Fine structure and enzymatic degradation of poly[(R)-3-hydroxybutyrate] and stereocomplexed poly(lactide) nanofibers

Fiber morphology and crystalline structure of poly[(R)-3-hydroxybutyrate] (P(3HB)) and stereocomplexed poly(lactide) (PLA) nanofibers were investigated by using scanning and transmission electron microscopies and X-ray and electron diffractions. In the P(3HB) nanofibers spun from less than 1 wt% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solution, planar zigzag conformation (beta-form) as well as 2(1) helix conformation (alpha-form) structure was formed. Based on the electron diffraction measurement of single P(3HB) nanofiber, it was revealed that the molecular chains of P(3HB) align parallel to the fiber direction. From the enzymatic degradation test of P(3HB) nanofiber, it was shown that beta-form molecular chains are degraded more preferentially than alpha-form chains. Stereocomplexed PLA nanofibers were electrospun from 1 wt% poly(l-lactide)/poly(d-lactide) (PLLA/PDLA) solution in HFIP, which contains equal amounts of PLLA and PDLA. While as-spun stereocomplexed PLA nanofiber was amorphous, PLA nanofiber annealed at 100 degrees C contained only racemic crystal. It was supposed that the crystallization behavior of stereocomplexed PLA in the nanofiber is affected by the electrospinning process, which forcibly exerts the strain onto the polymer chains.     

Enzymatic hydrolysis of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4

Enzymatic degradability has been investigated for a series of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s (P(3HB-co-3HP)s) with 3-hydroxypropionate (3HP) unit contents from 11 to 86 mol % as well as poly(3-hydroxybutyrate) (P(3HB)) and chemosynthesized poly(3-hydroxypropionate) (P(3HP)). The behavior of degradation by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pikettii T1 and Acidovorax Sp. TP4, defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were compared in relation to the thermal properties and crystalline structures of the PHA samples elucidated by differential scanning calorimetry and wide-angle X-ray diffraction. The degradation products were characterized by high-performance liquid chromatography and one- (1D) and two-dimension (2D) (1)H NMR spectroscopy. It was found that the PHA depolymerase of Acidovorax Sp. TP4 showed degradation behavior different from that shown by depolymerase of R. pikettii T1. PHA depolymerase from Acidovorax Sp. TP4 degraded the P(3HB-co-3HP) films with lower crystallinity in higher rates than those with higher crystallinity, no matter what kinds of crystalline structures they formed. In contrast, PHA depolymerase from R. pikettii T1 degraded P(3HB-co-3HP) films forming P(3HB) crystalline structure in higher rates than those forming P(3HP)s. The increase in amorphous nature of the P(3HB-co-3HP) films with P(3HB)-homopolymer-like crystalline structure increases and then decreases the rate of degradation by depolymerase from R. pikettii T1. The 3-hydroxybutyrate (3HB) monomer was produced as a major product by the hydrolysis of P(3HB) film by PHA depolymerase from Acidovorax Sp. TP4. The P(3HB-co-3HP) films could be degraded into 3HB and 3-hydroxypropionate (3HP) monomer at last, indicating that the catalytic domain of the enzyme recognized at least two monomeric units as substrates. While the PHA depolymerase from R. pikettii T1 hydrolyzed P(3HB) film into 3HB dimer as a major product, and the catalytic domain recognized at least three monomeric units. The degradation behavior of P(3HB-co-3HP) films by the PHA depolymerase of Acidovorax Sp. TP4 could be distinguished from that by the depolymerase of R. pikettii T1.     

Enzymatic degradation processes of lamellar crystals in thin films for poly[(R)-3-hydroxybutyric acid] and its copolymers revealed by real-time atomic force microscopy

Enzymatic degradation processes of flat-on lamellar crystals in melt-crystallized thin films of poly[(R)-3-hydroxybutyric acid] (P(3HB)) and its copolymers were characterized by real-time atomic force microscopy (AFM) in a phosphate buffer solution containing PHB depolymerase from Ralstonia pickettii T1. Fiberlike crystals with regular intervals were generated along the crystallographic a axis at the end of lamellar crystals during the enzymatic degradation. The morphologies and sizes of the fiberlike crystals were markedly dependent on the compositions of comonomer units in the polyesters. Length, width, interval, and thickness of the fiberlike crystals after the enzymatic degradation for 2 h were measured by AFM, and the dimensions were related to the solid-state structures of P(3HB) and its copolymers. The width and thickness decreased at the tip of fiberlike crystals, indicating that the enzymatic degradation of crystals takes place not only along the a axis but also along the b and c axes. These results from AFM measurement were compared with the data on crystal size by wide-angle X-ray diffraction, and on lamellar thickness and long period by small-angle X-ray scattering. In addition, the enzymatic erosion rate of flat-on lamellar crystals along the a axis was measured from real-time AFM height images. A schematic glacier model for the enzymatic degradation of flat-on lamellar crystals of P(3HB) by PHB depolymerase has been proposed on the basis of the AFM observations.     

