Doi Y

References (30)

Title : Prescription of Antidementia Drugs and Antipsychotics for Elderly Patients in Japan: A Descriptive Study Using Pharmacy Prescription Data - Tsukada_2018_J.Geriatr.Psychiatry.Neurol__891988718785775
Author(s) : Tsukada T , Sato I , Matsuoka N , Imai T , Doi Y , Arai M , Fujii Y , Matsunaga T , Kawakami K
Ref : J Geriatr Psychiatry Neurol , :891988718785775 , 2018
Abstract : BACKGROUND: Antipsychotics are commonly used for managing behavioral and psychological symptoms of dementia among elderly patients with dementia receiving antidementia drugs (ADDs). However, the use of antipsychotics among these patients has not been investigated since 3 ADDs were approved in 2011 in Japan. METHOD: We conducted a descriptive study using pharmacy prescription data and identified patients aged >/=65 years who were newly prescribed donepezil, memantine, rivastigmine, and galantamine between January 1, 2012, and September 30, 2014. We determined the proportion of antipsychotic prescription and the factors affecting antipsychotic prescription using multivariable Cox proportional hazard models. RESULT: Of 13 876 patients, 1705 were memantine users, and the proportion of antipsychotic prescription among them was the highest (11.1%). Adjusted hazard ratios for donepezil, rivastigmine, and galantamine were 0.66, 0.56, and 0.66, respectively, relative to that for memantine. CONCLUSION: Compared to other ADD users, new memantine users were most likely to be prescribed antipsychotics.
ESTHER : Tsukada_2018_J.Geriatr.Psychiatry.Neurol__891988718785775
PubMedSearch : Tsukada_2018_J.Geriatr.Psychiatry.Neurol__891988718785775
PubMedID: 30016896

Title : Donepezil can improve ischemic muscle atrophy by activating angiomyogenic properties of satellite cells - Noguchi_2014_Circ.J_78_2317
Author(s) : Noguchi T , Kakinuma Y , Arikawa M , Okazaki K , Hoshino E , Iiyama T , Kubo T , Kitaoka H , Doi Y , Sato T
Ref : Circ J , 78 :2317 , 2014
Abstract : BACKGROUND: Saving more limbs of patients with peripheral arterial disease (PAD) from amputation by accelerating angiogenesis in affected limbs has been anticipated for years. We hypothesized that an anti-Alzheimer drug, donepezil (DPZ), can activate angiomyogenic properties of satellite cells, myogenic progenitors, and thus be an additional pharmacological therapy against PAD.Methods and Results:In a murine hindlimb ischemia model, we investigated the angiogenic effects of a clinical dose of DPZ (0.2 and its combination with cilostazol, a platelet aggregation inhibitor and a conventional therapeutic drug against PAD. The combination therapy most effectively improved skin coldness and most effectively upregulated vascular endothelial growth factor (VEGF)-producing satellite cells in ischemic hindlimbs. Computed tomography revealed that DPZ remarkably attenuated ischemic muscle atrophy and induced super-restoration in affected hindlimbs. The in vitro study with human aortic endothelial cells showed that DPZ or its combination with cilostazol effectively upregulated the expression of pAkt, hypoxia inducible factor-1alpha, and VEGF protein. Likewise, in primary cultured satellite cells, DPZ, alone or in combination, upregulated the expression of VEGF, interleukin-1beta, and fibroblast growth factor 2 protein.
CONCLUSIONS: The present results suggest that a clinical dosage of DPZ accelerates angiomyogenesis by directly acting on both endothelial and satellite cells. Therefore, DPZ is a potential additional choice for conventional drug therapy against PAD. (Circ J 2014; 78: 2317-2324).
ESTHER : Noguchi_2014_Circ.J_78_2317
PubMedSearch : Noguchi_2014_Circ.J_78_2317
PubMedID: 25070503

Title : Whole-genome assembly of Klebsiella pneumoniae coproducing NDM-1 and OXA-232 carbapenemases using single-molecule, real-time sequencing - Doi_2014_Antimicrob.Agents.Chemother_58_5947
Author(s) : Doi Y , Hazen TH , Boitano M , Tsai YC , Clark TA , Korlach J , Rasko DA
Ref : Antimicrobial Agents & Chemotherapy , 58 :5947 , 2014
Abstract : The whole-genome sequence of a carbapenem-resistant Klebsiella pneumoniae strain, PittNDM01, which coproduces NDM-1 and OXA-232 carbapenemases, was determined in this study. The use of single-molecule, real-time (SMRT) sequencing provided a closed genome in a single sequencing run. K. pneumoniae PittNDM01 has a single chromosome of 5,348,284 bp and four plasmids: pPKPN1 (283,371 bp), pPKPN2 (103,694 bp), pPKPN3 (70,814 bp), and pPKPN4 (6,141 bp). The contents of the chromosome were similar to that of the K. pneumoniae reference genome strain MGH 78578, with the exception of a large inversion spanning 23.3% of the chromosome. In contrast, three of the four plasmids are unique. The plasmid pPKPN1, an IncHI1B-like plasmid, carries the blaNDM-1, armA, and qnrB1 genes, along with tellurium and mercury resistance operons. blaNDM-1 is carried on a unique structure in which Tn125 is further bracketed by IS26 downstream of a class 1 integron. The IncFIA-like plasmid pPKPN3 also carries an array of resistance elements, including blaCTX-M-15 and a mercury resistance operon. The ColE-type plasmid pPKPN4 carrying blaOXA-232 is identical to a plasmid previously reported from France. SMRT sequencing was useful in resolving the complex bacterial genomic structures in the de novo assemblies.
ESTHER : Doi_2014_Antimicrob.Agents.Chemother_58_5947
PubMedSearch : Doi_2014_Antimicrob.Agents.Chemother_58_5947
PubMedID: 25070096

