Updated on 2024/07/16

写真a

 
OIKAWA,Tadao
 
Organization
Faculty of Chemistry, Materials and Bioengineering Professor
Title
Professor
Contact information
メールアドレス
External link

Degree

  • Doctor of Agriculture ( 1992.7 )

Research Interests

  • 酵素工学

Research Areas

  • Life Science / Applied biochemistry

  • Life Science / Functional biochemistry

Education

  • Kyoto University   Graduate School, Division of Agriculture   Agricultural Chemistry

    1991 - 1992

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    Country: Japan

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  • University of Tsukuba   Second Cluster of College   College of Agriculture and Forestry

    - 1986

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    Country: Japan

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Research History

  • Kansai University/Professor

    2008.4

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  • Kansai University/Associate Professer

    1998.4

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  • Kansai University/Instructor

    1993.4 - 1998.3

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  • Kansai University/Assistant

    1991.4 - 1994.3

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Professional Memberships

Committee Memberships

  •   英文誌論文審査員  

    2005.5   

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Papers

  • Constitutive and high gene expression in the diaminopimelate pathway accelerates ε-poly-L-lysine production in Streptomyces albulus. International journal

    Fumihito Hasebe, Kazuya Adachi, Kazuya Yamanaka, Tadao Oikawa, Chitose Maruyama, Yoshimitsu Hamano

    The Journal of antibiotics   76 ( 9 )   522 - 531   2023.6

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    Language:English   Publishing type:Research paper (scientific journal)  

    Streptomyces albulus NBRC14147 produces a homopoly(amino acid), ε-poly-L-lysine (ε-PL). Due to its antibiotic activity, thermostability, biodegradability, and non-toxicity to humans, ε-PL is used as a food preservative. In this study, homology searches of diaminopimelate (DAP) pathway genes (dapB and dapE), in an S. albulus genome database, were shown to encode predicted enzymes using dapB or dapE in Escherichia coli strain complementation assays. We observed that dapB and dapE transcriptional levels were weak during ε-PL production stages. Therefore, we strengthened this expression using an ermE constitutive promoter. Engineered strains generated faster growth and ε-PL production rates when compared with the control strain. Moreover, maximum ε-PL yields in S. albulus, where dapB was constitutively expressed, were approximately 14% higher when compared with the control strain. These findings showed that enhanced lysine biosynthetic gene expression generated faster and higher ε-PL production levels.

    DOI: 10.1038/s41429-023-00636-9

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  • First enzymological characterization of selenocysteine β-lyase from a lactic acid bacterium, Leuconostoc mesenteroides Reviewed

    Tadao Oikawa, Kouhei Okajima, Kazuya Yamanaka, Shiro Kato

    Amino Acids   2022.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media {LLC}  

    DOI: 10.1007/s00726-022-03133-9

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  • Discovery of a Polyamino Acid Antibiotic Solely Comprising <scp>l</scp>-β-Lysine by Potential Producer Prioritization-Guided Genome Mining

    Kazuya Yamanaka, Hibiki Fukumoto, Naoki Yoshimura, Kenji Arakawa, Yasuo Kato, Yoshimitsu Hamano, Tadao Oikawa

    ACS Chemical Biology   17 ( 1 )   171 - 180   2022.1

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    Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

    DOI: 10.1021/acschembio.1c00832

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  • ポジティブネットワーク形成の主軸となる地域産品の特性、機能、条件―「天満天神の水」を対象とした計量分析的整理― Reviewed

    与謝野 有紀, 林 直保子, 老川 典夫, 山本 秀樹

    社会的信頼研究   2巻, 25-42   2021.8

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  • Molecular and Mechanistic Characterization of PddB, the First PLP-Independent 2,4-Diaminobutyric Acid Racemase Discovered in an Actinobacterial D-Amino Acid Homopolymer Biosynthesis Reviewed

    山中 一也, 老川 典夫

    Frontiers in Microbiology   12, 686023   2021.6

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  • The Stereocontrolled Biosynthesis of Mirror-Symmetric 2,4-Diaminobutyric Acid Homopolymers Is Critically Governed by Adenylation Activations. Reviewed International journal

    Kazuya Yamanaka, Hibiki Fukumoto, Munenori Takehara, Yoshimitsu Hamano, Tadao Oikawa

    ACS chemical biology   2020.6

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    Language:English   Publishing type:Research paper (scientific journal)  

    Among the four bioactive cationic homo-poly(amino acids) discovered in nature, two are mirror-image isomers of poly(2,4-diaminobutyric acid) (poly-Dab) whose biosynthesis has long been unexplained. Their structural analogy plausibly suggested that they could share a common biosynthetic pathway utilizing ε-poly(l-lysine) synthetase-like enzymology but with an unprecedented process for enantiomeric inversion of polymer building blocks. To investigate this possibility, we comparatively explored the biosynthesis of poly-l-Dab and its mirror-image isomer poly-d-Dab in Streptomyces celluloflavus USE31 and Streptoalloteichus hindustanus NBRC15115, respectively, through genome mining, genetic inactivation, and heterologous expression combined with biochemical assays. While they shared the same biosynthetic pathway, the poly-d-Dab biosynthetic gene cluster additionally harbored the racemase gene. The critical finding that poly-d-Dab synthetase, in contrast to the synthetase generating the l-isomer, selectively activated d-Dab through adenylation conclusively demonstrated that free diffusible d-Dab preactivationally generated by the racemase is directly activated to be incorporated into the polymer. Our study thus represents the first demonstration of the stereoselective biosynthesis of a nonribosomal peptide governed by adenylation activity for a d-amino acid other than alanine. In silico sequence comparison between poly-Dab synthetases allowed us to identify amino acid residues potentially responsible for the discrimination of Dab enantiomers. Our results will provide significant insight not only for the future discovery of novel bioactive cationic poly(amino acids) but also for the creation of designer nonribosomal peptides with d-configuration.

