the blend containing 10% (w/w) PCL, revealing domains around 30 nm, where the spin diffusion process was extremely close to that observed in the pure polymers. Other than that, the transversal relaxation showed that the partial miscibility of this composition occurs predominantly in the chain segments located in the interphase intercalation of the rigid regions, reducing the systems' crystallinity. These results are in accordance with the findings obtained through the WAXD analysis.     

Crystallization behavior, thermal property and biodegradation of poly(3-hydroxybutyrate)/poly(ethylene glycol) grafting copolymer

The poly(3-hydroxybutyrate)(PHB)/poly(ethylene glycol)(PEG) grafting copolymer was successfully prepared by PHB and acrylate groups ended PEGM using AIBN as initiator. The crystallization behavior, thermal stability and environmental biodegradability of PHB/PEG grafting copolymers were investigated with differential scanning calorimetry (DSC), Thermogravimetric analysis (TGA), wide angle X-ray diffraction (WAXD), scanning electron microscopy (SEM), and Biodegradation test in vitro. In the results, all the grafting copolymers were found to show the X-ray diffraction arising from the PHB crystal lattice, while none of the PEG crystallized peaks could be found even though the graft percent reached 20%. This result indicated that PEG molecules were randomly grafted onto PHB chain. The thermal properties measured by DSC showed that the melting temperature(T_m) and glass transition temperature (T_g) were both shifted to lower temperature with the graft percent increasing, and this broadened the narrow processability window of PHB. According to TGA results, the thermal stability of the grafting copolymers is not changed compared to pure PHB. From the biodegradation test, it could be concluded that degradation occurred gradually from the surface to the inside and that the degradation rate could be adjusted by the PEG grafting ratio. In another words, the biodegradation profiles of PHB/PEG grafting copolymer can be controlled. These properties make PHB/PEG grafting copolymer have promising potential applications especially in agriculture fields.     

Catalytically enhanced endocellulase cel5a from Acidothermus cellulolyticus

When Tyr245 in endocellulase Cel5A from Acidothermus cellulolyticus was changed to Gly (Y245G) by designed mutation, the value of K _i for inhibition of the enzyme by the product cellobiose was increased more than 1480%. This reduction in product inhibition enabled the mutant enzyme (used in conjunction with Trichoderma reesei cellobiohydrolase-I) to release soluble sugars from biomass cellulose at a rate as much as 40% greater than that achieved by the wild-type (WT) enzyme. The mutant was designed on the basis of the previously published crystal structure of the WT enzyme/substrate complex (at a resolution of 2.4 Å), which provided insights into the enzyme mechanism at the atomic level and identified Tyr245 as a key residue interacting with a leaving group. To determine the origin of the change in activity, the crystal structure of Y245G was solved at 2.4-Å resolution to an R-factor of 0.19 (R-free=0.25). To obtain additional information on the enzyme-product interactions, density functional calculations were performed on representative fragments of the WT Cel5A and Y245G. The combined results indicate that the loss of the platform (Y245G) and of a hydrogen bond (from a conformational change in Gln247) reduces the binding energy between product and enzyme by several kilo calories per mole. Both kinetic and structural analyses thus relate the increased enzymatic activity to reduced product inhibition.     

Attempt to control monomer sequences in copolycondensations of IPA,TPA, BPA,and PHB

Copolycondensations of IPA, TPA, BPA, and PHB were studied to investigate how PHB, which can form mesogenic segments, should be incorporated into the amorphous IPA/TPA–BPA polyester to obtain the thermotropic copolyester, unlike other copolymerizations studied so far by randomly introducing nonmesogenic components into the liquid crystalline polyesters. Random and controlled copolycondensations were attempted to regulate the segment length of mesogenic PHB units by stepwise addition of BPA and PHB through the two- and three-stage reactions using TsCl/DMF/Py as a condensing agent. Thermotropic copolyesters with ca. 40 mol % PHB could be obtained by a three-stage reaction, despite that more than 70 mol % PHB is needed to prepare by usual random copolymerization with PHB. The segment length of the PHB unit was indirectly studied from IPA/TPA–BPA oligomer distribution at initial reaction by means of GPC and from the NMR analysis of the resulting copolymers. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 2371–2377, 1999     

Production of Polyhydroxyalkanoates (PHAs) by Vibrio alginolyticus Strains Isolated from Salt Fields

Vibrio alginolyticus is a halophilic organism usually found in marine environments. It has attracted attention as an opportunistic pathogen of aquatic animals and humans, but there are very few reports on polyhydroxyalkanoate (PHA) production using V. alginolyticus as the host. In this study, two V. alginolyticus strains, LHF01 and LHF02, isolated from water samples collected from salt fields were found to produce poly(3-hydroxybutyrate) (PHB) from a variety of sugars and organic acids. Glycerol was the best carbon source and yielded the highest PHB titer in both strains. Further optimization of the NaCl concentration and culture temperature improved the PHB titer from 1.87 to 5.08 g/L in V. alginolyticus LHF01. In addition, the use of propionate as a secondary carbon source resulted in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). V. alginolyticus LHF01 may be a promising host for PHA production using cheap waste glycerol from biodiesel refining.     

Cofactor Biogenesis in Cysteamine Dioxygenase: C−F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine

Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein‐derived cofactor. Reported herein is the discovery and elucidation of a Cys‐Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C?S) bond. By genetically incorporating an unnatural amino acid, 3,5‐difluoro‐tyrosine (F₂‐Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon–fluorine bond activation and fluoride release were identified by mass spectrometry and ¹⁹F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low‐resolution techniques. Finally, a new sequence motif, C‐X‐Y‐Y(F), is proposed for identifying the Cys‐Tyr crosslink.