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Terumi Saito - One of the best experts on this subject based on the ideXlab platform.

  • Secretion pathway for the poly(3-hydroxybutyrate) depolymerase in Ralstonia pickettii T1.
    Antonie van Leeuwenhoek, 2008
    Co-Authors: Akiko Sugimoto, Mari Shiraki, Sachie Hatakeyama, Terumi Saito
    Abstract:

    The extracellular poly(3-hydroxybutyrate) depolymerase from Ralstonia pickettii T1 has been purified, its function and character investigated in detail, and its gene cloned and sequenced. However, the mechanism by which this enzyme is secreted has not been elucidated. A mutant unable to degrade poly(3-hydroxybutyrate), N17, was obtained with the random insertion of a mini-transposon, Tn5. Western analysis using antiserum against the poly(3-hydroxybutyrate) depolymerase of Ralstonia pickettii T1, revealed that N17 accumulated the poly(3-hydroxybutyrate) depolymerase in the periplasm and cytoplasm, and did not secrete the enzyme into the external medium. It was also found that 3-hydroxybutyrate-oligomer hydrolase was secreted but inactive. The disrupted gene in N17, depO, was analyzed by Southern hybridization and its nucleotide sequence was determined. One complete open reading frame was found in the cloned 2.3-kbp DNA fragment. From a BLAST search, this gene product was found to be homologous to PulO of Ralstonia eutropha JMP134 (60% identity) and XcpA of Pseudomonas aeruginosa (60% identity). These proteins are prepilin peptidase/N-metyltransferases, a component of the Type II secretion pathway. DepO also had the four cysteines highly conserved in most prepilin peptidases at the same positions. The transcript of depO was examined by Northern hybridization using depO as a probe. In the total RNA of Ralstonia pickettii T1 in the early stationary phase, a band at 2.6-kb was detected, suggesting depO to be a functional gene. In this study, it was found that poly(3-hydroxybutyrate) depolymerase was secreted by the Type II pathway.

  • Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16
    Journal of bacteriology, 2005
    Co-Authors: Teruyuki Kobayashi, Keiichi Uchino, Tomoko Abe, Yuya Yamazaki, Terumi Saito
    Abstract:

    Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.

  • Biopolymers Online - Intracellular Degradation of Polyhydroxyalkanoates (PHAs)
    Biopolymers Online, 2002
    Co-Authors: Terumi Saito, Teruyuki Kobayashi
    Abstract:

    Introduction Historical Outline Intracellular PHA Depolymerase Endogenous Degradation of PHA Intracellular mcl-PHA Depolymerase Intracellular Poly(3HB) Depolymerase Intracellular d(–)-3-Hydroxybutyrate Oligomer Hydrolase Other Enzymes Related to PHA Degradative Metabolism d(–)-3-Hydroxybutyrate Dehydrogenase Acetoacetyl-CoA Transferase and Acetoacetyl-CoA Synthetase 3-Ketothiolase Outlook and Perspectives Patents Keywords: intracellular poly(3HB) depolymerase; intracellular PHA depolymerase; poly(3HB) depolymerase; 3-hydroxybutyrate dehydrogenase; phaZ; succinyl-CoA transferase; acetoacetyl-CoA synthetase; 3-hydroxybutyrate oligomer hydrolase; 3-hydroxybutyrate dimer hydrolase; 3-ketothiolase; Ralstonia eutropha; Azotobacter beijerinkii; Zoogloea ramigera; Legionella pneumophila; Pseudomonas oleovorans; Pseudomonas aeruginosa; Hydrogenophaga pseudoflava; Rhodospirillum rubrum; Bacillus megaterium.

  • Determination of the active sites serine of the poly (3-hydroxybutyrate) depolymerases of Pseudomonas lemoignei (PhaZ5) and of Alcaligenes faecalis
    FEMS microbiology letters, 1996
    Co-Authors: Takeyuki Shinohe, Terumi Saito, Masaki Nojiri, Thomas Stanislawski, Dieter Jendrossek
    Abstract:

    Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S138 ( P. lemoignei) and S139 ( A. faecalis) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase ☐ of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S195 ( P. lemoignei) and S196 ( A. faecalis), resulted in mutant proteinswith almost full activity and proved that S195 and S196 are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.