    DOI: 10.1021/acschembio.0c00321

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  • The Stereocontrolled Biosynthesis of Mirror-Symmetric 2,4-Diaminobutyric Acid Homopolymers Is Critically Governed by Adenylation Activations Reviewed

    山中 一也, 福本 響, 濱野 吉十, 老川 典夫

    ACS Chem. Biol.   5, 7, 1964–1973   2020.6

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  • Enhancement of metabolic flux toward ε-poly-l-lysine biosynthesis by targeted inactivation of concomitant polyene macrolide biosynthesis in Streptomyces albulus Reviewed

    山中 一也, 濱野 吉十, 老川 典夫

    J. Biosci. Bioeng   129, 558-564   2020.5

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  • 哺乳動物におけるD-アミノ酸代謝

    加藤 志郎, 老川 典夫

    微量栄養素研究   36, 95-101   2019.12

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  • Application of L-methionine γ-lyase in chiral amino acid analysis

    Shiro Kato, Kenji Inagaki, Tadao Oikawa

    Analytical Biochemistry   580   56 - 61   2019.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Academic Press Inc.  

    Here, a conventional chiral amino acid analysis method using high-performance liquid chromatography was coupled with a sample pretreatment using L-methionine γ-lyase from Pseudomonas putida ICR 3460 for the selective analysis of L-methionine and L-tryptophan. The sample was analyzed after the degradation of L-methionine with L-methionine γ-lyase, as L-methionine coelutes with L-tryptophan under the standard chiral amino acid analytical conditions used for precolumn derivatization with o-phthalaldehyde and N-acetyl-L-cysteine. The L-tryptophan in the sample was then eluted as a clearly separated peak and analyzed further. Since the L-methionine γ-lyase did not act on L-tryptophan, we were able to calculate the L-methionine or L-tryptophan concentration based on the data obtained from 2 individual runs: the sample with and without L-methionine γ-lyase pretreatment. The concentration of L-tryptophan was calculated from the data obtained from the sample with L-methionine γ-lyase pretreatment, while the concentration of L-methionine was calculated using the following equation: L-methionine concentration = {the data from the sample without L-methionine γ-lyase pretreatment}-{the data from the sample with L-methionine γ-lyase pretreatment}. Model samples containing authentic amino acids and a fermented food sample were analyzed by our method, and the calculated concentrations of L-methionine and L-tryptophan were consistently in agreement with the theoretical values.

    DOI: 10.1016/j.ab.2019.05.018

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  • Application of L-methionine gamma-lyase in chiral amino acid analysis Reviewed

    加藤志郎, 稲垣賢二, 老川 典夫

    Analytical Biochemistry   580, 56-61   2019.9

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  • 放線菌Streptoalloteichus hindustanusに見出した新規PLP非依存型Diaminobutyric acidラセマーゼの機能解析 Reviewed

    尾崎 僚, 福本 響, 濱野 吉十, 老川 典夫, 山中 一也

    日本生物工学会大会講演要旨集   2019年   261 - 261   2019.8

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    Language:Japanese   Publisher:(公社)日本生物工学会  

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  • シロイヌナズナ(Arabidopsis thaliana)における亜セレン酸ナトリウムおよびセレノ-L-メチオニン曝露後の発現変動遺伝子の網羅的解析 Reviewed

    大塚 政志, 細見 亮太, 老川 典夫, 福永 健治, 吉田 宗弘

    Trace Nutrients Research   35, 21-27   2018.12

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  • 乳酸菌によるセレンの代謝と蓄積

    岡島 浩平, 老川 典夫

    微量栄養素研究   35, 87-91   2018.12

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  • The first identification and characterization of a histidine-specific amino acid racemase, histidine racemase from a lactic acid bacterium, Leuconostoc mesenteroides subsp. sake NBRC 102480 Reviewed

    老川 典夫

    Amino Acids   2018.10

  • A novel bifunctional amino acid racemase with multiple substrate specificity, malY from Lactobacillus sakei LT-13: Genome-based identification and enzymological characterization

    Shiro Kato, Tadao Oikawa

    Frontiers in Microbiology   9   2018.3

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Frontiers Media S.A.  

    The Lactobacillus sakei strain LK-145 isolated from Moto, a starter of sake, produces potentially large amounts of three D-amino acids, D-Ala, D-Glu, and D-Asp, in a medium containing amylase-digested rice as a carbon source. The comparison of metabolic pathways deduced from the complete genome sequence of strain LK-145 to the type culture strain of Lactobacillus sakei strain LT-13 showed that the L- and D-amino acid metabolic pathways are similar between the two strains. However, a marked difference was observed in the putative cysteine/methionine metabolic pathways of strain LK-145 and LT-13. The cystathionine β-lyase homolog gene malY was annotated only in the genome of strain LT-13. Cystathionine β-lyase is an important enzyme in the cysteine/methionine metabolic pathway that catalyzes the conversion of L-cystathionine into L-homocysteine. In addition to malY, most genome-sequenced strains of L. sakei including LT-13 lacked the homologous genes encoding other putative enzymes in this pathway. Accordingly, the cysteine/methionine metabolic pathway likely does not function well in almost all strains of L. sakei. We succeeded in cloning and expressing the malY gene from strain LT-13 (Ls-malY) in the cells of Escherichia coli BL21 (DE3) and characterized the enzymological properties of Ls-MalY. Spectral analysis of purified Ls-MalY showed that Ls-MalY contained a pyridoxal 5'-phosphate (PLP) as a cofactor, and this observation agreed well with the prediction based on its primary structure. Ls-MalY showed amino acid racemase activity and cystathionine β-lyase activity. Ls-MalY showed amino acid racemase activities in various amino acids, such as Ala, Arg, Asn, Glu, Gln, His, Leu, Lys, Met, Ser, Thr, Trp, and Val. Mutational analysis revealed that the ε-amino group of Lys233 in the primary structure of Ls-MalY likely bound to PLP, and Lys233 was an essential residue for Ls-MalY to catalyze both the amino acid racemase and β-lyase reactions. In addition, Tyr123 was a catalytic residue in the amino acid racemase reaction but strongly affected β-lyase activity. These results showed that Ls-MalY is a novel bifunctional amino acid racemase with multiple substrate specificity
    both the amino acid racemase and β-lyase reactions of Ls-MalY were catalyzed at the same active site.

