Ken-ichi Kasuya

Biodegradabilities of various aliphatic polyesters in natural waters

Eight types of aliphatic polyesters were prepared by both biosynthetic and chemosynthetic methods, and their biodegradation tests were carried out at 25 °C for 28 days under aerobic conditions in different environmental natural waters. Biodegradabilities of melt-crystallized polyester films were evaluated in a temperature-controlled reactor by monitoring the time-dependent changes in the biochemical oxygen demand (BOD) and weight loss (erosion) of polyester film. The biosynthetic poly(3-hydroxybutyrate-co-14% 3-hydroxyvalerate) was degraded at a rapid rate in all natural waters used, and the weight-loss and BOD biodegradabilities of the films were 100% and 78 ± 8% for 28 days, respectively. By contrast, the films of chemosynthetic poly(ethylene succinate) were eroded completely in freshwater within 10 days, whereas the films were hardly eroded after 28 days in seawater.

Evaluation of biodegradabilities of biosynthetic and chemosynthetic polyesters in river water

Abstract Biodegradabilities of 21 samples of biosynthetic and chemosynthetic polyesters were measured at 25 °C under aerobic conditions in a temperature-controlled reactor containing 200 ml of natural water from the river Arakawa (Saitama, Japan) as an inoculum, by monitoring the time-dependent changes in the biochemical oxygen demand (BOD), weight loss (erosion) of polyester film and dissolved organic carbon concentration (DOC) of the test solution. The microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxypropionate), were degraded in the river water at a rapid rate, and the weight-loss- and BOD-biodegradabilities of the majority of biosynthetic polyesters were 100 and 80% ± 5% for 28 days, respectively. In contrast, the biodegradabilities of chemosynthetic polyesters were strongly dependent on the chemical structure and decreased in the following order: poly(ethylene succinate) > poly(ϵ-caprolactone) > poly(ethylene adipate) > poly(butylene adipate) > poly(butylene sebacate) > poly(ethylene sebacate) = poly(butylene succinate) = poly(hexylene succinate) = poly(β-propiolactone).

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.

Enzymatic degradation of poly[(R)-3-hydroxybutyrate] by Comamonas testosteroni ATSU of soil bacterium

Abstract A bacterium capable of degrading poly(3-hydroxybutyrate) (P(3HB)) was isolated from the soil and identified as Comamonas testosteroni . The strain C.testosteroni ATSU excreted an extracellular PHB depolymerase and grew on P(3HB) as a sole carbon source. C.testosteroni also grew on 3-hydroxybutyrate P(3HB), malate, and poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (P(3HB- co -3HV)). The secretion of PHB depolymerase was induced in the presence of P(3HB) and P(3HB- co -3HV). The PHB depolymerase was purified to electrophoretic homogeneity from the culture medium by hydrophobic column chromatography, preparative isoelectric focusing and gel filtration, and its molecular weight was determined as 49 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The optimum activity of degrading P(3HB) by the depolymerase was observed at pH 8·5 and 70°C. The enzymatic hydrolysis of P(3HB) film produced water-soluble products composed of the monomer and dimer of 3-hydroxybutyric acid. The rate of production of the monomer and dimer increased to a maximum value with the concentration of PHB depolymerases, followed by a gradual decrease. The properties of PHB depolymerase from C.testosteroni ATSU were compared with those of PHB depolymerase from Alcaligenes faecalis TI, Pseudomonas pickettii YM-b and Comamonas testosteroni YM1004.

