Y443F mutation in the substrate-binding domain of extracellular PHB depolymerase enhances its PHB adsorption and disruption abilities

Extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ_RpiT1) from Ralstonia pickettii T1 adsorbs to the PHB surface via its substrate-binding domain (SBD) and cleaves the PHB chain using its catalytic domain. Our previous study (Biomacromolecules 2010; 11: 113-119) has suggested that the hydrophobic interaction between the amino acid residues at positions 441, 443, and 445 in the SBD and the PHB surface plays a crucial role in facilitating the association phase of the enzyme adsorption process. In the present study, in order to improve PhaZ_RpiT1 for effective PHB degradation, we targeted Tyr at position 443 for substitution with a more highly hydrophobic amino acid residue because its hydrophobicity shows medium to high degree compared to those of general naturally occurring amino acid residues. We designed a mutant enzyme with an amino acid substitution at this position, taking the following factors into consideration: (1) to achieve higher hydrophobicity than the original residue, (2) to retain the β-sheet structure, and (3) to change as little as possible the volume of the amino acid residue after the substitution. As a result, the substitution of Tyr443 with Phe (Y443F) was considered to be appropriate. The purified Y443F enzyme showed identical CD spectrum and hydrolysis activity for a water-soluble substrate with the wild type, indicating that the mutation had no influence on the structure and the ester bond cleavage activity. In contrast, the Y443F enzyme had higher PHB degradation activity than the wild type. Kinetic analysis of PHB degradation suggests that this amino acid substitution promoted not only the adsorption of the mutant enzyme to PHB, but also the disruption of the PHB surface to enhance the hydrolysis of the PHB polymer chain.     

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.     

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.     

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.     

Adsorption effects of poly(hydroxybutyric acid) depolymerase on chain-folding surface of polyester single crystals revealed by mutant enzyme and frictional force microscopy

Adsorption effects of poly(hydroxybutyric acid) (PHB) depolymerase from Ralstonia pickettii T1 on various polymer single crystals were studied using a catalytically inactive mutant of PHB depolymerase by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), and frictional force microscopy (FFM). Six types of polymer single crystals, poly[(R)-3-hydroxybutyric acid] (P(3HB)), poly[(R)-3-hydroxybutyric acid-co-6 mol% (R)-3-hydroxyvaleric acid] (P(3HB-co-6 mol% 3HV)), poly[(R)-3-hydroxybutyric acid-co-8 mol% (R)-3-hydroxyhexanoic acid] (P(3HB-co-8 mol% 3HH)), poly(l-lactic acid) (PLLA), poly(d-lactic acid) (PDLA), and polyethylene (PE), were prepared to examine the influence of an ester bond and stereoregularity of a polymer on the enzymatic adsorption. The numbers of PHB depolymerase enzymes adsorbed on P(3HB) and P(3HB-co-6 mol% 3HV) single crystals were determined as 171 and 183 enzymes/μm² by AFM, respectively. AFM observation revealed that the concentration of PHB depolymerase enzymes adsorbed onto PLLA and PDLA single crystals is much higher compared to those on a P(3HB) single crystal, whereas the concentration of enzyme adsorbed onto PE and P(3HB-co-8 mol% 3HH) single crystals is much less. In addition, the single crystals of each polymer were characterized by TEM and FFM before and after enzymatic treatment by mutant for 1 h at 37 °C. The surface properties of P(3HB), P(3HB-co-6 mol% 3HV), and P(3HB-co-8 mol% 3HH) single crystals were changed by the enzymatic adsorption, whereas the internal structures were not affected. On the basis of these results, the properties of the binding domain of PHB depolymerase to polymer chain-folding surfaces have been discussed.     

Microarray of DNA–protein complexes on poly-3-hydroxybutyrate surface for pathogen detection

A novel strategy was developed for the specific immobilization of DNA probes on poly-3-hydroxybutyrate (PHB) surface by using the substrate-binding domain (SBD) of PHB depolymerase as an active binding motif. To demonstrate whether this method can be used for the detection of clinical pathogens, the pathogen-specific biotin-labeled DNA probes were immobilized via core streptavidin (cSA) fused to the SBD. The pathogen-specific 15-mer oligonucleotide probes were designed for four model pathogens, while the target DNAs were prepared by PCR using universal primers. The complex of pathogen-specific probes and cSA-SBD fusion protein was immobilized on the PHB-coated slide by microspotting. This DNA–protein complex microarray was able to successfully diagnose Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, the specific pathogens could be diagnosed in the presence of other microorganisms. Thus, the DNA–protein complex microarray platform technology employing PHB and the SBD reported here can be widely used for the detection of DNA–DNA and DNA–biomolecule interactions without synthetic or chemical modification of biomolecules or solid surface.     