    DOI: 10.3389/fmicb.2018.00403

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  • Thermostable and highly specific l-aspartate oxidase from Thermococcus litoralis DSM 5473: cloning, overexpression, and enzymological properties

    Tsubasa Washio, Tadao Oikawa

    Extremophiles   22 ( 1 )   59 - 71   2018.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer Tokyo  

    We successfully expressed the l-aspartate oxidase homolog gene (accession no: OCC_06611) of Thermococcus litoralis DSM 5473 in the soluble fraction of Escherichia coli BL21 (DE3) using a pET21b vector with 6X His tag at its C-terminus. The gene product (Tl-LASPO) showed l-aspartate oxidase activity in the presence of FAD in vitro, and this report is the first that details an l-aspartate oxidase derived from a Thermococcus species. The homologs of Tl-LASPO existed mainly in archaea, especially in the genus of Thermococcus, Pyrococcus, Sulfolobus, and Halobacteria. The quaternary structure of Tl-LASPO was homotrimeric with a subunit molecular mass of 52 kDa. The enzyme activity of Tl-LASPO increased with temperature up to 70 °C. Tl-LASPO was active from pH 6.0 to 9.0, and its highest activity was at pH 8.0. Tl-LASPO was stable at 80 °C for 1 h. The highest kcat/Km value was observed in assays at 70 °C. Tl-LASPO was highly specific for l-aspartic acid. Tl-LASPO utilized fumaric acid, 2,6-dichlorophenolindophenol, and ferricyanide in addition to FAD as a cofactor under anaerobic conditions. The absorption spectrum of holo-Tl-LASPO exhibited maxima at 380 and 450 nm. The FAD dissociation constant, Kd, of the FAD-Tl-LASPO complex was determined to be 5.9 × 10−9 M.

    DOI: 10.1007/s00792-017-0977-4

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  • シロイヌナズナのセレンの取り込み、輸送、耐性機構に関する研究の現状と展望

    老川 典夫

    微量栄養素研究   34,114-118   2017.12

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  • 無機および有機セレン化合物の曝露がシロイヌナズナ(Arabidopsis thaliana)の生長と遺伝子発現量に及ぼす影響 Reviewed

    大塚 政志, 廣瀬 侑太郞, 細見 亮太, 老川 典夫, 吉田 宗弘

    微量栄養素研究   34, 8-13   2017.12

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  • 超好熱アーキアThermococcus litoralis DSM5473の耐熱性アスパラギン酸ラセマーゼ及び耐熱性L-アスパラギン酸オキシダーゼを用いたD-及びL-アスパラギン酸の新規酵素定量法 Reviewed

    鷲尾 翼, 老川 典夫

    微量栄養素研究   34, 1-7   2017.12

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  • Genome sequence of lactobacillus sakei LK-145 isolated from a Japanese sake cellar as a high producer of D-amino acids

    Shiro Kato, Tadao Oikawa

    Genome Announcements   5 ( 33 )   2017.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    This announcement reports the complete genome sequence of strain LK-145 of Lactobacillus sakei isolated from a Japanese sake cellar as a potent strain for the production of large amounts of D-amino acids. Three putative genes encoding an amino acid racemase were identified.

    DOI: 10.1128/genomeA.00656-17

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  • Whole-genome sequence of Leuconostoc mesenteroides LT-38, a non-spore-forming gram-positive lactic acid bacterium

    Shiro Kato, Tadao Oikawa

    Genome Announcements   5 ( 31 )   2017.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    The present study reports the complete genome sequence of Leuconostoc mesenteroides strain LT-38, which is a non-spore-forming Gram-positive lactic acid bacterium. The genome is composed of a 2,022,184-bp circular chromosome and contains 2,005 putative protein-coding genes.

    DOI: 10.1128/genomeA.00670-17

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  • Whole genome sequence of Lactobacillus sakei LT-13 isolated from moto starter of sake Reviewed

    加藤 志郎, 老川 典夫

    Genome Announcements   5   2017.8

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  • Genome sequence of Leuconostoc mesenteroides LK-151 isolated from a Japanese sake cellar as a high producer of D-amino acids

    Shiro Kato, Tadao Oikawa

    Genome Announcements   5 ( 30 )   2017.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    Here, we report the complete genome sequence of strain LK-151 of Leuconostoc mesenteroides, which was isolated from a Japanese sake cellar and has the potential to produce large amounts of D-amino acids, namely, D-Ala and D-Glu. The genome contains 4 genes related to D-amino acid production.

    DOI: 10.1128/genomeA.00661-17

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  • シロイヌナズナのD-アミノ酸代謝関連酵素

    加藤 志郎, 老川 典夫

    微量栄養素研究   33,118-121   2016.12

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  • The crystal structure of maleylacetate reductase from Rhizobium sp strain MTP-10005 provides insights into the reaction mechanism of enzymes in its original family

    Tomomi Fujii, Ai Sato, Yuko Okamoto, Takae Yamauchi, Shiro Kato, Masahiro Yoshida, Tadao Oikawa, Yasuo Hata

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   84 ( 8 )   1029 - 1042   2016.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H-dependent reduction of maleylacetate, at a carbon-carbon double bond, to 3-oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP-10005, GraC, has been elucidated by the X-ray diffraction method at 1.5 angstrom resolution. GraC is a homodimer, and each subunit consists of two domains: an N-terminal NADH-binding domain adopting an alpha/beta structure and a C-terminal functional domain adopting an alpha-helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase-like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate-binding state and in the ligand-free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase-like superfamily. (C) 2016 Wiley Periodicals, Inc.

    DOI: 10.1002/prot.25046

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  • Molecular cloning and enzymological characterization of pyridoxal 5'-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473 Reviewed

    鷲尾 翼, 加藤 志郎, 老川 典夫

    Extremophiles   20, 711-721   2016.7

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  • Enantioselective analysis of D- and L-amino acids from mouse macrophages using high performance liquid chromatography

    Shiro Kato, Yuki Masuda, Morichika Konishi, Tadao Oikawa

    JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS   116   101 - 104   2015.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE BV  

    The intrinsic D-amino acid profile of mouse macrophages extracted from the peritoneal cavity was analyzed using high performance liquid chromatography. Six D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu, D-Gln and D-Lys) were detected in cell lysates of mouse macrophages. The content and the D/D + L ratio differed depending on the type of D-amino acid and were approximately 3.5-22 nmol/g cells, and approximately 1-20%, respectively. The D-amino acid composition of RAW 264.7 cells, which is a model macrophage cell line, was similar to that of the mouse macrophage. These results suggest that macrophages and RAW 264.7 cells with macrophage-like functions have a similar D-amino acid profile. (C) 2015 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.jpba.2015.04.028

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  • Crystallographic studies of aspartate racemase from Lactobacillus sakei NBRC 15893

    Tomomi Fujii, Takae Yamauchi, Makoto Ishiyama, Yoshitaka Gogami, Tadao Oikawa, Yasuo Hata

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   71   1012 - 1016   2015.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:INT UNION CRYSTALLOGRAPHY  

    Aspartate racemase catalyzes the interconversion between L-aspartate and D-aspartate and belongs to the PLP-independent racemases. The enzyme from the lactic acid bacterium Lactobacillus sakei NBRC 15893, isolated from kimoto, is considered to be involved in D-aspartate synthesis during the brewing process of Japanese sake at low temperatures. The enzyme was crystallized at 293K by the sitting-drop vapour-diffusion method using 25%(v/v) PEG MME 550, 5%(v/v) 2-propanol. The crystal belonged to space group P3(1)21, with unit-cell parameters a = b = 104.68, c = 97.29 angstrom, and diffracted to 2.6 angstrom resolution. Structure determination is under way.