Side-chain effect of second monomer units on crystalline morphology, thermal properties, and enzymatic degradability for random copolyesters of (R)-3-hydroxybutyric acid with (R)-3-hydroxyalkanoic acids

Three types of random copolymers with 94 mol % (R)-3-hydroxybutyric acid (3HB) and 6 mol % (R)-3-hydroxyalkanoic acids with different side-chain lengths, (R)-3-hydroxypentanoic acid (3HV), (R)-3-hydroxyhexanoic acid (3HHx), and medium-chain-length (R)-3-hydroxyalkanoic acids (mcl-3HA, C8-C12), were prepared by biological synthetic techniques. The solid-state structure and thermal properties of melt-crystallized films for copolymers were characterized by means of wide-angle X-ray diffraction, small-angle X-ray scattering, differential scanning calorimetry, and optical microscopy. The randomly distributed second monomer units, except for 3HV in copolyesters, act as defects of the P(3HB) crystal and are excluded from the P(3HB) crystalline lamellae. The lamellar thickness of copolymers decreased with an increase in the side-chain length of second monomer units. In addition, the growth rate of spherulites decreased with an increase in the carbon numbers of second monomer units at an identical crystallization temperature. These results indicate that a steric bulkiness of the second monomer unit affects the crystallization of (R)-3HB segments in random copolyesters. An enzymatic degradation test of melt-crystallized copolymer films was carried out in the presence of PHB depolymerase from Alcaligenes faecalis T1. Erosion rate of copolyesters was dependent on both the crystallinity and the lamellar thickness of samples. As the result, the rate of enzymatic degradation for copolymer films increased with an increase in the carbon numbers of second monomer units.     

Time-resolved X-ray diffraction study on poly[(R)-3-hydroxybutyrate] films during two-step-drawing: generation mechanism of planar zigzag structure

Uniaxially oriented films with high tensile strength were processed from ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] (P(3HB)) by a method combining hot-drawing near the melting point of the polymer and two-step-drawing at room temperature. In a two-step-drawn and subsequently annealed film, P(3HB) molecular chains fall into two states: 2/1 helix (alpha-form) and planar zigzag (beta-form) conformations. The mechanism for generating the beta-form during two-step-drawing was investigated by time-resolved synchrotron wide- and small-angle X-ray scattering measurements (WAXD and SAXS), together with the measurement of stress-strain curves. It was found that the improvement of mechanical properties is due to not only the orientation of molecular chains but also the generation of the beta-form during the drawing. The crystal and molecular structures of the alpha-form remained unchanged until the yield point of the stress-strain curve. At the yield point, the long period obtained from SAXS doubled and a new reflection indicative of the beta-form was observed on the equatorial line in WAXD. The intensity of the reflection from the beta-form increased with an increase in the two-step-drawing ratio at room temperature. The SAXS pattern changed from a two-point reflection along the meridian to a cross pattern with streaking on the equatorial line, demonstrating the close alignment of shish-kebab structures. The reflection intensity, crystal orientation and crystal size of the alpha-form decreased during two-step-drawing. Based on these results, the beta-form is mainly introduced from the orientation of free molecular chains in the amorphous regions between alpha-form lamellar crystals, but the structural transformation of molecular chains also occurs from the alpha-form to the beta-form at the deformed lamellar crystals.     

Nonhydrolytic fragmentation of a poly[(R)-3-hydroxybutyrate] single crystal revealed by use of a mutant of polyhydroxybutyrate depolymerase