Title : Cloning of poly(aspartic acid) (PAA) hydrolase-1 gene from Pedobacter sp. KP-2 and hydrolysis of thermally synthesized PAA by its gene product - Hiraishi_2009_Macromol.Biosci_9_10
Author(s) : Hiraishi T , Masuda E , Kanayama N , Nagata M , Doi Y , Abe H , Maeda M
Ref : Macromol Biosci , 9 :10 , 2009
Abstract : Pedobacter sp. KP-2 can degrade and metabolize thermally synthesized alpha,beta-poly(D,L-aspartic acid) (tPAA), which contains 70% of unnatural beta-amide units, with high-molecular-weight. In this study, gene cloning and molecular characterization of PAA hydrolase-1 from KP-2 was carried out. Gene analysis reveals that deduced amino acid sequence of the enzyme shows a similarity to only that of PAA hydrolase-1 from Sphingomonas sp. KT-1. GPC and NMR analyses of the hydrolyzed products of tPAA by PAA hydrolase-1 of KP-2 indicate that this enzyme cleaves the beta-beta amide linkage via endo-mode to yield oligo(aspartic acid) from tPAA. Taking the composition of tPAA and the substrate specificity of PAA hydrolase-1 into consideration, the enzyme possibly plays a crucial role in tPAA biodegradation by KP-2.
ESTHER : Hiraishi_2009_Macromol.Biosci_9_10
PubMedSearch : Hiraishi_2009_Macromol.Biosci_9_10
PubMedID: 18756460
Gene_locus related to this paper: 9sphi-b6vqa9

Title : Combination of N149S and D171G mutations in Aeromonas caviae polyhydroxyalkanoate synthase and impact on polyhydroxyalkanoate biosynthesis - Tsuge_2007_FEMS.Microbiol.Lett_277_217
Author(s) : Tsuge T , Watanabe S , Shimada D , Abe H , Doi Y , Taguchi S
Ref : FEMS Microbiology Letters , 277 :217 , 2007
Abstract : Aeromonas caviae polyhydroxyalkanoate synthase (PhaC(Ac)) is an important biocatalyst for the synthesis of practically useful two-component polyhydroxyalkanoate copolymer, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. In a previous study, two PhaC(Ac) mutants that have a single amino acid substitution of either asparagine 149 by serine (N149S) or aspartate 171 by glycine (D171G) were isolated as higher active enzymes by means of evolutionary engineering. In this study, the synergistic effects of N149S and D171G double mutation (NSDG) in PhaC(Ac) on polyhydroxyalkanoate biosynthesis were investigated in recombinant Ralstonia eutropha. The PhaC(Ac) NSDG mutant showed enhanced incorporation of longer 3-hydroxyalkanoate (3HA) units into the polyhydroxyalkanoate copolymer from octanoate (3HA fraction: 18.5 mol%) and soybean oil (5.4 mol%) as a carbon source. Besides, the NSDG mutant synthesized P(3HB) homopolymer with a very high molecular weight (M(w)=368 x 10(4)) when fructose was used as a carbon source. Thus, a combination of the beneficial mutations synergistically altered enzymatic properties, leading to synthesis of a polyhydroxyalkanoate copolymer with enhanced 3HA fraction and increased molecular weight.
ESTHER : Tsuge_2007_FEMS.Microbiol.Lett_277_217
PubMedSearch : Tsuge_2007_FEMS.Microbiol.Lett_277_217
PubMedID: 18031343
Gene_locus related to this paper: aerca-PHAC

Title : Branched poly(lactide) synthesized by enzymatic polymerization: effects of molecular branches and stereochemistry on enzymatic degradation and alkaline hydrolysis - Numata_2007_Biomacromolecules_8_3115
Author(s) : Numata K , Srivastava RK , Finne-Wistrand A , Albertsson AC , Doi Y , Abe H
Ref : Biomacromolecules , 8 :3115 , 2007
Abstract : In this article the effects of the number of molecular branches (chain ends) and the stereochemistry of poly(lactide)s (PLAs) on the enzymatic degradation and alkaline hydrolysis are studied. Various linear and branched PLAs were synthesized using lipase PS (Pseudomonas fluorescens)-catalyzed ring-opening polymerization (ROP) of lactide monomers having different stereochemistries (L-lactide, D-lactide, and D,L-lactide). Five different alcohols were used as initiators for the ROP, and the monomer-to-initiator molar feed ratio was varied from 10 to 100 and 1000 for each branch in the polymer architecture. The properties of branched PLAs that would affect the enzymatic and alkaline degradations, i.e., the glass transition temperature, the melting temperature, the melting enthalpy, and the advancing contact angle, were determined. The PLA films were degraded using proteinase K or 1.0 M NaOH solution, and the weight loss and changes in the number average molecular weight (Mn) of the polymer were studied during 12 h of degradation. The results suggest that an increase in the number of molecular branches of branched PLAs enhances its enzymatic degradability and alkali hydrolyzability. Moreover, the change in Mn of the branched poly(L-lactide) (PLLA) by alkaline hydrolysis indicated that the decrease in Mn was in the first place dependent on the number of molecular branches and thereafter on the length of the molecular branch of branched PLA. The branched PLLA, poly(D-lactide) (PDLA), and poly(D,L-lactide) (PDLLA) differed in weight loss and change in Mn of the PLA segment during the enzymatic degradation. It is suggested that the branched PDLLA was degraded preferentially by proteinase K.
ESTHER : Numata_2007_Biomacromolecules_8_3115
PubMedSearch : Numata_2007_Biomacromolecules_8_3115
PubMedID: 17722879