    DOI: 10.1107/S2053230X15010572

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  • 乳酸菌のゲノム解析:現状とD-アミノ酸に着目したゲノム情報の活用に向けて Reviewed

    老川 典夫, 加藤 志郎

    微量栄養素研究   32, 78-82   2015

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  • クエン酸が乳酸の生育と代謝に及ぼす影響 Reviewed

    老川 典夫, 森田 朱香

    微量栄養素研究   32, 86-89   2015

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  • Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp CKU-1

    Yuki Uchida, Hideyuki Hayashi, Tsubasa Washio, Ryo Yamasaki, Shiro Kato, Tadao Oikawa

    EXTREMOPHILES   18 ( 3 )   589 - 602   2014.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER JAPAN KK  

    We successfully cloned a novel branched-chain amino acid aminotransferase (Ts-BcAT; EC 2.6.1.42) gene from the Thermococcus sp. CKU-1 genome and expressed it in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Ts-BcAT is a homodimer with an apparent molecular mass of approximately 92 kDa. The primary structure of Ts-BcAT showed high homology with the fold-type I, subgroup I aminotransferases, but showed little homology with BcATs known to date, i.e., those of Escherichia coli and Salmonella typhimurium, which belong to the fold-type IV, subgroup III aminotransferases. The maximum enzyme activity of Ts-BcAT was detected at 95 A degrees C, and Ts-BcAT did not lose any enzyme activity, even after incubation at 90 A degrees C for 5 h. Ts-BcAT was active in the pH range from 4.0 to 11.0, the optimum pH was 9.5, and the enzyme was stable between pH 6 and 7. The exceptionally low pK (a) of the nitrogen atom in the Lys258 epsilon-amino group in the internal aldimine bond of Ts-BcAT was determined to be 5.52 +/- A 0.05. Ts-BcAT used 21 natural and unnatural amino acids as a substrate in the overall transamination reaction. l-Leucine and other aliphatic amino acids are efficient substrates, while polar amino acids except glutamate were weak substrates. Phylogenetic analysis revealed that Ts-BcAT is a novel fold-type I, subgroup I branched-chain aminotransferase.

    DOI: 10.1007/s00792-014-0642-0

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  • D-及びL-アミノ酸添加がArabidopsis thaliana芽生えの生育に及ぼす影響 Reviewed

    老川 典夫, 加藤志郎

    微量栄養素研究   32, 78-82   2014

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  • Crystal Structure Analyses of Oxygenase Component of Resorcinol Hydroxylase Reviewed

    藤井知実, 小林 一隆, 吉田 雅博, 老川 典夫

    Acta Cryst   C448   2014

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  • Crystal Structures of Bacterial Flavin Reductase GraD and Its Complex with NADH Reviewed

    山内 貴恵, 藤井, 吉田 雅博, 老川 典夫, 畑 安雄

    Acta Cryst.   A70 C448   2014

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  • 食品に関連する乳酸菌のD-アミノ酸代謝関連酵素

    老川典夫

    バイオインダストリー   31, 33-40   2014

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  • Structural Features and Low-Temperature Adaptation of Aspartate Racemase Reviewed

    畑 安雄, 山内 貴恵, 郷上 佳孝, 老川 典夫

    Acta Cryst   A70 C105   2014

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  • Principal component analysis of the relationship between the d-amino acid concentrations and the taste of the sake

    Kaori Okada, Yoshitaka Gogami, Tadao Oikawa

    Amino Acids   44 ( 2 )   489 - 498   2013.2

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    We performed sensory evaluations on 141 bottles of sake and analyzed the relationship between the d-amino acid concentrations, and the taste of the sake using principal component analysis, which yielded seven principal components (PC1-7) that explained 100 % of the total variance in the data. PC1, which explains 33.6 % of the total variance, correlates most positively with strong taste and most negatively with balanced tastes. PC2, which explains 54.4 % of the total variance, correlates most positively with a sweet taste and most negatively with bitter and sour tastes. Sakes brewed with "Kimoto yeast starter" and "Yamahaimoto" had high scores for PC1 and PC2, and had strong taste in comparison with sakes brewed with "Sokujo-moto". When present at concentrations below 50 μM, d-Ala did not affect the PC1 score, but all the sakes showed a high PC1 score, when the d-Ala was above 100 μM. Similar observations were found for the d-Asp and d-Glu concentrations with regard to PC1, and the threshold concentrations of d-Asp and d-Glu that affected the taste were 33.8 and 33.3 μM, respectively. Certain bacteria present in sake, especially lactic acid bacteria, produce d-Ala, d-Asp and d-Glu during storage, and these d-amino acids increased the PC1 score and produced a strong taste (Nojun). When d- and l-Ala were added to the sakes, the value for the umami taste in the sensory evaluation increased, with the effect of d-Ala being much stronger than that of l-Ala. The addition of 50-5,000 μM dl-Ala did not effect on the aroma of the sakes at all. © 2012 Springer-Verlag.