This paper reports the initial process of the enzymatic degradation of solution-grown lamellar single crystals of bacterial poly[(R)-3-hydroxybutyrate] (P(3HB)) with an extracellular polyhydroxybutyrate (PHB) depolymerase purified from Alcaligenes faecalis T1. We used a hydrolytic-activity-disrupted mutant of the PHB depolymerase in order to avoid the influence of hydrolytic reaction in the system. The effect of addition of the mutant enzyme upon the P(3HB) single crystals was investigated by turbidimetric assay, high-performance liquid chromatography (HPLC), and atomic force microscopy (AFM). Suspension turbidity of the P(3HB) single crystals increased after addition of the mutant enzyme having no hydrolytic activity. No soluble product from the P(3HB) single crystals with the mutant enzyme was detected by HPLC. AFM observation of the P(3HB) single crystals adsorbed on highly ordered pyrolytic graphite revealed that the mutant enzyme yielded a lot of lengthwise crystal fragments from the P(3HB) single crystals. On the basis of these results, we concluded that the mutant enzyme disturbs the molecular packing of the P(3HB) polymer chain around the loose chain packing region in the single crystal, resulting in the fragmentation. Therefore, it is suggested that the enzymatic degradation of P(3HB) single crystals with a wild-type PHB depolymerase progresses via three steps: (1) adsorption of the enzyme onto the surface of the single crystal; (2) disturbance of the molecular packing of P(3HB) polymer chain in the single crystal by the adsorbed enzyme; and (3) hydrolysis of the disturbed polymer chain by the adsorbed enzyme.     

Morphology and enzymatic degradation of oriented thin film of ultrahigh molecular weight poly[(R)-3-hydroxybutyrate

Thin films of ultrahigh molecular weight poly[(R)-3-hydroxybutyrate] (P(3HB)) were sheared and isothermally crystallized at 100 degrees C. Transmission electron microscopy and atomic force microscopy (AFM) observations revealed that thick fibrous textures, on which lamellae are overgrown normal to the long axis of the fibril, run parallel to the shearing direction. A selected area electron diffraction pattern taken from the fibrils exhibits a fiber pattern of P(3HB) alpha-modification, and the crystallographic c-axis (chain axis) of P(3HB) is set parallel to the long axis of the fibril. In situ AFM observations of enzymatic degradation for the thin film were performed with an extracellular P(3HB) depolymerase from Ralstonia pickettii T1 in a buffer solution. The film surface and thickness became rougher and thinner, respectively, with time after adding the enzyme. During the degradation, fine shish-kebab structures appeared gradually. This fact supports that the amorphous region in the film is preferentially degraded rather than the crystalline one by the depolymerase. The in situ AFM observations also revealed that one thick fibril in the original film is composed of three different states, namely, finer fibril (shish), stacked lamellae (kebab) in edge-on state, and the surrounding amorphous phase.     

Local sequence dependence of polyhydroxyalkanoic acid degradation in Hydrogenophaga pseudoflava

The first order intracellular degradation of various polyhydroxyalkanoic acid (PHA) inclusions in Hydrogenophaga pseudoflava cells was investigated by analyzing the compositional and microstructural changes of the PHA using gas chromatography, (13)C NMR spectroscopy, and differential scanning calorimetry. Two types of PHA, copolymers and blend-type polymers, were separately accumulated in cells for comparison. The constituent monomers were 3-hydroxybutyric acid (3HB), 4-hydroxybutyric acid (4HB), and 3-hydroxyvaleric acid (3HV). It was found that the 3HB-4HB copolymer was degraded only when the polymer contained a minimal level of 3HB units. With the cells containing a 3HB/4HB blend-type polymer, only poly(3HB) was degraded, whereas poly(4HB) was not degraded, indicating the totally inactive nature of the intracellular depolymerase against poly(4HB). On the basis of the magnitude of the first order degradation rate constants, the relative substrate specificity of the depolymerase toward the constituting monomer units was determined to decrease in the order 3HB > 3HV > 4HB. (13)C NMR resonances of the tetrad, triad, and dyad sequences were analyzed for the samples isolated before and after degradation experiments. The results showed that the intracellular degradation depended on the local monomer sequence of the copolymers. The relative substrate specificity of the depolymerase determined from the NMR local sequence analysis agreed well with that obtained from the kinetics analysis. It is suggested that, without isolation and purification of the intracellular PHA depolymerase and "native" PHA substrates, the relative specificity of the enzyme as well as the microstructural heterogeneity of the PHA could be determined by measuring in situ the first order degradation rate constants of the PHA in cells.     

Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei.

Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source.     

Production of poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudoflava

A Hydrogenophaga pseudoflava strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) [P(3HB-co-4HB)] having a high level of 4-hydroxybutyric acid monomer unit (4HB) from gamma-butyrolactone. In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) [P(3HB)] and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step. Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution 13C nuclear magnetic resonance spectroscopy. In order to synthesize 3HB-free poly(4-hydroxybutyric acid) [P(4HB)] homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB). The resulting polymer was free of 3HB. However, when the strain was grown on gamma-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced. One-step cultivation on gamma-butyrolactone required a rather long induction time (3 to 4 days). On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A. Thus, 3HB formation from gamma-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to gamma-butyrolactone, as is the case for a one-step culture. In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases. Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study.     