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.     

Synergized Antimicrobial Activity of Eugenol Incorporated Polyhydroxybutyrate Films Against Food Spoilage Microorganisms in Conjunction with Pediocin

Biopolymers and biopreservatives produced by microorganisms play an essential role in food technology. Polyhydroxyalkanoates and bacteriocins produced by bacteria are promising components to safeguard the environment and for food preservation applications. Polyhydroxybutyrate (PHB)-based antimicrobial films were prepared incorporating eugenol, from 10 to 200 μg/g of PHB. The films were evaluated for antimicrobial activity against foodborne pathogens, spoilage bacteria, and fungi such as Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Bacillus cereus, Aspergillus flavus, Aspergillus niger, Penicillium sp., and Rhizopus sp. The synergistic antimicrobial activity of the films in the presence of crude pediocin was also investigated. The broth system containing pediocin (soluble form) as well as antimicrobial PHB film demonstrated an extended lag phase and a significant growth reduction at the end of 24 h against the bacteria. Crude pediocin alone could not elicit antifungal activity, while inhibition of growth and sporulation were observed in the presence of antimicrobial PHB film containing eugenol (80 μg/g) until 7 days in the case of molds, i.e., A. niger, A. flavus, Penicillium sp., and Rhizopus sp. in potato dextrose broth. In the present study, we identified that use of pediocin containing broth in conjunction with eugenol incorporated PHB film could function in synergized form, providing effective hurdle toward food contaminating microorganisms. Furthermore, tensile strength, percent crystallinity, melting point, percent elongation to break, glass transition temperature, and seal strength of the PHB film with and without eugenol incorporation were investigated. The migration of eugenol on exposure to different liquid food simulants was also analyzed using Fourier transform infrared spectroscopy. The study is expected to provide applications for pediocin in conjunction with eugenol containing PHB film to enhance the shelf life of foods in the food industry.     

Gamma irradiation effects on poly(hydroxybutyrate)

Brazilian poly(hydroxybutyrate), PHB, as well as its copolymer, poly(hydroxybutyrate-co-valerate), P(HB-co-HV), containing 6.3 mol% of valerate, were irradiated with γ radiation (⁶⁰Co) at ambient temperature and in the presence of oxygen. The viscosity-average molar mass (M_v) was analyzed by the viscosity technique using an Ostwald-type capillary viscometer. The polymers showed a decrease in molar mass with the increase in dose, reflecting the scissions that occurred at random in the main chain. The value G (scissions/100 eV of energy transferred to the system) and the parameter α (scissions per original molecule) were also obtained by the viscosity technique. The melting temperature (T_m) was determined by differential scanning calorimetry (DSC) and showed a decrease with increasing irradiation dose. Analyses of DSC also revealed double endothermic peaks, associated with the polymorphic transitions, which became a single peak with increased dose. Thermogravimetry analysis (TGA) revealed small differences between the decomposition temperatures of the irradiated and non-irradiated samples. The degree of crystallinity of PHB samples, on the other hand, which were obtained by the DSC and X-ray diffraction techniques, increased with the irradiation dose. Changes in the lattice parameter of the irradiated samples and in the size of the crystallites were also observed by X-ray diffraction. The samples used in this work did not pass through any purification process and were analyzed in powder form, exactly as they arrived from the factory.     

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.     