    DOI: 10.1007/s00726-012-1359-y

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  • 環境アポトジェンを含む環境汚染科学物質の作用動態解析と化学生態学的防除法の開発研究プロジェクト

    土戸哲明, 池内俊彦, 上里新一, 下家浩二, 吉田宗弘, 福永健治, 安原裕紀, 長谷川喜衛, 老川典夫, 松村吉信, 岩木宏明

    技苑   3-10   2013

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  • ゲノム・エピゲノム

    老川典夫, 土戸哲明, 松村吉信, 吉田宗弘, 池内俊彦, 下家浩二

    技苑   136, 137-143   2013

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  • 黒酢及び米酢中のD- 及びL-アミノ酸の定量的解析 Reviewed

    岡田かおり, 郷上佳孝, 竹下義隆, 老川典夫

    微量栄養素研究   29, 62-66   2012

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  • D-Amino acid in food: occurrence, production mechanism, and function

    Tadao Oikawa

    VIVA ORIGINO   40, 47-56   2012

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  • 環境アポトジェンを含む環境汚染科学物質の作用動態解析と化学生態学的防除法の開発研究プロジェクト

    土戸哲明, 池内俊彦, 上里新一, 下家浩二, 吉田宗弘, 福永健治, 安原裕紀, 長谷川喜衛, 老川典夫, 松村吉信, 岩木宏明

    技苑   134, 3-11   2012

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  • 生酛,乳酸菌添加生酛,速醸酛造りの日本酒醸造工程中のD-アミノ酸の定量的解析 Reviewed

    郷上佳孝, 岡田かおり, 森山昌和, 溝口晴彦, 老川典夫

    微量栄養素研究   29, 1-6   2012

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  • High-performance liquid chromatography analysis of naturally occurring D-amino acids in sake

    Yoshitaka Gogami, Kaori Okada, Tadao Oikawa

    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES   879 ( 29 )   3259 - 3267   2011.11

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    We measured all of the D- and L-amino acids in 141 bottles of sakes using HPLC. We used two precolumn derivatization methods of amino acid enantiomer detection with o-phthalaldehyde and N-acetyl-L-cysteine, as well as (+)-1-(9-fluorenyl)ethyl chloroformate/1-aminoadamantane and one postcolumn derivatization method with o-phthalaldehyde and N-acetyl-L-cysteine. We found that the sakes contained the D-amino acids forms of Ala, Asn, Asp, Arg, Glu, Gin, His, Ile, Leu, Lys, Ser, Tyr, Val, Phe, and Pro. We were not able to detect D-Met, D-Thr D-Trp in any of the sakes analyzed. The most abundant D-Ala, D-Asp, and D-Glu ranged from 66.9 to 524.3 mu M corresponding to relative 34.4, 12.0, and 14.6% D-enantiomer. The basic parameters that generally determine the taste of sake such as the sake meter value (SMV; "Nihonshudo"), acidity ("Sando"), amino acid value ("Aminosando"), alcohol content by volume, and rice species of raw material show no significant relationship to the D-amino acid content of sake. The brewing water ("Shikomimizu") and brewing process had effects on the D-amino acid content of the sakes: the D-amino acid contents of the sakes brewed with deep-sea water "Kaiyoushinosousui", "Kimoto yeast starter", "Yamahaimoto", and the long aging process "Choukijukusei" are high compared with those of other sakes analyzed. Additionally, the D-amino acid content of sakes that were brewed with the adenine auxotroph of sake yeast ("Sekishoku seishu kobo", Saccharomyces cerevisiae) without pasteurization ("Hiire") increased after storage at 25 degrees C for three months. (C) 2011 Elsevier B.V. All rights reserved.

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  • Corynebacterium glutamicum as a Host for Synthesis and Export of D-Amino Acids

    Norma Staebler, Tadao Oikawa, Michael Bott, Lothar Eggeling

    JOURNAL OF BACTERIOLOGY   193 ( 7 )   1702 - 1709   2011.4

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    A number of D-amino acids occur in nature, and there is growing interest in their function and metabolism, as well as in their production and use. Here we use the well-established L-amino-acid-producing bacterium Corynebacterium glutamicum to study whether D-amino acid synthesis is possible and whether mechanisms for the export of these amino acids exist. In contrast to Escherichia coli, C. glutamicum tolerates D-amino acids added extracellularly. Expression of argR (encoding the broad-substrate-specific racemase of Pseudomonas taetrolens) with its signal sequence deleted results in cytosolic localization of ArgR in C. glutamicum. The isolated enzyme has the highest activity with lysine (100%) but also exhibits activity with serine (2%). Upon overexpression of argR in an L-arginine, L-ornithine, or L-lysine producer, equimolar mixtures of the D-and L-enantiomers accumulated extracellularly. Unexpectedly, argR overexpression in an L-serine producer resulted in extracellular accumulation of a surplus of D-serine (81 mM D-serine and 37 mM L-serine) at intracellular concentrations of 125 mM D-serine plus 125 mM L-serine. This points to a nonlimiting ArgR activity for intracellular serine racemization and to the existence of a specific export carrier for D-serine. Export of D-lysine relies fully on the presence of lysE, encoding the exporter for L-lysine, which is apparently promiscuous with respect to the chirality of lysine. These data show that D-amino acids can also be produced with C. glutamicum and that in special cases, due to specific carriers, even a preferential extracellular accumulation of this enantiomer is possible.

    DOI: 10.1128/JB.01295-10

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  • 環境アポトジェンを含む環境汚染科学物質の作用動態解析と化学生態学的防除法の開発研究プロジェクト

    土戸哲明, 池内俊彦, 上里 新一, 下家浩二, 吉田宗弘, 福永健治, 安原裕紀, 長谷川喜衛, 老川典夫, 松村吉信, 岩木宏明

    技苑   No.132   2011

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  • Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate

    Osamu Kato, Jung-Won Youn, K. Corinna Stansen, Daisuke Matsui, Tadao Oikawa, Volker F. Wendisch

    BMC MICROBIOLOGY   10   2010.12

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    Background: Corynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032.
    Results: Here, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer.
    Conclusions: Cg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer.

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  • Crystal structure of UDP-galactose 4-epimerase-like l-threonine dehydrogenase belonging to the intermediate short-chain dehydrogenase-reductase superfamily

    Kazunari Yoneda, Haruhiko Sakuraba, Ikuo Muraoka, Tadao Oikawa, Toshihisa Ohshima

    FEBS JOURNAL   277 ( 24 )   5124 - 5132   2010.12

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    The crystal structure of a L-threonine dehydrogenase (L-ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1, which shows no sequence similarity to conventional L-ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two-fold rotation axis. Each monomer consisted of a Rossmann-fold domain and a carboxyl-terminal catalytic domain. The overall fold of F. frigidimaris L-ThrDH showed significant similarity to that of UDP-galactose 4-epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD-binding loop) around the substrate and NAD binding sites. In F. frigidimaris L-ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP-glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimaris L-ThrDH. Consequently, the cofactor binds to F. frigidimaris L-ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the beta-hydroxyl hydrogen of L-threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE-like L-ThrDHs from GalEs.