Structural effects on enzymatic degradabilities for poly[(R)-3-hydroxybutyric acid] and its copolymers.

Poly[(R)-3-hydroxybutyric acid] and its copolymers were prepared by biosynthetic and chemosynthetic methods. The films of polyesters were prepared by both the solution-cast and melt-crystallized techniques. The enzymatic degradation of polyester films was carried out at 37 degrees C in an aqueous solution (pH 7.4) of PHB depolymerase from Alcaligenes faecalis. The rate of enzymatic erosion on the solution-cast films increased markedly with an increase in the fraction of second monomer units up to 10-20 mol% to reach a maximum value followed by a decrease in the erosion rate. Analysis of the water-soluble products liberated during the enzymatic degradation of polyester films showed the formation of a mixture of monomers and oligomers of (R)-3HB and hydroxyalkanoic acids units, suggesting that the active site of PHB depolymerase recognizes at least three monomeric units as substrate for the hydrolysis of ester bonds in a polymer chain. The rate of enzymatic erosion of melt-crystallized polyester films decreased with an increase in crystallinity. PHB depolymerase predominantly hydrolyzed the polymer chains in the amorphous phase and subsequently eroded crystalline phase. In addition, the enzymatic degradation of crystalline phase by PHB depolymerase progressed from the edges of crystalline lamellar stacks. The enzymatic erosion rate of crystalline phase in polyester films decreased with an increase in the lamellar thickness.     

Kinetics and mechanism of heterogeneous hydrolysis of poly[(R)-3-hydroxybutyrate] film by PHA depolymerases

The kinetics and mechanism of enzymatic degradation on the surface of poly[(R)-3-hydroxybutyrate] (P[(R)-3HB]) film have been studied using three types of extracellular poly(hydroxyalkanoate) (PHA) depolymerases from Alcaligenes faecalis, Pseudomonas pickettii and Comamonas testosteroni. The monomer and dimer of 3-hydroxybutyric acid were produced during the course of the enzymatic degradation of P[(R)-3HB] film, and the rate of production was determined by monitoring the increase in absorbance at 210 nm on a spectrophotometer. The rate of enzymatic degradation increased to a maximum value with the concentration of PHA depolymerase, followed by a gradual decrease. The kinetic data were accounted for in terms of a heterogeneous enzymatic reaction, involving enzymatic degradation on the surface of P[(R)-3HB] film via two steps of adsorption and hydrolysis by a PHA depolymerase with binding and catalytic domains. The kinetic results suggest that the properties of the catalytic domains are very similar among the three PHA depolymerases, but that those of the binding domains are strongly dependent on the type of depolymerase.     

Degradation of poly(3-hydroxyoctanoic acid) [P(3HO)] by bacteria: purification and properties of a P(3HO) depolymerase from Pseudomonas fluorescens GK13.

Twenty-five gram-negative bacteria and one gram-positive bacterium capable of growing on poly(3-hydroxyoctanoic acid) [P(3HO)] as the sole source of carbon and energy were isolated from various soils, lake water, and activated sludge. Most of the isolates degraded only P(3HO) and copolymers of medium-chain-length (MCL) hydroxyalkanoic acids (HA). Except for the gram-positive strain, which was able to hydrolyze P(3HO) and poly(3-hydroxybutyric acid) [P(3HB)], no isolate was able to degrade polymers of short-chain-length HA, such as P(3HB) or poly(3-hydroxyvalerate) [P(3HV)]. All strains utilized a large variety of monomeric substrates for growth. All gram-negative strains, but not the gram-positive strain, accumulated poly(hydroxyalkanoic acids) (PHA), consisting of MCL HA, if they were cultivated under accumulation conditions. One strain, which was identified as Pseudomonas fluorescens GK13 (biovar V), was selected and the extracellular P(3HO) depolymerase of this strain was purified from the culture medium of P(3HO)-grown cells by chromatography with Octyl-Sepharose CL4B and by gel filtration with Superose 12. The relative molecular weights of the native and sodium dodecyl sulfate-treated enzymes were 48,000 and 25,000, respectively. The purified enzyme hydrolyzed P(3HO), copolymers of MCL HA, and para-nitrophenyl esters of fatty acids. P(3HB), P(3HV), and characteristic substrates for lipases, such as Tween 80 or triolein, were not hydrolyzed. The P(3HO) depolymerase of P. fluorescens GK13 was insensitive to phenylmethylsulfonyl fluoride and dithioerythritol, unlike other PHA depolymerases. The dimeric ester of 3-hydroxyoctanoic acid was identified as the main product of enzymatic hydrolysis of P(3HO).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the intracellular polyhydroxyalkanoate depolymerase gene of Paracoccus denitrificans and some properties of the gene product