Title : The crystal structure of polyhydroxybutyrate depolymerase from Penicillium funiculosum provides insights into the recognition and degradation of biopolyesters - Hisano_2006_J.Mol.Biol_356_993
Author(s) : Hisano T , Kasuya K , Tezuka Y , Ishii N , Kobayashi T , Shiraki M , Oroudjev E , Hansma H , Iwata T , Doi Y , Saito T , Miki K
Ref : Journal of Molecular Biology , 356 :993 , 2006
Abstract : Polyhydroxybutyrate is a microbial polyester that can be produced from renewable resources, and is degraded by the enzyme polyhydroxybutyrate depolymerase. The crystal structures of polyhydroxybutyrate depolymerase from Penicillium funiculosum and its S39 A mutant complexed with the methyl ester of a trimer substrate of (R)-3-hydroxybutyrate have been determined at resolutions of 1.71 A and 1.66 A, respectively. The enzyme is comprised of a single domain, which represents a circularly permuted variant of the alpha/beta hydrolase fold. The catalytic residues Ser39, Asp121, and His155 are located at topologically conserved positions. The main chain amide groups of Ser40 and Cys250 form an oxyanion hole. A crevice is formed on the surface of the enzyme, to which a single polymer chain can be bound by predominantly hydrophobic interactions with several hydrophobic residues. The structure of the S39A mutant-trimeric substrate complex reveals that Trp307 is responsible for the recognition of the ester group adjacent to the scissile group. It is also revealed that the substrate-binding site includes at least three, and possibly four, subsites for binding monomer units of polyester substrates. Thirteen hydrophobic residues, which are exposed to solvent, are aligned around the mouth of the crevice, forming a putative adsorption site for the polymer surface. These residues may contribute to the sufficient binding affinity of the enzyme for PHB granules without a distinct substrate-binding domain.
ESTHER : Hisano_2006_J.Mol.Biol_356_993
PubMedSearch : Hisano_2006_J.Mol.Biol_356_993
PubMedID: 16405909
Gene_locus related to this paper: penfu-PHAZ

Title : Enzymatic transformation of bacterial polyhydroxyalkanoates into repolymerizable oligomers directed towards chemical recycling - Kaihara_2005_Macromol.Biosci_5_644
Author(s) : Kaihara S , Osanai Y , Nishikawa K , Toshima K , Doi Y , Matsumura S
Ref : Macromol Biosci , 5 :644 , 2005
Abstract : The enzymatic transformation into an oligomer was carried out with the objective of developing the chemical recycling of bacterial polyesters. Poly(R-3-hydroxyalkanoate)s (PHAs), such as poly[(R-3-hydroxybutyrate)-co-12%(R-3-hydroxyhexanoate)] and poly[(R-3-hydroxybutyrate)-co-12%(R-3-hydroxyvalerate)], were degraded by granulated Candida antarctica lipase B immobilized on hydrophilic silica (lipase GCA) in a diluted organic solvent at 70 degrees C. The degradation products were cyclic oligomers having a molecular weight of a few hundreds. The obtained cyclic oligomer was readily repolymerized by the same lipase (lipase GCA) to produce the corresponding polyester in a concentrated solution. The cyclic oligomer was copolymerized with epsilon-caprolactone using lipase to produce the corresponding terpolymers having an Mw of 21,000. This is the first example of the enzymatic chemical recycling of bacterial PHAs using lipase. Poly(R-3-hydroxybutyrate) [P(3HB)] was also degraded into the linear-type R-3HB monomer to trimer by P(3HB)-depolymerase (PHBDP) in phosphate buffer at 37 degrees C. The degradation using PHBDP required a longer reaction time compared with the lipase-catalyzed degradation in organic solvent. The monomer composition of the oligomer depended on the origin of the PHBDP. The R-3HB monomer was predominately produced by PHBDP from Pseudomonas stutzeri, while the R-3HB dimer was produced by PHBDP from Alcaligenes faecalis T1. Repolymerization of these oligomers by lipase in concentrated organic solvent produced a relatively low-molecular-weight P(3HB) (e.g., Mw=2,000). Degradation of P(3HB) by lipase in organic solvent into repolymerizable cyclic oligomer and degradation of P(3HB) by PHBDP in buffer into hydroxy acid type R-3HB dimer.
ESTHER : Kaihara_2005_Macromol.Biosci_5_644
PubMedSearch : Kaihara_2005_Macromol.Biosci_5_644
PubMedID: 15988790