    DOI: 10.1111/j.1742-4658.2010.07916.x

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  • Detection of D-Ornithine Extracellularly Produced by Corynebacterium glutamicum ATCC 13032::argF

    Daisuke Matsui, Tadao Oikawa

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   74 ( 12 )   2507 - 2510   2010.12

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    We found that Corynebacterium glutamicum ATCC 13032::argF extracellularly produced a large amount of D-ornithine when cultivated in a CGXII medium containing 1 mm L-arginine. This is the first report that C. glutamicum ATCC 13032 or its mutant produces a D-amino acid extracellularly. C. glutamicum ATCC 13032::argF produced 13 mm D-ornithine in 45 h of cultivation.

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  • X線構造解析による代謝酵素の反応機構解明

    老川典夫

    京都大学化学研究所共同利用・共同研究拠点 化学関連分野の深化・連携を基軸とする先端・学際研究拠点 平成22年成果報告書   p1-2   2010

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  • Detection and Function of the Intramolecular Disulfide Bond in Arginine Racemase: An Enzyme with Broad Substrate Specificity

    Daisuke Matsui, Tadao Oikawa

    CHEMISTRY & BIODIVERSITY   7 ( 6 )   1591 - 1602   2010

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    We found that a single intramolecular disulfide bond between the cysteines C47 and C73 exists in the primary structure of arginine racemase (ArgR) from Pseudomonas taetrolens NBRC 3460, and this is the first example of a pyridoxal phosphate (PLP)-dependent amino acid racemase that contains a disulfide bond. The amino acid racemase activity was still detected, when the disulfide bond of ArgR was disrupted by site-directed mutagenesis or reduced with dithiothreitol (DTT). The thermal and pH profiles and the quaternary structure of ArgR did not change when the disulfide bond of ArgR was disrupted by site-directed mutagenesis. The substrate specificity and the overall structure did not change when the disulfide bond of ArgR was reduced with DTT after the protein was matured. However, these properties changed when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before protein maturation. The total activity of ArgR decreased when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before the protein was matured or when ArgR was expressed in the cytoplasm. Based on these results, we can conclude that the disulfide bond of ArgR is essential for ArgR to fold and mature as an amino acid racemase with broad substrate specificity.

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  • Site-Directed Mutagenesis of Rice Serine Racemase: Evidence That Glu219 and Asp225 Mediate the Effects of Mg2+ on the Activity

    Yoshitaka Gogami, Ai Kobayashi, Toshihiko Ikeuchi, Tadao Oikawa

    CHEMISTRY & BIODIVERSITY   7 ( 6 )   1579 - 1590   2010

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    We succeeded in constructing the Glu219Ala/Asp225Ala (i.e., E219A/D225A) serine racemase (SerR) by site-directed mutagenesis, and the effects of Mg2+ on the catalytic efficiency and the structure were compared between the E219A/D225A-SerR and the wild-type protein. This is the first example of a serine racemase whose amino acid residues in the Mg2+-binding site were replaced with other amino acids by site-directed mutagenesis. Neither the serine racemase nor the dehydratase activities of the E219A/D225A-SerR were affected by the addition of Mg2+, and Glu219 and Asp225 of the SerR are the essential amino acid residues for Mg2+ to affect both kinds of enzyme activities. Therefore, Glu219 and Asp225 mediate the effects of Mg2+ on the activity and are important for the SerR to form the Mg2+- binding site. Judging from the difference of the K-eq values between the E219A/D225A-SerR and the SerR. Mg2+ might affect the equilibrium states in the racemase reaction. The fluorescence quenching analysis of the E219A/D225A-SerR showed that Me2+ hound to Glu219 and Asp225 of the SerR probably causes a conformational change in the ternary structure of the SerR.

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  • Characterization of Structure of Arginine Racemase, the Essential Enzyme for Microorganism to Utilize D-Lysine as a Carbon Source Reviewed

    D. Matsui, T. Oikawa

    Trance Nutrients Research   27, p47-51   2010

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  • 環境アポトジェンを含む環境汚染科学物質の作用動態解析と化学生態学的防除法の開発研究プロジェクト

    土戸哲明, 池内俊彦, 下家浩二, 上里新一, 吉田宗弘, 福永健治, 安原裕紀, 長谷川喜衛, 岩木宏明, 老川典夫, 松村吉信

    技苑   №130 p3-10   2010

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  • A periplasmic, pyridoxal-5&apos;-phosphate-dependent amino acid racemase in Pseudomonas taetrolens Reviewed

    Daisuke Matsui, Tadao Oikawa, Noriaki Arakawa, Shintaro Osumi, Frank Lausberg, Norma Staebler, Roland Freudl, Lothar Eggeling

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   83 ( 6 )   1045 - 1054   2009.7

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    The pyridoxal-5&apos;-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of d-arginine and d-lysine by P. taetrolens.

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  • Occurrence of D-serine in rice and characterization of rice serine racemase

    Yoshitaka Gogami, Katsuyoshi Ito, Yuji Kamitani, Yuki Matsushima, Tadao Oikawa

    PHYTOCHEMISTRY   70 ( 3 )   380 - 387   2009.2

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    Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D-and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 degrees C and pH 9.0) and for the dehydratase reaction (35 degrees C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg2+ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg2+, and this structural change probably regulates the two enzymatic activities. (C) 2009 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.phytochem.2009.01.003

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  • イネのセリンラセマーゼ:マグネシウム (Ⅱ) イオンによる構造変化と2酵素活性の制御 Reviewed

    郷上佳孝, 松島由貴, 老川典夫

    微量栄養素研究   25巻, 69-71   2008

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  • Building an international newwork exchange program of education and research for graduate course students in life science and biotechnology

    T. Oikawa, S. Uesato, H. Tamura, H. Kawahara, Y. Nagaoka, K. Shimoke, T. Tsuchido, L. Eggeling, V. F, Wendisch, J. Buchs, R. Rujiravanit, N. Najimudin, C. L. Keng

    Science and Technology Reports of Kansai University   No.50, p83-94   2008

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  • 高等植物及び食品中のD-アミノ酸とその代謝関連酵素

    老川典夫

    生化学   第80巻 第4号,pp.300-307   2008

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  • Crystallization and preliminary X-ray diffraction studies of tetrameric malate dehydrogenase from the novel Antarctic psychrophile Flavobacterium frigidimaris KUC-1

    Tomomi Fujii, Tadao Oikawa, Ikuo Muraoka, Kenji Soda, Yasuo Hata

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   63   983 - 986   2007.11

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    Flavobacterium frigidimaris KUC-1 is a novel psychrotolerant bacterium isolated from Antarctic seawater. Malate dehydrogenase (MDH) is an essential metabolic enzyme in the citric acid cycle and has been cloned, overexpressed and purified from F. frigidimaris KUC-1. In contrast to the already known dimeric form of MDH from the psychrophile Aquaspirillium arcticum, F. frigidimaris MDH exists as a tetramer. It was crystallized at 288 K by the hanging-drop vapour-diffusion method using ammonium sulfate as the precipitating agent. The crystal diffracted to a maximum resolution of 1.80 angstrom. It contains one tetrameric molecule in the asymmetric unit.