  • Radiation-induced degradation of poly(3-hydroxybutyrate) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
    Polymer Degradation and Stability, 1994
    Co-Authors: Hiroshi Mitomo, Yuhei Watanabe, Isao Ishigaki, Terumi Saito
    Abstract:

    Poly(3-hydroxybutyrate) {P(3HB)} and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) {P(3HB-co-3HV)} were irradiated with γ-rays at 25°C in air and in vacuum. Melting points (Tm) and glass-transition temperatures (Tg) were measured by differential scanning calorimetry. Number-average molecular weights (Mn) were analyzed by gel permeation chromatography. No significant differences were observed between Tm values of P(3HB) and P(3HB-co-3HV) irradiated in air and in vacuum, which decreased almost linearly with increasing irradiation dose. The Mn values of both samples decreased sharply with increasing dose, reflecting typical random chain scission. The decrease in Mn of the sample irradiated in vacuum was smaller than that irradiated in air with the same dose, implying the occurrence of crosslinking. The Tg values for both polymers irradiated in vacuum remained almost unchanged over a wide dose range, while those irradiated in air decreased as the irradiation dose increased. Both the Tm and Tg of samples irradiated in air were inversely proportional to Mn. Biodegradability was clearly promoted with decreasing molecular weight.

Guo-qiang Chen - One of the best experts on this subject based on the ideXlab platform.

  • Microbial production of 4-hydroxybutyrate, poly-4-hydroxybutyrate, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by recombinant microorganisms
    Applied microbiology and biotechnology, 2009
    Co-Authors: Lei Zhang, Zhen-yu Shi, Guo-qiang Chen
    Abstract:

    4-Hydroxybutyrate (4HB) was produced by Aeromonas hydrophila 4AK4, Escherichia coli S17-1, or Pseudomonas putida KT2442 harboring 1,3-propanediol dehydrogenase gene dhaT and aldehyde dehydrogenase gene aldD from P. putida KT2442 which are capable of transforming 1,4-butanediol (1,4-BD) to 4HB. 4HB containing fermentation broth was used for production of homopolymer poly-4-hydroxybutyrate [P(4HB)] and copolymers poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)]. Recombinant A. hydrophila 4AK4 harboring plasmid pZL-dhaT-aldD containing dhaT and aldD was the most effective 4HB producer, achieving approximately 4 g/l 4HB from 10 g/l 1,4-BD after 48 h of incubation. The strain produced over 10 g/l 4HB from 20 g/l 1,4-BD after 52 h of cultivation in a 6-L fermenter. Recombinant E. coli S17-1 grown on 4HB containing fermentation broth was found to accumulate 83 wt.% of intracellular P(4HB) in shake flask study. Recombinant Ralstonia eutropha H16 grew to over 6 g/l cell dry weight containing 49 wt.% P(3HB-13%4HB) after 72 h.

  • Physical properties and biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) blended with poly(3-hydroxybutyrate-co-4-hydroxybutyrate).
    Journal of biomaterials science. Polymer edition, 2009
    Co-Authors: Ling Luo, Xing Wei, Guo-qiang Chen
    Abstract:

    Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was blended with poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) to improve physical properties and biocompatibility of PHBHHx for a wide range of biomedical applications. PHBHHx was completely miscible with P3HB4HB in their blends. All the PHBHHx/P3HB4HB blends showed improved physical properties compared with PHBHHx, including higher thermal stability, flexibility and mechanical strength. All the blends had more hydrophilic surface, higher polar component and rougher surface than PHBHHx. The PHBHHx/P3HB4HB blend in 4:2 weight ratio showed the roughest surface and also had the highest chondrocyte viability among all the blends and the polymers tested, which was 59% higher than that on PHBHHx and 32% higher than that on P3HB4HB. The blend with 4:2 weight ratio also had the maximum cartilage-specific collagen II mRNA expression among all the blends and the polymers tested, which was 9-times higher than that on PHBHHx and 8-times higher than that on P3HB4HB. These results demonstrated that PHBHHx had improved physical properties and biocompatibility after blending with P3HB4HB. The blends could be used for cartilage tissue engineering.