Title : Biosynthesis and compositional regulation of poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] in recombinant ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genes - Tsuge_2004_Macromol.Biosci_4_238
Author(s) : Tsuge T , Saito Y , Kikkawa Y , Hiraishi T , Doi Y
Ref : Macromol Biosci , 4 :238 , 2004
Abstract : A new strategy for bacterial polyhydroxyalkanoate (PHA) production by recombinant Ralstonia eutropha PHB(-)4 harboring mutated PHA synthase genes (phaC(Ac)) from Aeromona caviae was investigated. The strain harboring wild-type phaC(Ac) gene produced a PHA copolymer consisting of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate [P(3HB-co-3HHx)] with 3.5 mol-% of 3HHx fraction from soybean oil. When the mutants of phaC(Ac) gene were applied to this production system, 3HHx fraction in copolymers was varied in the range of 0-5.1 mol-%. Thus, the regulation of PHA copolymer compositions has been achieved by the use of mutated PHA synthase genes.
ESTHER : Tsuge_2004_Macromol.Biosci_4_238
PubMedSearch : Tsuge_2004_Macromol.Biosci_4_238
PubMedID: 15468213
Gene_locus related to this paper: aerca-PHAC

Title : An extra large insertion in the polyhydroxyalkanoate synthase from Delftia acidovorans DS-17: its deletion effects and relation to cellular proteolysis - Tsuge_2004_FEMS.Microbiol.Lett_231_77
Author(s) : Tsuge T , Imazu S , Takase K , Taguchi S , Doi Y
Ref : FEMS Microbiology Letters , 231 :77 , 2004
Abstract : The polyhydroxyalkanoate (PHA) synthase (PhaC(Da)) from Delftia acidovorans DS-17 (formerly Comamonas acidovorans) has a unique large insertion consisting of 40 amino acid residues in the alpha/beta hydrolase fold region. In order to examine whether this insertion is necessary for enzyme function, we generated a mutant gene where the nucleotides encoding the insertion sequence were deleted [phaC(Da)del(342-381)]. The ability of the mutant PhaC(Da) lacking the insertion sequence to produce PHA in recombinant Escherichia coli JM109 was compared with that of wild-type PhaC(Da). The results revealed that the mutant enzyme had approximately one fourth the activity of the wild-type enzyme. However, there was no significant difference in PHA content accumulated in cells harboring either the mutant PhaC(Da) or wild-type PhaC(Da) nor were there any differences in the molecular masses of the produced polymers. Therefore, we have concluded that the characteristic insertion is not indispensable for PHA synthesis. Also, slight cellular proteolysis in E. coli was found specifically for wild-type PhaC(Da) by Western blot analysis. This result prompted us to further examine the proteolytic stability of PhaC(Da) in D. acidovorans. Consequently, it has been suggested that the insertion region of PhaC(Da) is susceptible to cellular proteolysis during accumulation of PHA.
ESTHER : Tsuge_2004_FEMS.Microbiol.Lett_231_77
PubMedSearch : Tsuge_2004_FEMS.Microbiol.Lett_231_77
PubMedID: 14769470

Title : Genetic Analysis and Characterization of Poly(aspartic acid) Hydrolase-1 from Sphingomonas sp. KT-1 - Hiraishi_2003_Biomacromolecules_4_80
Author(s) : Hiraishi T , Kajiyama M , Tabata K , Yamato I , Doi Y
Ref : Biomacromolecules , 4 :80 , 2003
Abstract : Sphingomonas sp. KT-1 hydrolyzes poly(aspartic acid) (PAA) containing alpha- and beta-amide units and has at least two different types of PAA hydrolases. The PAA hydrolase-1 hydrolyzes selectively beta-beta amide units in PAA. Molecular cloning of PAA hydrolase-1 from Sphingomonas sp. KT-1 has been carried out to characterize its gene products. Genetic analysis shows that the deduced amino acid sequence of PAA hydrolase-1 has a similarity with those of the catalytic domain of poly(3-hydroxybutyric acid) (PHB) depolymerases from Alcaligenes faecalis AE122 and Pseudomonas lemoignei. Site-specific mutation analysis indicates that (176)Ser is a part of a strictly conserved pentapeptide sequence (Gly-Xaa-Ser-Xaa-Gly), which is the lipase box, and plays as an active residue.
ESTHER : Hiraishi_2003_Biomacromolecules_4_80
PubMedSearch : Hiraishi_2003_Biomacromolecules_4_80
PubMedID: 12523851
Gene_locus related to this paper: 9sphn-q7wsc1