Paracoccus denitrificans degraded poly(3-hydroxybutyrate) (PHB) in the cells under carbon source starvation. Intracellular poly(3-hydroxyalkanoate) (PHA) depolymerase gene (phaZ) was identified near the PHA synthase gene (phaC) of P. denitrificans. Cell extract of Escherichia coli carrying lacZ--phaZ fusion gene degraded protease-treated PHB granules. Reaction products were thought to be mainly D(--)-3-hydroxybutyrate (3HB) dimer and 3HB oligomer. Diisopropylfluorophosphonate and Triton X-100 exhibited an inhibitory effect on the degradation of PHB granules. When cell extract of the recombinant E. coli was used, Mg(2+) ion inhibited PHB degradation. However, the inhibitory effect by Mg(2+) ion was not observed using the cell extract of P. denitrificans.     

Degradation of poly(3-hydroxybutyrate) by soil streptomycetes

The ability of 64 soil streptomycetes to degrade poly(3-hydroxybutyrate) [P(3HB)] was evaluated on Pridham and Lyons mineral salts agar medium overlayered with the same medium containing 0.2% P(3HB). The streptomycete isolates were grown on this overlayered medium and the degradation was detected by the formation of clear zone surrounding the growth. Four potent degrader isolates identified as species of Streptomyces were selected. Degradation of P(3HB) by these isolates was studied for a period of 8 days. The rate of degradation increased with increase in concentration of P(3HB) in the medium while it decreased with the supplementation of readily utili- zable carbon sources like glucose, fructose and sucrose. All four isolates also degraded the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB–co–3HV)] in solid medium but to a lesser extent. However, the isolates were equally efficient in degrading P(3HB) in liquid medium.     

Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases

The substrate specificities of three extracellular polyhydroxybutyrate (PHB) depolymerases from Alcaligenes faecalis (PhaZ_Afa), Pseudomonas stutzeri (PhaZ_Pst), and Comamonas acidovorans (PhaZ_Cac), which are grouped into types A and B based on the position of a lipase box sequence in the catalytic domain, were examined for films of 12 different aliphatic polyesters. Each of these PHB depolymerases used was capable of hydrolyzing poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxypropionate) (P(3HP)), poly(4-hydroxybutyrate) (P(4HB)), poly(ethylene succinate) (PESU), and poly(ethylene adipate) (PEA) but could not hydrolyze another seven polyesters. In addition, the binding characteristics of substrate binding domains from PhaZ_Afa, PhaZ_Cac, and PHB depolymerase from Comamonas testosteroni (PhaZ_Cte) were studied by using fusions with glutathione S-transferase (GST). All of fusion proteins adsorbed strongly on the surfaces of polyester granules of P(3HB), P(3HP), and poly(2-hydroxypropionate) (P(2HP)) which was not hydrolyzed by the PHB depolymerases used in this study, while they did not bind on Avicel and chitin granules. The adsorption kinetics of the fusion proteins to the surface of P(3HB) and P(2HP) granules were found to obey the Langmuir isotherm. The cross-area per molecule of fusion protein bound to P(3HB) granules was estimated to be 12±4 nm²/molecule. It has been suggested that the active sites in catalytic domains of PHB depolymerases have a similar conformational structure, and that several amino acids in substrate-binding domains of PHB depolymerases interact specifically with the surface of polyesters.     

Adsorption and hydrolysis reactions of poly(hydroxybutyric acid) depolymerases secreted from Ralstonia pickettii T1 and Penicillium funiculosum onto poly[(R)-3-hydroxybutyric acid