  • Study on the biocompatibility of novel terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate).
    Journal of biomedical materials research. Part A, 2008
    Co-Authors: Yansheng Liang, Wei Zhao, Guo-qiang Chen
    Abstract:

    Terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) containing 5.3 mol % 3-hydroxyvalerate (3-HV) and 10.2 mol % 3-hydroxyhexanoate was obtained via microbial synthesis using recombinant Aeromonas hydrophila. For the first time in vitro biocompatibility of the terpolyester was evaluated in comparison with poly(L-lactic acid), poly (3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Scanning electron microscopy showed whorls-like morphology only on PHBVHHx film surface. Methylthiazol tetrazolium assay demonstrated that PHBVHHx was better than the above four materials in promoting cell attachment and proliferation of fibroblast cell line L929 and osteoblast cell line MC3T3, respectively. Histological study using rabbits proved that PHBVHHx was a fairly harmless implantable biomaterial. Thus, PHBVHHx with an adjustable mechanical properties, combined with its biocompatibility, are in the process of developing into a new generation of bioimplant material. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2008

  • Processability Modifications of Poly(3-hydroxybutyrate) by Plasticizing, Blending, and Stabilizing
    Journal of Applied Polymer Science, 2007
    Co-Authors: Liang Wang, Xiaojuan Wang, Wenfu Zhu, Xianyu Chen, Guo-qiang Chen
    Abstract:

    Poly(3-hydroxybutyrate) (PHB) was plasti- cized with dioctyl (o-)phthalate, dioctyl sebacate, and ace- tyl tributyl citrate (ATBC). The thermal properties, me- chanical properties, and melt flow ability were studied with differential scanning calorimetry, thermogravimetric analysis, a universal material testing machine, and a melt flow indexer. ATBC was revealed to be an efficient plasti- cizer, reducing the glass-transition temperature and increasing the thermoplasticization ability of PHB. We also blended poly(3-hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) and poly(3-hydroxybutyrate-co-4-hydroxybuty- rate) (P(3/4HB)) with PHB, ATBC, and antioxidant 1010 to overcome the brittleness of PHB and improve the melt flow stability of the materials. PHBHHx did little to improve the thermal processing but increased the fluidity of PHB, and P(3/4HB) toned the toughness of PHB. The addition of antioxidant 1010 enhanced the thermal stabili- zation of PHB. 2007 Wiley Periodicals, Inc. J Appl Polym Sci 107: 166-173, 2008

  • the application of polyhydroxyalkanoates as tissue engineering materials
    Biomaterials, 2005
    Co-Authors: Guo-qiang Chen, Qiong Wu
    Abstract:

    Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth conditions. They are generally biodegradable and thermoprocessable, making them attractive as biomaterials for applications in both conventional medical devices and tissue engineering. Over the past years, PHA, particularly poly 3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO) and its composites have been used to develop devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings. The changing PHA compositions also allow favorable mechanical properties, biocompatibility, and degradation times within desirable time frames under specific physiological conditions. This paper reviews what have been achieved in the PHA tissue engineering area and concluded that the PHA prospective will look very bright in the near future.

Luiz A. F. Coelho - One of the best experts on this subject based on the ideXlab platform.

  • Phase behavior of poly(3-hydroxybutyrate)/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) blends
    Fluid Phase Equilibria, 2007
    Co-Authors: Denise S. Conti, Maria Irene Yoshida, Sérgio Henrique Pezzin, Luiz A. F. Coelho
    Abstract:

    Miscibility, molecular interactions and crystallinity of blends of poly(3-hydroxybutyrate) [P(3HB)] with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] were studied in this work. P(3HB)/P(3HB-co-3HV)-6%3HV blends were prepared by casting and characterized by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The results for the glass transition temperatures (T g ) and the melting temperatures (T m ) showed that most of the blends are miscible in amorphous and melting phases. A good agreement between values of experimental T g S for miscible blends and those predicted by Fox equation was achieved. XRD analyses confirmed the crystallinity degrees found by DSC. Measurement of T m depression for the blends allowed to determine the Flory-Huggins interaction parameter (Χ 12 ) using the Nishi-Wang equation. The value of Χ 12 obtained is negative, indicating miscibility of the components of the blends, which is in agreement with DSC experiments.