Title : Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis, saturation mutagenesis, and in vitro recombination of the type II polyhydroxyalkanoate synthase gene - Takase_2003_J.Biochem_133_139
Author(s) : Takase K , Taguchi S , Doi Y
Ref : J Biochem , 133 :139 , 2003
Abstract : Type II synthase (PhaC1(Ps)) for polyhydroxyalkanoate (PHA) from Pseudomonas sp. 61-3 was subjected to an in vitro evolution system including PCR-mediated mutagenesis in order to improve the function of PhaC1(Ps) in terms of its ability to produce poly(3-hydroxybutyrate) [P(3HB)] in recombinant Escherichia coli. Based on our established in vivo assay system, two positions (Ser325 and Gln481) where mutations provided remarkable increases in P(3HB) synthesis were identified. Saturation mutagenesis at these positions was carried out to explore whether there might be more beneficial sequences for P(3HB) synthesis than those identified in the point mutation library. As a result, five single mutants [S325C (T) and Q481M (K, R)] gave rise to highly enhanced P(3HB) synthesis. Drastically enhanced P(3HB) synthesis (up to 340- to 400-fold the amount of that of the wild type) was further achieved by generation of all five variants of the double mutants combining the codons for residues 325/481. It is feasible that the replacement of Ser (specific for type II synthase) by Thr (specific for type I synthase) at position 325 resulted in acquiring greater P(3HB) synthesis ability as exhibited by type I synthases. The other hot spot, 481, that positively contributes to enhanced P(3HB) synthesis is located adjacent to a His479, a residue that forms a putative catalytic diad that can be inferred by sequence alignment.
ESTHER : Takase_2003_J.Biochem_133_139
PubMedSearch : Takase_2003_J.Biochem_133_139
PubMedID: 12761209
Gene_locus related to this paper: psesp-PHAC1

Title : Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1 - Kasuya_2003_Int.J.Biol.Macromol_33_221
Author(s) : Kasuya K , Takano T , Tezuka Y , Hsieh WC , Mitomo H , Doi Y
Ref : Int J Biol Macromol , 33 :221 , 2003
Abstract : A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) (P(3HB)) depolymerase was cloned from the genomic DNA of Marinobacter sp. DNA sequencing analysis revealed that the Marinobacter sp. P(3HB) depolymerase gene is composed of 1734bp and encodes 578 amino acids with a molecular mass of 61,757Da. A sequence homology search showed that the deduced protein contains the signal peptide, catalytic domain (CD), cadherin-type linker domain (LD), and two substrate-binding domain (SBD). The fusion proteins of glutathione S-transferase (GST) with the CD showed the hydrolytic activity for denatured P(3HB) (dP(3HB)), P(3HB) emulsion (eP(3HB)) and p-nitrophenylbutyrate. On the other hand, the fusion proteins lacking the SBD showed much lower hydrolytic activity for dP(3HB) compared to the proteins containing both CD and SBD. In addition, binding tests revealed that the SBDs are specifically bound not to eP(3HB) but dP(3HB). These suggest that the SBDs play a crucial role in the enzymatic hydrolysis of dP(3HB) that is a solid substrate.
ESTHER : Kasuya_2003_Int.J.Biol.Macromol_33_221
PubMedSearch : Kasuya_2003_Int.J.Biol.Macromol_33_221
PubMedID: 14607367
Gene_locus related to this paper: marsp-PHAZ

Title : Correlation between structure of the lactones and substrate specificity in enzyme-catalyzed polymerization for the synthesis of polyesters - Suzuki_2003_Biomacromolecules_4_537
Author(s) : Suzuki Y , Taguchi S , Hisano T , Toshima K , Matsumura S , Doi Y
Ref : Biomacromolecules , 4 :537 , 2003
Abstract : Small-size (4-membered) and medium-size (5-, 6-, and 7-membered) unsubstituted lactones as well as unsubstituted macrolides (12 and 13 membered) were subjected to the ring-opening polymerization using the extracellular PHB depolymerase from Alcaligenes faecalis T1 (PhaZ(Afa)). The characteristic reactivities of the lactones were discussed based on a tertiary structure model of the active site of the PhaZ(Afa). With respect to the ring-size of the lactones, the 4-membered beta-propiolactone and 6-membered delta-valerolactone (delta-VL) showed the highest polymerization activity, and delta-VL seemed to be the upper size limit for the molecular recognition of the narrow active site cleft of PhaZ(Afa). On the other hand, epsilon-caprolactone, 11-undecanolide, and 12-dodecanolide, which showed excellent polymerization activities by lipases, were scarcely polymerized by PhaZ(Afa). This was ascribed to the difference in the recognition sites between PhaZ(Afa) and lipase. In addition, the effect of the substrate-binding domain of PhaZ(Afa) and the enantioselective ring-opening polymerization of (R,S)-beta-butyrolactone ((R,S)-beta-BL) were studied. The substrate-binding domain lacking PhaZ(Afa) showed higher reactivities than PhaZ(Afa) for the polymerization of the lactones and that a significant enantioselectivity was observed at the early stage of the polymerization of (R,S)-beta-BL to produce the (R)-enriched optically active poly(3-hydroxybutyrate).
ESTHER : Suzuki_2003_Biomacromolecules_4_537
PubMedSearch : Suzuki_2003_Biomacromolecules_4_537
PubMedID: 12741767

Title : Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase - Kichise_2002_Appl.Environ.Microbiol_68_2411
Author(s) : Kichise T , Taguchi S , Doi Y
Ref : Applied Environmental Microbiology , 68 :2411 , 2002
Abstract : By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC(Ac), was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC(Ac) gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) [by LS5218 [fadR601 atoC(Con)] strain]. The expression vector for the phaC(Ac) gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).
ESTHER : Kichise_2002_Appl.Environ.Microbiol_68_2411
PubMedSearch : Kichise_2002_Appl.Environ.Microbiol_68_2411
PubMedID: 11976116
Gene_locus related to this paper: aerca-PHAC

