PHB Surface Research Articles

Nanofiber Material with Hydrophobic-Hydrophilic Properties Based on Poly(3-Hydroxybutyrate) and Poly(2-Hydroxyethyl Methacrylate)

A new material with hydrophobic-hydrophilic properties is obtained based on ultrathin poly(3-hydroxybutyrate) (PHB) fibers modified with poly(2-hydroxyethyl methacrylate). The supramolecular structure of the samples is investigated by structurally dynamic methods: scanning electron microscopy, differential scanning calorimetry, and electron paramagnetic resonance. The equilibrium-kinetic characteristics of water sorption by the material are determined. Evidence is obtained by the method of 1H NMR (proton nuclear magnetic resonance spectroscopy) for the chemical modification of the PHB surface due to the participation of its terminal groups in the transesterification reaction of 2-hydroxyethyl methacrylate.

Poly(3-hydroxybutyrate) Modified by Nanocellulose and Plasma Treatment for Packaging Applications.

In this work, a new eco-friendly method for the treatment of poly(3-hydroxybutyrate) (PHB) as a candidate for food packaging applications is proposed. Poly(3-hydroxybutyrate) was modified by bacterial cellulose nanofibers (BC) using a melt compounding technique and by plasma treatment or zinc oxide (ZnO) nanoparticle plasma coating for better properties and antibacterial activity. Plasma treatment preserved the thermal stability, crystallinity and melting behavior of PHB‒BC nanocomposites, regardless of the amount of BC nanofibers. However, a remarkable increase of stiffness and strength and an increase of the antibacterial activity were noted. After the plasma treatment, the storage modulus of PHB having 2 wt % BC increases by 19% at room temperature and by 43% at 100 °C. The tensile strength increases as well by 21%. In addition, plasma treatment also inhibits the growth of Staphylococcus aureus and Escherichia coli by 44% and 63%, respectively. The ZnO plasma coating led to important changes in the thermal and mechanical behavior of PHB‒BC nanocomposite as well as in the surface structure and morphology. Strong chemical bonding of the metal nanoparticles on PHB surface following ZnO plasma coating was highlighted by infrared spectroscopy. Moreover, the presence of a continuous layer of self-aggregated ZnO nanoparticles was demonstrated by scanning electron microscopy, ZnO plasma treatment completely inhibiting growth of Staphylococcus aureus. A plasma-treated PHB‒BC nanocomposite is proposed as a green solution for the food packaging industry.

Chitosan-based Silver Nanoparticles Incorporated at the Surface of Plasma-treated PHB Films

This study focused on the incorporation of silver nanoparticles and chitosan at poly(hydroxybutyrate) (PHB) film surfaces by a two-step method: helium plasma followed by wet chemical treatments. Silver nanoparticles were obtained by a facile green synthesis in a chitosan solution, in which the plasma-treated PHB samples were immersed. The increase in plasma treatment times provided an increase at PHB surface of the silver nanoparticles content, which proved to have a core–shell shape and diameters between 14–16 nm.

Antibacterial properties of modified biodegradable PHB non-woven fabric

The antibacterial properties of poly(hydroxybutyrate) (PHB) non-woven fabric were explored in this study. The PHB was activated by plasma modification and subsequently processed with either immersion into a solution of nanoparticles or direct metallization. The wettability and surface chemistry of the PHB surface was determined. The thickness of the sputtered nanolayer on PHB fabric was characterized. It was found that plasma modification led to a formation of strongly hydrophilic surface, while the subsequent metallization by silver or gold resulted in a significantly increased water contact angle. Further, it was found that antibacterial activity may be controlled by the type of a metal and deposition method used. The immersion of plasma modified fabric into Ag nanoparticle solution led to enhanced antibacterial efficiency of PHB against Escherichia coli (E. coli). Direct silver sputtering on PHB fabric was proved to be a simple method for construction of a surface with strong antibacterial potency against both Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epidermidis). We demonstrated the antibacterial activity of PHB fabric modified by plasma activation and consecutive selection of a treatment method for an effective antibacterial surface construction.

Superhydrophobic to Superhydrophylic Biomimetic Poly(3-Hydroxybutyrate) Surfaces Made by Phase Inversion

Biodegradable and biocompatible materials have gained increased attentions because of their applications in biomedicine and tissue engineering. Among them, poly(3-hydroxybutyrate) (PHB), a natural origin polymer, has been employed in biomedical applications as a suitable substitute of synthetic polymers for preparing scaffolds and other devices. In this context it is relevant to understand the interactions between the cells and PHB surfaces, which are governed by aspects such as surface topography and chemical composition. Both, surface topography and chemistry determine surface wettability. The aim of this work is to process PHB surfaces exhibiting extreme wettablility properties, ranging from superhydrophobic (surface contact angle higher than 150º) to superhydrophilic (surface contact angle lower than 5º). A simple phase inversion method was used to fabricate surperhydrophobic PHB surface. Chloroform was used as solvent, and a mixture of water and ethanol as the coagulation bath. The water contact angle was found to be 154.3±2.9º. Furthermore the surface was treated by argon plasma for different times, permitting the control of wettability of the PHB surface from the superhydrophobic to superhydrophilic regime.