Reaction processes of poly[(R)-3-hydroxybutyric acid] (P(3HB)) with two types of poly(hydroxybutyric acid) (PHB) depolymerases secreted from Ralstonia pickettii T1 and Penicillium funiculosum were characterized by means of atomic force microscopy (AFM) and quartz crystal microbalance (QCM). The PHB depolymerase from R. pickettii T1 consists of catalytic, linker, and substrate-binding domains, whereas the one from P. funiculosum lacks a substrate-binding domain. We succeeded in observing the adsorption of single molecules of the PHB depolymerase from R. pickettii T1 onto P(3HB) single crystals and the degradation of the single crystals in a phosphate buffer solution at 37 degrees C by real-time AFM. On the contrary, the enzyme molecule from P. funiculosum was hardly observed at the surface of P(3HB) single crystals by real-time AFM, even though the enzymatic degradation of the single crystals was surely progressed. On the basis of the AFM observations in air of the P(3HB) single crystals after the enzymatic treatments, however, not only the PHB depolymerase from R. pickettii T1 but also that from P. funiculosum adsorbed onto the surface of P(3HB) crystals, and both concentrations of the enzymes on the surface were nearly identical. This means both enzymes were adsorbed onto the surface of P(3HB) single crystals. Moreover, QCM measurements clarified quantitatively the differences in detachment behavior between two types of PHB depolymerases, namely the enzyme from R. pickettii T1 was hardly detached but the enzyme from P. funiculosum was released easily from the surface of P(3HB) crystals under an aqueous condition.     

Production of Poly(3-Hydroxybutyric Acid-Co-4-Hydroxybutyric Acid) and Poly(4-Hydroxybutyric Acid) without Subsequent Degradation by Hydrogenophaga pseudoflava

A Hydrogenophaga pseudoflava strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) [P(3HB-co-4HB)] having a high level of 4-hydroxybutyric acid monomer unit (4HB) from γ-butyrolactone. In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) [P(3HB)] and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step. Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution ¹³C nuclear magnetic resonance spectroscopy. In order to synthesize 3HB-free poly(4-hydroxybutyric acid) [P(4HB)] homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB). The resulting polymer was free of 3HB. However, when the strain was grown on γ-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced. One-step cultivation on γ-butyrolactone required a rather long induction time (3 to 4 days). On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A. Thus, 3HB formation from γ-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to γ-butyrolactone, as is the case for a one-step culture. In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases. Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study.     

Regulating the molar fraction of 4-hydroxybutyrate in Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by biological fermentation and enzymatic degradation

The regulation of 4-hydroxybutyrate (4HB) molar fraction in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] of a local isolate Cupriavidus sp. USMAA1020 was attempted by employing a feeding strategy through fed-batch fermentation in 100-L fermenter. The growth of Cupriavidus sp. USMAA1020 was enhanced by frequently feeding carbon and nitrogen at a ratio of 5 (C/N 5) using a DO-stat with cascade mode at 20% (v/v) dissolved oxygen (DO). The feeding of C/N 5 and the use of the DO-stat mode were able to regulate the 4HB composition from 0–67 mol% by sequential feeding of γ-butyrolactone and supplementing oleic acid. A high 4HB molar fraction of 67 mol% with a PHA concentration of 5.2 g/L was successfully obtained by employing this feeding strategy. Notably, enzymatic degradation carried out enhanced the 4HB composition of the copolymer synthesized. PHB depolymerase enzyme from Acidovorax sp. was used to degrade this P(3HB-co-70-mol%4HB) copolymer and the 4HB composition could be increased up to 83 mol%. The degradation process was observed by monitoring the time-dependent change in the weight loss of copolymer films. The percentage of weight loss of solvent-cast film increased proportionally up to 19% within 3 h, whereas salt-leached films showed 90% of weight loss within 3 h of incubation and were completely degraded by 4 h. The molecular weight (M _n ) of the films treated with enzyme demonstrated a slight decrease. SEM observation exhibited a rough surface morphology of the copolymer degraded with depolymerase enzyme.     

Expression of a novel hyaluronidase from Streptococcus zooepidemicus in Escherichia coli and its application for the preparation of HA oligosaccharides

Hyaluronan (HA) oligosaccharides which can stimulate angiogenesis and suppress the growth of tumors have attracted more and more attention. In order to prepare pure and well-defined oligosaccharides from high-molecular-weight HA in a rapid and simple manner, an enzymatic degradation method was developed, which included degradation with a novel recombinant hyaluronan lyase (HA lyase, hyaluronidase, or HAase) and gel permeation chromatography. The HAase protein was expressed in Escherichia coli with the expression vector pBV220. The HAase was purified and refolded, and specific activity of the enzyme solution was 3800 U/mg. HA was degraded with HAase at the optimized conditions, yielding 46% and 31% of HA disaccharides and HA tetrasaccharides, respectively. These HA oligosaccharides were conveniently separated by consecutive column chromatography on Bio-gel P6 and were identified by HPLC–MS.