Title : A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA - Maehara_2002_J.Bacteriol_184_3992
Author(s) : Maehara A , Taguchi S , Nishiyama T , Yamane T , Doi Y
Ref : Journal of Bacteriology , 184 :3992 , 2002
Abstract : Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.
ESTHER : Maehara_2002_J.Bacteriol_184_3992
PubMedSearch : Maehara_2002_J.Bacteriol_184_3992
PubMedID: 12081972
Gene_locus related to this paper: parde-Q9WX79

Title : Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer - Fukui_2002_Biomacromolecules_3_618
Author(s) : Fukui T , Abe H , Doi Y
Ref : Biomacromolecules , 3 :618 , 2002
Abstract : Recombinant Ralstonia eutropha capable of producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer [P(3HB-co-3HHx)] from fructose was engineered by introduction of genes for crotonyl-CoA reductase (CCR) from Streptomyces cinnamonensis (ccrSc) and for PHA synthase and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (phaC-JAc). In this recombinant strain, C6-acyl-CoA intermediates were provided via beta-ketothiolase-mediated elongation of butyryl-CoA, which was generated from crotonyl-CoA by the function of CCR. The recombinant strain could accumulate the copolyester up to 48 wt % of dry cell weight with 1.5 mol % of 3HHx fraction from fructose, when the expression of ccrSc under the control of the PBAD promoter was induced with 0.01% L-arabinose. The absence of L-arabinose or the deletion of ccrSc from the plasmid resulted in accumulation of poly(3-hydroxybutyrate) homopolymer, indicating the critical role of CCR in the formation of the 3-hydroxyhexanoate unit. Higher CCR activity obtained by the addition of a larger amount of L-arabinose did not affect the composition but reduced the intracellular content of the copolyester. The P(3HB-co-1.5 mol % 3HHx) copolyester produced from fructose by the recombinant R. eutropha showed relatively lower melting temperatures (150 degrees C and 161 degrees C) and lower crystallinity (48 +/- 5%) compared to those (175 degrees C and 60 +/- 5%) of P(3HB) homopolymer. It has been found that the incorporation of a small amount (1.5 mol %) of 3HHx units into P(3HB) sequences leads to a remarkable change in the solid-state properties of P(3HB) crystals. The present study demonstrates the potential of the engineered pathway for the production of copolyesters having favorable characteristics from inexpensive carbon resources.
ESTHER : Fukui_2002_Biomacromolecules_3_618
PubMedSearch : Fukui_2002_Biomacromolecules_3_618
PubMedID: 12005535

Title : In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis - Taguchi_2002_J.Biochem_131_801
Author(s) : Taguchi S , Nakamura H , Hiraishi T , Yamato I , Doi Y
Ref : J Biochem , 131 :801 , 2002
Abstract : In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Re)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.
ESTHER : Taguchi_2002_J.Biochem_131_801
PubMedSearch : Taguchi_2002_J.Biochem_131_801
PubMedID: 12038975
Gene_locus related to this paper: ralso-PHBC

Title : Analysis of mutational effects of a polyhydroxybutyrate (PHB) polymerase on bacterial PHB accumulation using an in vivo assay system - Taguchi_2001_FEMS.Microbiol.Lett_198_65
Author(s) : Taguchi S , Maehara A , Takase K , Nakahara M , Nakamura H , Doi Y
Ref : FEMS Microbiology Letters , 198 :65 , 2001
Abstract : Polymerase is a central enzyme involved in the biosynthesis of polyhydroxybutyrate (PHB), a well-known bacterial biodegradable polyester. In this study, we have established an in vivo assay system to analyze mutational effects of Ralstonia eutropha polymerase (termed PhbC(Re)) on the level of PHB accumulation in recombinant strains of Escherichia coli. This in vitro evolution system consists of a polymerase chain reaction-mediated random mutagenesis and two assay procedures, a plate assay using a PHB-staining dye and a high-pressure liquid chromatographic assay based on the converting reaction from PHB to crotonic acid. The distribution pattern of the PHB accumulation level of the mutant population using 378 clones arbitrarily selected, suggested that the present level of PhbC(Re) is high and well-optimized. It is noteworthy that many of the amino acid substitutions affecting the PHB accumulation occurred in the conserved positions or regions within an 'alpha/beta hydrolase fold' which is commonly found among hydrolytic enzymes. From a good correlation with the level of PHB accumulation, an activity estimation of the PhbC(Re) would be efficiently achieved by monitoring the level of PHB accumulation using the in vivo assay system established here.
ESTHER : Taguchi_2001_FEMS.Microbiol.Lett_198_65
PubMedSearch : Taguchi_2001_FEMS.Microbiol.Lett_198_65
PubMedID: 11325555

Title : Purification and characterization of poly(aspartic acid) hydrolase from Sphingomonas sp. KT-1 - Tabata_2001_Biomacromolecules_2_1155
Author(s) : Tabata K , Kajiyama M , Hiraishi T , Abe H , Yamato I , Doi Y
Ref : Biomacromolecules , 2 :1155 , 2001
Abstract : Poly(aspartic acid) (PAA) hydrolase was purified from Sphingomonas sp. KT-1 (JCM10459). The purified hydrolase degraded thermally synthesized PAA to oligomers. The molecular mass of PAA hydrolase was 30 kDa and the isoelectric point was 8.9. The optimum values of pH and temperature for PAA degradation were 10.0 and 40 degrees C, respectively. The investigation of the effect of inhibitors for the PAA-degrading activities has revealed that the PAA hydrolase is a serine-type hydrolase. The structural analysis of PAA-degraded products using (1)H and (13)C nuclear magnetic resonances has indicated that the purified enzyme hydrolyzes selectively the beta-amide linkage connecting with beta-aspartic acid units in PAA.
ESTHER : Tabata_2001_Biomacromolecules_2_1155
PubMedSearch : Tabata_2001_Biomacromolecules_2_1155
PubMedID: 11777387