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  • A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 Reviewed

    T. Kazuoka, T. Oikawa, I. Muraoka, I. Kuroda, K. Soda

    Extremophiles   11, 257-267   2007.10

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    An NAD(+)-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD(+) as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD(+). F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85 degrees C and the most active at 70 degrees C. The half-life time and k (cat) value at 60 degrees C were calculated to be 50 min and 27,400 min(-1), respectively. The enzyme also shows high catalytic efficiency at low temperatures (0-20 degrees C) (k (cat)/K (m) at 10 degrees C; 12,600 mM(-1 )min(-1)) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.

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  • The two carboxylases of Corynebacterium glutamicum essential for fatty acid and mycolic acid synthesis

    Roland Gande, Lynn G. Dover, Karin Krumbach, Gurdyal S. Besra, Hermann Sahm, Tadao Oikawa, Lothar Eggeling

    JOURNAL OF BACTERIOLOGY   189 ( 14 )   5257 - 5264   2007.7

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    The suborder Corynebacterianeae comprises bacteria like Mycobacterium tuberculosis and Corynebacterium glutamicum, and these bacteria contain in addition to the linear fatty acids, unique alpha-branched beta-hydroxy fatty acids, called mycolic acids. Whereas acetyl-coenzyme A (CoA) carboxylase activity is required to provide malonyl-CoA for fatty acid synthesis, a new type of carboxylase is apparently additionally present in these bacteria. It activates the alpha-carbon of a linear fatty acid by carboxylation, thus enabling its decarboxylative condensation with a second fatty acid to afford mycolic acid synthesis. We now show that the acetyl-CoA carboxylase of C. glutamicum consists of the biotinylated alpha-subunit AccBC, the beta-subunit AccD1, and the small peptide AccE of 8.9 kDa, forming an active complex of approximately 812,000 Da. The carboxylase involved in mycolic acid synthesis is made up of the two highly similar beta-subunits AccD2 and AccD3 and of AccBC and AccE, the latter two identical to the subunits of the acetyl-CoA carboxylase complex. Since AccD2 and AccD3 orthologues are present in all Corynebacterianeae, these polypeptides are vital for mycolic acid synthesis forming the unique hydrophobic outer layer of these bacteria, and we speculate that the two beta-subunits present serve to lend specificity to this unique large multienzyme complex.

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  • Biochemical and genetic analysis of the gamma-resorcylate (2,6-dihydroxybenzoate) catabolic pathway in Rhizobium sp strain MTP-10005: Identification and functional analysis of its gene cluster

    Masahiro Yoshida, Tadao Oikawa, Hitoshi Obata, Katsumasa Abe, Hisaaki Mihara, Nobuyoshi Esaki

    JOURNAL OF BACTERIOLOGY   189 ( 5 )   1573 - 1581   2007.3

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    We identified a gene cluster that is involved in the gamma-resorcylate (2,6-dihydroxybenzoate) catabolism of the aerobic bacterium Rhizobium sp. strain MTP-10005. The cluster consists of the graPDAFCBEK genes, and graA, graB, graC, and graD were heterologously expressed in Escherichia coli. Enzymological studies showed that graD, graA, graC, and graB encode the reductase (GraD) and oxygenase (GraA) components of a resorcinol hydroxylase (EC 1.14.13.x), a maleylacetate reductase (GraC) (EC 1.3.1.32), and a hydroxyquinol 1,2-dioxygenase (GraB) (EC 1.13.11.37). Bioinformatic analyses suggested that graE, graR, and graK encode a protein with an unknown function (GraE), a MarR-type transcriptional regulator (GraR), and a benzoate transporter (GraK). Quantitative reverse transcription-PCR of graF, which encodes gamma-resorcylate decarboxylase, revealed that the maximum relative mRNA expression level ([5.93 +/- 0.82] X 10(-4)) of graF was detected in the total RNA of the cells after one hour of cultivation when gamma-resorcylate was used as the sole carbon source. Reverse transcription-PCR of graDAFCBE showed that these genes are transcribed as a single mRNA and that the transcription of the gene cluster is induced by gamma-resorcylate. These results suggested that the graDAFCBE genes are responsible as an operon for the growth of Rhizobium sp. strain MTP-10005 on gamma-resorcylate and are probably regulated by GraR at the transcriptional level. This is the first report of the gamma-resorcylate catabolic pathway in an aerobic bacterium.

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  • イネのセリンデヒドラターゼ/ラセマーゼ:Mg2+による酵素反応の制御機構の発見

    郷上佳孝, 伊藤克佳, 老川典夫

    微量栄養素研究   24巻,110-112   2007

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  • 野菜および果物中のD-アミノ酸の定量的解析と植物におけるD-アミノ酸の生合成機構 Reviewed

    郷上佳孝, 伊藤克佳, 老川典夫

    微量栄養素研究   23, 1-4   2006.12

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  • Crystal structures of nonoxidative zinc-dependent 2,6-dihydroxybenzoate (gamma-resorcylate) decarboxylase from Rhizobium sp strain MTP-10005

    Masaru Goto, Hideyuki Hayashi, Ikuko Miyahara, Ken Hirotsu, Masahiro Yoshida, Tadao Oikawa

    JOURNAL OF BIOLOGICAL CHEMISTRY   281 ( 45 )   34365 - 34373   2006.11

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    Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-angstrom resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one (alpha beta)(8) triose-phosphate isomerase-barrel domain with three functional linkers and one C-terminal tail. The native enzyme possesses one Zn2+ ion liganded by Glu(8), His(10), His(164), Asp(287), and a water molecule at the active site center, although the enzyme has been reported to require no cofactor for its catalysis. The substrate carboxylate takes the place of the water molecule and is coordinated to the Zn2+ ion. The 2-hydroxy group of the substrate is hydrogen-bonded to Asp(287), which forms a triad together with His(218) and Glu(221) and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn2+-dependent decarboxylation.