Kinetics of surface hydrolysis of poly[(R)-3-hydroxybutyrate] film by PHB depolymerase from Alcaligenes faecalis T1

The kinetics and mechanism of surface hydrolysis of poly[(R)-3-hydroxybutyrate] (P(3HB)) film have been studied with an extracellular polyhydroxybutyrate (PHB) depolymerase purified from Alcaligenes faecalis T1, The PHB depolymerase was stable at temperatures up to 40 °C in aqueous solution at pH values of 5.0–8.0. The HPLC analysis of water-soluble products showed that the primary product of the enzymatic hydrolysis of P(3HB) film was the dimer of 3-hydroxybutyric acid. The dimer was subsequently hydrolyzed to monomer by the PHB depolymerase in the aqueous solution. A kinetic study of the enzymatic hydrolysis of P(3HB) film was carried out at temperatures in the range 25–37°C and pH values in the range 6.0–8.0 in 0.1–1.0 m potassium phosphate buffer. The kinetic data were accounted for in tersm of a two-step enzymatic reaction involving surface hydrolysis of the P(3HB) film which takes place via the two steps of adsorption and hydrolysis by the PHB depolymerase with the binding and catalytic domains. The heat of adsorption of the enzyme on the P(3HB) surface was as large as 43 kJmol, indicative of a strong interaction between the binding domain of the enzyme and the hydrophobic surface of the P(3HB) film. The activation energy of hydrolysis of the P(3HB) chain by the adsorbed enzyme was determined as 82 kJmol.

Adsorption kinetics of bacterial PHB depolymerase on the surface of polyhydroxyalkanoate films

The kinetics of adsorption and hydrolysis by an extracellular PHB depolymerase from Alcaligenes faecalis were studied at 37 degrees C on the surface of five types of polyhydroxyalkanoate (PHA) films. The films of poly[(R)-3-hydroxybutyrate] (P(3HP)), poly(3-hydroxypropionate) (P(3HP)), and poly(4-hydroxybutyrate)(P(4HB)) were hydrolyzed by the enzyme, while the films of poly[(S)-2-hydroxypropionate)(P(2HP)) and poly(6-hydroxyhexanoate)(P(6HH)) were not eroded. The PHB depolymerase with binding and catalytic domains adsorbed on the surface of all PHA films used, and the adsorption kinetics were found to obey the Langmuir isotherm. The cross-area per one molecule of enzyme binding to the surface of PHA film was estimated to be 17 +/- 8 (nm2/molecule). It has been concluded that the binding domain of enzyme is non-specific for the binding to the surface of PHA film, while the active site in a catalytic domain is specific for the hydrolysis of PHA molecules.

Biodegradation of poly(3-hydroxyalkanoic acids) fibers and isolation of poly(3-hydroxybutyric acid)-degrading microorganisms under aquatic environments

Biodegradabilities of poly[(R)-3-hydroxybutyric acid-co-14mol%(R)-3-hydroxyvaleric acid] (P(3HB-co-14%3HV)) monofilament fibers were evaluated at 25C for 28 days by monitoring the time-dependent changes in the biochemical oxygen demand (BOD) and weight-loss (erosion) of fibers under aerobic conditions in a temperature-controlled reactor containing natural waters from various aquatic environments (seawater, lake freshwater and river freshwater in Japan). Two types of fibers with diAerent diameters (213 and 493m) were used in this test. The biodegradabilities of fibers decreased in the following order: river freshwater > lake freshwater > seawater. By analyses of scanning electron microscopy and X-ray diAraction of eroded fibers, it has been concluded that the biodegradation proceeded from amorphous regions on the surface of fibers by the function of microorganisms in freshwater or seawater. In addition, 13 strains of P(3HB)-degrading bacteria were isolated from diAerent sources of seawater and identified. Majority of isolates grew well on P(3HB) or P(3HB-co-14%3HV) as sole carbon source and excreted extracellular polyhydroxybutyrate (PHB) depolymerases. # 1998 Elsevier Science Ltd. All rights reserved.

Synthesis and Verification of Biobased Terephthalic Acid from Furfural

Exploiting biomass as an alternative to petrochemicals for the production of commodity plastics is vitally important if we are to become a more sustainable society. Here, we report a synthetic route for the production of terephthalic acid (TPA), the monomer of the widely used thermoplastic polymer poly(ethylene terephthalate) (PET), from the biomass-derived starting material furfural. Biobased furfural was oxidised and dehydrated to give maleic anhydride, which was further reacted with biobased furan to give its Diels-Alder (DA) adduct. The dehydration of the DA adduct gave phthalic anhydride, which was converted via phthalic acid and dipotassium phthalate to TPA. The biobased carbon content of the TPA was measured by accelerator mass spectroscopy and the TPA was found to be made of 100% biobased carbon.