Title : PhaR, a protein of unknown function conserved among short-chain-length polyhydroxyalkanoic acids producing bacteria, is a DNA-binding protein and represses Paracoccus denitrificans phaP expression in vitro - Maehara_2001_FEMS.Microbiol.Lett_200_9
Author(s) : Maehara A , Doi Y , Nishiyama T , Takagi Y , Ueda S , Nakano H , Yamane T
Ref : FEMS Microbiology Letters , 200 :9 , 2001
Abstract : A putative regulatory protein, PhaR, which was identified in the polyhydroxyalkanoic acid synthetic locus (phaZCPR) in Paracoccus denitrificans, was investigated. The PhaR protein purified from a recombinant Escherichia coli was estimated to be 22 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being consistent with the mass calculated from the nucleotide sequence. The molecular mass was determined to be 93 kDa by size-exclusion chromatography, suggesting that the protein formed a tetramer. A gel mobility shift assay showed that PhaR specifically bound to the intergenic region of phaC--phaP. In a cell-free protein synthesis system using E. coli S30 extract, the expression of the phaP gene was repressed by the addition of purified PhaR. These results suggest that PhaR is a DNA-binding protein and may play a role in the regulation of phaP gene expression.
ESTHER : Maehara_2001_FEMS.Microbiol.Lett_200_9
PubMedSearch : Maehara_2001_FEMS.Microbiol.Lett_200_9
PubMedID: 11410342
Gene_locus related to this paper: parde-PHAC , parde-Q9WX79

Title : Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases - Kasuya_1999_Int.J.Biol.Macromol_24_329
Author(s) : Kasuya K , Ohura T , Masuda K , Doi Y
Ref : Int J Biol Macromol , 24 :329 , 1999
Abstract : 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.
ESTHER : Kasuya_1999_Int.J.Biol.Macromol_24_329
PubMedSearch : Kasuya_1999_Int.J.Biol.Macromol_24_329
PubMedID: 10408639
Gene_locus related to this paper: alcfa-PHAZ , psesp-PHBDP1

Title : Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains - Ohura_1999_Appl.Environ.Microbiol_65_189
Author(s) : Ohura T , Kasuya KI , Doi Y
Ref : Applied Environmental Microbiology , 65 :189 , 1999
Abstract : The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZPst) of Pseudomonas stutzeri was cloned and sequenced. phaZPst was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B from Clostridium paraputrificum. The binding characteristics of SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S-transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.
ESTHER : Ohura_1999_Appl.Environ.Microbiol_65_189
PubMedSearch : Ohura_1999_Appl.Environ.Microbiol_65_189
PubMedID: 9872779
Gene_locus related to this paper: psesp-PHBDP1

Title : Genetic analysis of Comamonas acidovorans polyhydroxyalkanoate synthase and factors affecting the incorporation of 4-hydroxybutyrate monomer - Sudesh_1998_Appl.Environ.Microbiol_64_3437
Author(s) : Sudesh K , Fukui T , Doi Y
Ref : Applied Environmental Microbiology , 64 :3437 , 1998
Abstract : The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaCCa) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp beta-ketothiolase gene (phaACa). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaACa. The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB-4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism's PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.
ESTHER : Sudesh_1998_Appl.Environ.Microbiol_64_3437
PubMedSearch : Sudesh_1998_Appl.Environ.Microbiol_64_3437
PubMedID: 9726894
Gene_locus related to this paper: delac-PHAC

Title : Cloning and molecular analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3 - Matsusaki_1998_J.Bacteriol_180_6459
Author(s) : Matsusaki H , Manji S , Taguchi K , Kato M , Fukui T , Doi Y
Ref : Journal of Bacteriology , 180 :6459 , 1998
Abstract : Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer (poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]) consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In the phb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), beta-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed by phbBPs-phbAPs-phbCPs. Upstream from phbBPs was found the phbRPs gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbRPs gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbRPs in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of beta-galactosidase activity from a transcriptional phb promoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBACPs in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 and phaDPs), and the gene order was ORF1, phaC1Ps, phaZPs, phaC2Ps, and phaDPs. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida and Ralstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.
ESTHER : Matsusaki_1998_J.Bacteriol_180_6459
PubMedSearch : Matsusaki_1998_J.Bacteriol_180_6459
PubMedID: 9851987
Gene_locus related to this paper: psepf-PHAZ , psesp-PHAC1 , psesp-PHAC2 , psesp-PHBC

Title : Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae - Fukui_1997_J.Bacteriol_179_4821
Author(s) : Fukui T , Doi Y
Ref : Journal of Bacteriology , 179 :4821 , 1997
Abstract : A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.
ESTHER : Fukui_1997_J.Bacteriol_179_4821
PubMedSearch : Fukui_1997_J.Bacteriol_179_4821
PubMedID: 9244271
Gene_locus related to this paper: aerca-PHAC