Interaction between poly[(R)-3-hydroxybutyrate] depolymerase and biodegradable polyesters evaluated by atomic force microscopy

Adsorption of PHB depolymerase from Ralstonia pickettii T1 to biodegradable polyesters such as poly[(R)-3-hydroxybutyrate] (PHB) and poly(l-lactic acid) (PLLA) was investigated by atomic force microscopy (AFM). The substrate-binding domain (SBD) with histidines within the N-terminus was prepared and immobilized on the AFM tip surface via a self-assembled monolayer with a nitrilotriacetic acid group. Using the functionalized AFM tips, the force-distance measurements for polyesters were carried out at room temperature in a buffer solution. In the case of AFM tips with immobilized SBD and their interaction with polyesters, multiple pull-off events were frequently recognized in the retraction curves. The single rupture force was estimated at approximately 100 pN for both PLLA and PHB. The multiple pull-off events were recognized even in the presence of a surfactant, which will prevent nonspecific interactions, but reduced when using polyethylene instead of polyesters as a substrate. The present results provide that the PHB depolymerase adsorbs specifically to the surfaces of polyesters and that the single unbinding event evaluated here is mainly associated with the interaction between one molecule of SBD and the polymer surface.     

Adaptive Laboratory Evolution of Halomonas bluephagenesis Enhances Acetate Tolerance and Utilization to Produce Poly(3-hydroxybutyrate)

Acetate is a promising economical and sustainable carbon source for bioproduction, but it is also a known cell-growth inhibitor. In this study, adaptive laboratory evolution (ALE) with acetate as selective pressure was applied to Halomonas bluephagenesis TD1.0, a fast-growing and contamination-resistant halophilic bacterium that naturally accumulates poly(3-hydroxybutyrate) (PHB). After 71 transfers, the evolved strain, B71, was isolated, which not only showed better fitness (in terms of tolerance and utilization rate) to high concentrations of acetate but also produced a higher PHB titer compared with the parental strain TD1.0. Subsequently, overexpression of acetyl-CoA synthetase (ACS) in B71 resulted in a further increase in acetate utilization but a decrease in PHB production. Through whole-genome resequencing, it was speculated that genetic mutations (single-nucleotide variation (SNV) in phaB, mdh, and the upstream of OmpA, and insertion of TolA) in B71 might contribute to its improved acetate adaptability and PHB production. Finally, in a 5 L bioreactor with intermittent feeding of acetic acid, B71 was able to produce 49.79 g/L PHB and 70.01 g/L dry cell mass, which were 147.2% and 82.32% higher than those of TD1.0, respectively. These results highlight that ALE provides a reliable method to harness H. bluephagenesis to metabolize acetate for the production of PHB or other high-value chemicals more efficiently.     

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.     

Production of L(+)-lactic acid using immobilized rhizopus oryzae reactor performance based on kinetic model and simulation

The production of L(+)-lactic acid using alginate immobilizedRhizopus oryzae in tapered-column fluidized-bed batch reactor was tested and simulated using the kinetic data taken independently in shake-flask cultures. The data show saturation kinetics with substrate and product inhibitions in linear form. Analysis of the kinetic data gave kinetic constants:V _m, 11.04 g lactic acid/(L-bead. h);K _m, 20.9 g glucose/L; andK _i, 365 g glucose/L for lactic acid production. The product inhibition constant,K _p, was found to be 316 g lactic acid/L. The simulation results showed a good agreement with the experimental results when the initial lag phase was taken into account in the simulation model. Without the adjustment for the initial lag period, the kinetic model showed higher conversion. Starting with a glucose concentration of 150 g/L, it was possible to produce 73 g/L of L(+)-lactic acid in 44.5 h. The lactic acid yield was 64.8% by weight based on the amount of glucose consumed.     

Synthesis of a Tyr-octreotate conjugated closo-carborane [HC2B10H10]: a potential compound for boron neutron capture therapy

A novel Tyr³-octreotate conjugated closo-carborane as a potential compound for boron neutron capture therapy was obtained via Fmoc solid phase peptide synthesis. The boron cluster [C₂B₁₀H₁₁] was introduced through the reaction of 6,9-bis(acetonitrile)decaborane and 5-hexynoic acid yielding a new closo-carborane conjugated carboxylic acid which was coupled subsequently with solid phase conjugated Tyr³-octreotate. The final boron-containing peptide was purified by preparative reverse phase HPLC and structural identity was proved applying MALDI-TOF mass spectrometry.     