  • Mechanical and Morphological Properties of Poly(3‐hydroxybutyrate)/Poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) Blends
    Macromolecular Symposia, 2006
    Co-Authors: Denise S. Conti, Sérgio Henrique Pezzin, Luiz A. F. Coelho
    Abstract:

    With the objective of developing new biodegradable materials, the mechanical properties and the morphology of blends of poly(3-hydroxybutyrate), P(3HB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), were studied in this work. P(3HB) (492 kg · mol−1)/P(3HB-co-3HV)-6%3HV (294.2 kg · mol−1) blends were prepared by injection in a wide range of proportions and characterized by mechanical behavior of tensile strength, Izod impact strength and hardness Shore D. According to the increase of the copolymer content in the blend, it was detected that the hardness Shore D and the maximum tensile strength presented a significant reduction, the elasticity modulus showed a significant reduction, the elongation at break presented a significant increase and the Izod impact strength practically remained constant. Scanning electronic microscopy (SEM) was carried out in the fractured surface of the samples obtained during the tests of tensile and impact strength. These analyses showed a morphology with fragile fracture for whole blends, agreeing with mechanical results previously reported.

  • Miscibility and crystallinity of poly(3-hydroxybutyrate)/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) blends
    Thermochimica Acta, 2006
    Co-Authors: Denise S. Conti, Maria Irene Yoshida, Sérgio Henrique Pezzin, Luiz A. F. Coelho
    Abstract:

    Abstract With the objective of developing new biodegradable materials, the miscibility and the crystallinity of blends of poly(3-hydroxybutyrate), P(3HB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), have been studied. P(3HB) (300 kg mol−1)/P(3HB-co-3HV)–10% 3HV (340 kg mol−1) blends were prepared by casting in a wide range of proportions, and characterized by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). The experimental values for the glass transition temperatures (Tg) are in good agreement with the values provided by the Fox equation, showing that the blends are miscible. It was observed that the Tg and the melting temperature (Tm) decreases with the increase in the P(3HB-co-3HV)–10% 3HV content, while the crystallization temperature (Tc) increases. FT-IR analyses confirmed the decrease on the crystallinity of P(3HB)/P(3HB-co-3HV)–10% 3HV blends with higher copolymer contents. Bands related to the crystallinity were changed, due to the copolymer content that produced miscible and less crystalline blends.

Zhiliu Feng - One of the best experts on this subject based on the ideXlab platform.

  • The kinetics of the thermal decomposition of poly(3-hydroxybutyrate) and maleated poly(3-hydroxybutyrate)
    Journal of Applied Polymer Science, 2002
    Co-Authors: Cheng Chen, Bin Fei, Shuwen Peng, Yugang Zhuang, Lisong Dong, Zhiliu Feng
    Abstract:

    The thermal decomposition mechanism of maleated poly(3-hydroxybutyrate) (PHB) was investigated by FTIR and H-1 NMR. The results of experiments showed that the random chain scission of maleated PHB obeyed the six-membered ring ester decomposition process. The thermal decomposition behavior of PHB and maleated PHB with different graft degree were studied by thermogravimetry (TGA) using various heating-up rates. The thermal stability of maleated PHB was evidently better than that of PHB. With increase in graft degree, the thermal decomposition temperature of maleated PHB gradually increased and then declined. Activation energy E. as a kinetic parameter of thermal decomposition was estimated by the Flynn-Wall-Ozawa and Kissinger methods, respectively. It could be seen that approximately equal values of activation energy were obtained by both methods.

  • Nonisothermal crystallization and melting behavior of poly(3-hydroxybutyrate) and maleated poly(3-hydroxybutyrate)
    European Polymer Journal, 2002
    Co-Authors: Cheng Chen, Bin Fei, Shuwen Peng, Yugang Zhuang, Lisong Dong, Zhiliu Feng
    Abstract:

    Nonisothermal crystallization and melting behavior of poly(3-hydroxybutyrate) (PHB) and maleated PHB were investigated by differential scanning calorimetry using various cooling rates. The results show that the crystallization behavior of maleated PHB from the melt greatly depends on cooling rates and its degree of grafting. With the increase in cooling rate, the crystallization process for PHB and maleated PHB begins at lower temperature. For maleated PHB, the introduction of maleic anhydride group hinders its crystallization, causing crystallization and nucleation rates to decrease, and crystallite size distribution becomes wider. The Avrami analysis, modified by Jeziorny, was used to describe the nonisothermal crystallization of PHB and maleated PHB. Double melting peaks for maleated PHB were observed, which was caused by recrystallization during the heating process.

Yasushi Shigeri - One of the best experts on this subject based on the ideXlab platform.