Title : Cloning of the gene for poly(3-hydroxybutyric acid) depolymerase of Comamonas testosteroni and functional analysis of its substrate-binding domain - Shinomiya_1997_FEMS.Microbiol.Lett_154_89
Author(s) : Shinomiya M , Iwata T , Kasuya K , Doi Y
Ref : FEMS Microbiology Letters , 154 :89 , 1997
Abstract : A poly(3-hydroxybutyric acid) (PHB) depolymerase gene of Comamonas testosteroni YM1004 was cloned on Sau3AI fragment from genomic DNA into Escherichia coli DH5. Nucleotide sequence analysis dedicated a 1539 bp open reading frame encoding a protein 513 amino acid with a putative 25 residue signal peptide for secretion. The deduced amino acid sequence was very similar to that of PHB depolymerase of Comamonas sp. In order to understand the characteristics of substrate-binding domain of the depolymerase, we constructed its glutathione S-transferase (GST) fusion protein and investigated the ability of adsorption on PHB single crystals by using gold-conjugated antibody and transmission electron microscopy. The fusion protein adsorbed on PHB single crystals tightly and homogeneously, suggesting that binding domain contributes to the adsorption of enzyme on solid PHB without site specificity.
ESTHER : Shinomiya_1997_FEMS.Microbiol.Lett_154_89
PubMedSearch : Shinomiya_1997_FEMS.Microbiol.Lett_154_89
PubMedID: 9297825
Gene_locus related to this paper: comte-PHAZ

Title : Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater - Kasuya_1997_Appl.Environ.Microbiol_63_4844
Author(s) : Kasuya K , Inoue Y , Tanaka T , Akehata T , Iwata T , Fukui T , Doi Y
Ref : Applied Environmental Microbiology , 63 :4844 , 1997
Abstract : Comamonas acidovorans YM1609 secreted a polyhydroxybutyrate (PHB) depolymerase into the culture supernatant when it was cultivated on poly(3-hydroxybutyrate) [P(3HB)] or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] as the sole carbon source. The PHB depolymerase was purified from culture supernatant of C. acidovorans by two chromatographic methods, and its molecular mass was determined as 45,000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures below 37 degrees C and at pH values of 6 to 10, and its activity was inhibited by diisopropyl fluorophosphonate. The liquid chromatography analysis of water-soluble products revealed that the primary product of enzymatic hydrolysis of P(3HB) was a dimer of 3-hydroxybutyric acid. Kinetics of enzymatic hydrolysis of P(3HB) film were studied. In addition, a gene encoding the PHB depolymerase was cloned from the C. acidovorans genomic library. The nucleotide sequence of this gene was found to encode a protein of 494 amino acids (M(r), 51,018 Da). Furthermore, by analysis of the N-terminal amino acid sequence of the purified enzyme, the molecular mass of the mature enzyme was calculated to be 48,628 Da. Analysis of the deduced amino acid sequence suggested a domain structure of the protein containing a catalytic domain, fibronectin type III module as linker, and a putative substrate-binding domain. Electron microscopic visualization of the mixture of P(3HB) single crystals and a fusion protein of putative substrate-binding domain with glutathione S-transferase demonstrated that the fusion protein adsorbed strongly and homogeneously to the surfaces of P(3HB) single crystals.
ESTHER : Kasuya_1997_Appl.Environ.Microbiol_63_4844
PubMedSearch : Kasuya_1997_Appl.Environ.Microbiol_63_4844
PubMedID: 9406404
Gene_locus related to this paper: comac-PHAZCAC

Title : Cytochemical and immunocytochemical demonstration of acetylcholinesterase of the prenatal rat lower limb - Umezu_1993_Arch.Hist.Cytol_56_217
Author(s) : Umezu Y , Nagata N , Doi Y , Furukawa H , Sagara T , Hayashida T , Ogata H , Fujimoto S
Ref : Archives of Histology & Cytology , 56 :217 , 1993
Abstract : Acetylcholinesterase (AChE) activities in the prenatal rat lower limb were investigated by both cytochemistry and immunocytochemistry. Results indicate that the epidermal cells show immunoreactions of AChE at a limited stage at prenatal day 15, and mesenchymal cells which are occasionally in contact with the basal lamina or with the adjacent myotubes begin to show AChE activities at prenatal day 17. Such AChE-positive mesenchymal cells, involved in the formation of the muscular tissues, have almost disappeared in the subepidermis by prenatal day 19. This suggests that AChE independent of the neuromuscular system may be involved in the mesenchymal cell differentiation especially in the inductive process during myogenesis.
ESTHER : Umezu_1993_Arch.Hist.Cytol_56_217
PubMedSearch : Umezu_1993_Arch.Hist.Cytol_56_217
PubMedID: 8373663

Title : Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei - Mukai_1992_Int.J.Biol.Macromol_14_235
Author(s) : Mukai K , Yamada K , Doi Y
Ref : Int J Biol Macromol , 14 :235 , 1992
Abstract : 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.
ESTHER : Mukai_1992_Int.J.Biol.Macromol_14_235
PubMedSearch : Mukai_1992_Int.J.Biol.Macromol_14_235
PubMedID: 1504046