In-situ atomic force microscopy observation of enzymatic degradation in poly(hydroxyalkanoic acid) thin films: normal and constrained conditions

The enzymatic degradation of lamellar crystals in poly(hydroxyalkanoic acid) thin films has been visualized by using in-situ dynamic force mode (tapping mode) atomic force microscopy (AFM) in buffer solution. It was found that poly(hydroxybutyric acid) (PHB) depolymerase from Ralstonia pickettii T1 degraded the thin surface layers formed at room temperature first, and that lamellar crystals formed at the crystallization temperature (110 degrees C) were eroded from the crystallographic a-axis to show splintered morphologies at the tips of the crystals. In some cases, lamellar crystals were hydrolyzed from the crystallographic b-axis, resulting in the formation of small crevices. These results suggest that disordered molecular chain-packing regions exist in the crystal along the crystallographic a- and b-axes, and that enzymatic degradation predominantly occurs from these defective regions. In addition, cantilever-tip-induced enzymatic degradation was carried out in the presence of PHB depolymerase. A concave area was artificially formed on the stacked lamellar crystals by the AFM tip. In-situ AFM observation has revealed that enzymatic degradation proceeds along both the longitudinal and lateral directions of the lamellae. At the same time, the PHB depolymerase preferentially eroded the concave area along the crystallographic c-axis. These results demonstrated that the PHB depolymerase predominantly degrades the less-ordered molecular chain-packing regions in the crystals.     

Catalytic Production of Alanine from Waste Glycerol

Chemical synthesis of amino acids directly from biomass feedstock is rare. Reported here is a one‐step protocol to convert crude glycerol, from the biodiesel industry, into 43 % alanine over a Ru₁Ni₇/MgO catalyst. The multifunctional catalytic system promotes glycerol conversion into lactic acid, and then into alanine. X‐ray absorption spectroscopy and scanning transmission electron microscopy revealed the existence of bimetallic RuNi species, whereas density‐functional theory calculations suggested Ni‐doped Ru substantially decreased the E_a of C?H bond dissociation of lactate alkoxide to form pyruvate, which is the rate‐determining step. The catalytic route established in this work creates new opportunities for glycerol utilization and enriches the substrate scope of renewable feedstock to access value‐added amino acids.     

TH‐11, a Streptomyces sp. Strain that Degrades Poly(3‐Hydroxybutyrate) and Poly(Ethylene Succinate)

TH‐11, a bacterial strain with strong depolymerase activity that breaks down aliphatic esters such as poly(3‐hydroxybutyrate) (PHB) and poly(ethylene succinate) (PES) was isolated from a soil sample collected from the sediment of Tou‐Chain River, Taiwan, R.O.C. It was phenotypically and genetically characterized to be a Streptomyces strain. The degradation of PHB and PES were tested both using emulsified polymers in solid agar and thin polymer films in liquid culture media. The degradations were measured by clear‐zone formation on solid agar plates, or direct weight measurements and electromicroscope inspection of the incubated polymer films in the liquid culture. The depolymerase activities can be detected in the cell‐free preparation of the culture medium, and can be enhanced by gelatin.     

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.     

Conformational studies of 3,4-dideoxy furanoid sugar amino acid containing analogs of the receptor binding inhibitor of vasoactive intestinal peptide

Conformational analysis of vasoactive intestinal peptide (VIP) receptor binding inhibitor Leu¹-Met²-Tyr³-Pro⁴-Thr⁵-Tyr⁶-Leu⁷-Lys⁸1 by various NMR techniques and constrained molecular dynamics (MD) simulation studies revealed that the molecule had a turn structure involving its Tyr³-Pro⁴-Thr⁵-Tyr⁶ moiety with intramolecular hydrogen bond between Tyr⁶NH→Tyr³CO. In order to mimic the structure of 1, peptidomimetic analogs 2-4 were synthesized using conformationally constrained scaffolds of 3,4-dideoxy furanoid sugar amino acids (2S,5R)-ddSaa1 5 and its enantiomer (2R,5S)-ddSaa2 6. All these analogs displayed well defined three-dimensional structures akin to that found in 1. Peptides 2 and 3, which differed only in the sugar amino acid stereochemistry, show propensity of structures with identical intramolecular hydrogen bonds between ThrNH→MetCO. A similar structure with a hydrogen bond between TyrNH→MetCO was observed in 4.