Plasma polymerization‐modified bacterial polyhydroxybutyrate nanofibrillar scaffolds

AbstractThe design and the development of novel scaffold materials for tissue engineering have attracted much interest in recent years. Especially, the prepared nanofibrillar scaffold materials from biocompatible and biodegradable polymers by electrospinning are promising materials to be used in biomedical applications. In this study, we propose to produce low‐cost and cell‐friendly bacterial electrospun PHB polymeric scaffolds by using Alcaligenes eutrophus DSM 545 strain to PHB production. The produced PHB was characterized by Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared Spectroscopy (FTIR). Nanofibrous scaffolds were fabricated via electrospinning method that has a fiber diameter approximately 700–800 nm. To investigate cell attachment, cell growth, and antioxidant enzyme activity on positively and negatively charged PHB scaffold, PHB surface was modified by plasma polymerization technique using polyethylene glycol (PEG) and ethylenediamine (EDA). According to the results of superoxide dismutase (SOD) activity study, PEG‐modified nanofibrillar scaffolds indicated more cellular resistance against oxidative stress compared to the EDA modification. As can be seen in cell proliferation results, EDA modification enhanced the cell proliferation more than PEG modification, while PEG modification is better as compared with nonmodified scaffolds. In general, through plasma polymerization technique, surface modified nanofibrillar structures are effective substrates for cell attachment and outgrowth. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Display of Functionally Active PHB Depolymerase onEscherichia ColiCell 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.

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.

Degradation and Adsorption Characteristics of PHB Depolymerase As Revealed by Kinetics of Mutant Enzymes with Amino Acid Substitution in Substrate-Binding Domain

Extracelluar Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ(RpiT1)) from Ralstonia pickettii T1 adsorbs to PHB surface via its substrate-binding domain (SBD) to enhance PHB degradation. Our previous study combining PCR random mutagenesis with the determination of PHB degradation levels of mutant enzymes suggested that Ser, Tyr, Val, Ala, and Leu residues in SBD are probably involved in the enzymatic adsorption to and degradation of PHB. In the present study, the effects of mutations at Leu441, Tyr443, and Ser445 on PHB degradation were investigated because these residues were predicted to form a beta-sheet structure and orient in the same direction to interact possibly directly with the PHB surface. Purified L441H, Y443H, and S445C mutant enzymes were prepared, and their CD spectra and hydrolytic activities for water-soluble substrates were found to be identical to those of wild-type enzyme, indicating that these mutations have no influence on their structures and their ability to cleave the ester bond. In contrast, the PHB-degrading activity of these mutants differed from that of the wild type: L441H and Y443H enzymes had lower PHB-degrading activity than their wild-type counterpart, whereas S445C had higher activity. Kinetic analysis of PHB degradation by the mutants suggested that the hydrophobic residues at these positions are important for the enzyme adsorption to the PHB surface, and such substitutions as Y443H and S445C may more effectively disrupt the PHB surface to enhance the hydrolysis of PHB polymer chains than the wild-type enzyme. Surface plasmon resonance (SPR) analysis revealed that the three substitutions mentioned above altered the association phase rather than the dissociation phase in the enzyme adsorption to the polymer surface.

Effect of composition of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) on growth of fibroblast and osteoblast

Films made of poly (3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx) consisting of 5%, 12% and 20% hydroxyhexanoate (HHx), respectively, were evaluated for biomedical application in comparison with poly ( l-Lactide) (PLA). With the increase of HHx content in PHBHHx, the polymer surface properties changed accordingly. P(HB-co-20%-HHx) had the smoothest surface while PHB surface was most hydrophilic among the evaluated PHB and all the PHBHHx. All PHBHHx also showed strong protein affinity and biocompatibility. It was found that fibroblast and osteoblast had different responses to these polymers: fibroblast cells favored P(HB-co-20%-HHx), yet osteoblast cells preferred P(HB-co-12%-HHx). PHB and all PHBHHx appeared to have better biocompatibility for fibroblast and osteoblast compared with PLA. Polymers possessing diferent surface properties may help meet different cellular requirements. Combined with their good mechanical properties for elongation and adjustable biocompatibility, PHBHHx may meet the needs of growth requirements of different tissues and cells.

Effects of higher-order structure of poly(3-hydroxybutyrate) on its biodegradation. II. Effects of crystal structure on microbial degradation

As one of a series of studies concerning the relationship between the higher-order structure and the biodegradability of a biodegradable plastic, the effects of the crystal structure of the plastic on microbial degradation were investigated. Bacterial poly(d-(−)-3-hydroxybutyrate) (PHB) films which had a wide range of crystallinity were prepared by the melt-quenching method. Results of the microbial degradation indicated that the development of crystallinity evidently depressed the microbial degradability. From scanning electron microscopy (SEM) observations, it is suggested that the microbial degradation proceeded in at least two manners. One was preferential degradation of the amorphous region leaving the crystalline lamellae intact, which was considered to be a homogeneous enzymatic degradation over the surface. The other was nonpreferential spherical degradation on the surface. The SEMs indicate that the spherical holes were the result of colonization by degrading bacteria. The holes varied in size and number with the change of crystal structure. Therefore, it is considered that the crystal structure of PHB also influenced the physiological behavior of the degrading bacteria on the PHB surface.

Effects of higher‐order structure of poly(3‐hydroxybutyrate) on its biodegradation. I. Effects of heat treatment on microbial degradation

AbstractTo clarify how the biodegradation of plastic is affected by physical properties based on the plastic's higher‐order structure, the microbial degradability of heat‐treated poly(3‐hydroxybutyrate) (PHB) samples was studied. Cast‐film samples and fibrous samples prepared with heat treatment were degraded by an isolated degrading bacterium: strain SC‐17. Consequently, it was found that the higher‐order structure properties such as crystallinity and modulus of elasticity of PHB acted as suppression factors. SEM observations of the degraded PHB films showed unique degradation patterns such as spherical and tubular holes on the surfaces and cells of SC‐17 in process of degrading the PHB surface. These patterns are thought to result from colonization of SC‐17 on the surfaces. From the results, it was suggested that the surface of the PHB was important as a growing field for the degrading bacterium and that the higher‐order structure properties of the PHB affected not only the degradation reaction but also the colonization of the degrading bacterium on the surface. © 1992 John Wiley & Sons, Inc.