Studies on comonomer compositional distribution of the bacterial poly(3-hydroxybutyric acid-co-3-hydroxypropionic acid)s and crystal and thermal characteristics of their fractionated component copolyesters

Abstract Two different kinds of natural poly(3-hydroxybutyric acid-co-3-hydroxypropionic acid)s [P(3HB-co-3HP)] with individual 3HP comonomer contents of 36.5 and 68.1 mol. % were biosynthesized by the bacteria Alcaligenes latus, fed on the cosubstrates of (R)-3-hydroxybutyric acid (3HBA) and 3-hydroxypropionic acid (3HPA). Employing the mixed chloroform/n-heptane solvent, the bacterial products were found to be fractionated into a number of fractions, and the presence of broad comonomer compositional distributions was unambiguously demonstrated. A deep insight into fractionation provided by means of g.p.c. and n.m.r. spectrometry confirmed that the fractionations were predominantly governed by the factor of comonomer composition, while the effect of molecular weight was not so significant. 13C n.m.r. investigation of carbonyl diad sequence distributions for the fractionated copolyesters along with the original bacterial product clearly revealed that the copolymerization occurring in the bacterial cell bodies was virtually in accordance with the random copolymerization model, and this feature was, however, obscured due to the presence of comonomer compositional distribution. Further, the crystal structures and thermal behaviors were investigated via WAXD and d.s.c. for the fractionated copolyesters with 3HP comonomer content spanning the whole comonomer composition. These investigations showed that both 3HB- and 3HP-rich copolyesters formed different 21 helix crystal structures, respectively, corresponding to the P(3HB) and P(3HP) types of lattice structures with distinctive fiber repeats, while those bearing intermediate 3HP content (about 48–75 mol.%) appeared as the amorphous state. The existence of the minor comonomer drastically retarded the crystallizability of either 3HB- or 3HP-rich copolyester chains; however, the glass transition behaviors reflected that the chain mobility in the amorphous state was enhanced linearly with the increase in the 3HP unit content. Moreover, the growth rates and morphologies of spherulites for the fractionated copolyesters revealed that the growth rates were markedly suppressed by the incorporated minor comonomer, and the uncrystallizable chains were likely trapped in the interfibrillar regions.

Enzymatic degradation of poly[(R)-3-hydroxybutyrate]: secretion and properties of PHB depolymerase from Pseudomonas stutzeri

Abstract The secretion mechanism and properties of an extracellular polyhydroxybutyrate (PHB) depolymerase of Pseudomonas stutzeri YM1006, which had been isolated from sea water, were studied in the culture containing poly [( R )-3-hydroxybutyrate] [P(3HB)], ( R )-3-hydroxybutyrate [( R )-3HB] or (S)-3-hydroxybutyrate [( S )-3HB] as sole carbon source. P. stutzeri YM1006 was able to grow on P(3HB), ( R )-3HB and ( S )-3HB. The bacterium secreted a PHB depolymerase into the culture supernatant during growth on P (3HB) and ( R )-3HB, while it did not secrete the enzyme on ( S )-3HB. The PHB depolymerase was purified to electrophoretic homogeneity from the culture medium of P. stutzeri by hydrophobic interaction chromatography and its molecular weight was determined as 60 kDa by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures up to 50 °C in aqueous solution at pH values of 6 to 12. The optimum activity of degrading P (3HB) by the enzyme was observed at pH 7.0. A kinetic study of enzymatic hydrolysis of P(3HB) film was carried out at temperatures from 30 to 45 °C at pH 7.4 in 0.1 M potassium phosphate buffer. The enzymatic degradation product contained monomer and dimer of 3-hydroxybutyric acid, and the monomer was a major product over the whole range of enzyme concentration.