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  • Crystal structures of nonoxidative zinc-dependent 2,6-dihydroxybenzoate (gamma-resorcylate) decarboxylase from Rhizobium sp strain MTP-10005

    Masaru Goto, Hideyuki Hayashi, Ikuko Miyahara, Ken Hirotsu, Masahiro Yoshida, Tadao Oikawa

    JOURNAL OF BIOLOGICAL CHEMISTRY   281 ( 45 )   34365 - 34373   2006.11

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    Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-angstrom resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one (alpha beta)(8) triose-phosphate isomerase-barrel domain with three functional linkers and one C-terminal tail. The native enzyme possesses one Zn2+ ion liganded by Glu(8), His(10), His(164), Asp(287), and a water molecule at the active site center, although the enzyme has been reported to require no cofactor for its catalysis. The substrate carboxylate takes the place of the water molecule and is coordinated to the Zn2+ ion. The 2-hydroxy group of the substrate is hydrogen-bonded to Asp(287), which forms a triad together with His(218) and Glu(221) and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn2+-dependent decarboxylation.

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  • Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase

    Tadao Oikawa, Andreas Tauch, Steffen Schaffer, Toru Fujioka

    JOURNAL OF BIOTECHNOLOGY   125 ( 4 )   503 - 512   2006.10

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    We constructed the high-expression system of the alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli BL21 (DE3) to characterize the enzymological and structural properties of the gene product, Alr. The Alr was expressed in the soluble fractions of the cell extract of the E. coli clone and showed alanine racemase activity. The purified Alr was a dimer with a molecular mass of 78 kDa. The Alr required pyridoxal 5'-phosphate (PLP) as a coenzyme and contained 2 mol of PLP per mol of the enzyme. The holoenzyme showed maximum absorption at 420 nm, while the reduced form of the enzyme showed it at 3 10 nm. The Alr was specific for alanine, and the optimum pH was observed at about nine. The Alr was relatively thermostable, and its half-life time at 60 degrees C was estimated to be 26 min. The K-m and V-max values were determined as follows: L-alanine to D-alanine, K-m (L-alanine) 5.01 mM and V-max 306 U/Mg; D-alanine to L-alanine, K-m (D-alanine) 5.24 mM and V-max 345 U/mg. The K-eq value was calculated to be 1.07 and showed good agreement with the theoretical value for the racemization reaction. The high substrate specificity of the Alr from C glutamicum ATCC 13032 is expected to be a biocatalyst for D-alanine production from the L-counter part. (c) 2006 Elsevier B.V. All rights reserved.

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  • Synthesis and cancer antiproliferative activity of new histone deacetylase inhibitors: hydrophilic hydroxamates and 2-aminobenzamide-containing derivatives

    Y. Nagaoka, T. Maeda, Y. Kawai, D. Nakashima, T. Oikawa, K. Shimoke, T. Ikeuchi, H. Kuwajima, S. Uesato

    EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY   41 ( 6 )   697 - 708   2006.6

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    New series historic deacetylase inhibitors comprising a hydroxamic acid or 2-aminobenzamide group as a zinc-chelating function were synthesized and evaluated for antiproliferative activities against a panel of human cancer cells. The 2-aminobenzamide series inhibitors generally had the potency in cell growth inhibitions comparable to that of MS-275. Among them, the compound having a (3,4-difluorobenzyl)(2-hydroxyethyl) amino group at one end and a 2-aminobenzamide group at the other of molecule showed the most promising profile as an anticancer drug candidate, since it had a comparatively low toxicity as did MS-275 against a normal fibroblast cell CCD-1059SK. Additionally, the derivative exhibited a high recovery in human plasma stability test. (c) 2006 Elsevier SAS. All rights reserved.

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  • Alanine racemase of alfalfa seedlings (Medicago sativa L.): First evidence for the presence of an amino acid racemase in plants

    K Ono, K Yanagida, T Oikawa, T Ogawa, K Soda

    PHYTOCHEMISTRY   67 ( 9 )   856 - 860   2006.5

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    We demonstrated several kinds Of D-amino acids in plant seedlings, and moreover alanine racemase (E.C.5.1.1.1) in alfalfa (Medicago sativa L.) seedlings. This is the first evidence for the presence of amino acid racemase in plant. The enzyme was effectively induced by the addition Of L- or D-alanine, and we highly purified the enzyme to show enzymological properties. The enzyme exclusively catalyzed racemization Of L- and D-alanine. The K-m and V-max values of enzyme for L-alanine were 29.6 x 10(-3) M and 1.02 mol/s/kg, and those for D-alanine are 12.0 x 10(-3) M and 0.44 mol/s/kg, respectively. The K-eq value was estimated to be about I and indicated that the enzyme catalyzes a typical racemization of both enantiomers of alanine. The enzyme was inactivated by hydroxylamine, phenylhydrazine and some other pyridoxal 5'-phosphate enzyme inhibitors. Accordingly, the enzyme required pyridoxal 5'-phosphate as a coenzyme, and enzymologically resembled bacterial alanine racemases studied so far. (c) 2006 Elsevier Ltd. All rights reserved.

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  • Molecular and biochemical characterization of a serine racemase from Arabidopsis thaliana

    Y Fujitani, N Nakajima, K Ishihara, T Oikawa, K Ito, M Sugimoto

    PHYTOCHEMISTRY   67 ( 7 )   668 - 674   2006.4

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    A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5'-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca2+, Mg2+, Mn2+, Fe2+, or Ni2+, at alkaline pH for both activities. The racemization process is highly specific toward L-serine, whereas L-alanine, L-arginine, and L-glutamine were poor substrates. The V-max/K-m values for racemase activity of L- and D-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for L- and D-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward L-serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana. (c) 2006 Elsevier Ltd. All rights reserved.

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  • 15.超好熱始原菌Thermococcus litoralis DAM5473の新規低基質特異性アミノトランスフェラーゼ : クローニングと基礎的性質(第403回ビタミンB研究協議会研究発表要旨,ビタミンB研究委員会)

    左右田 健次, 内田 悠喜, 老川 典夫

    ビタミン   80 ( 3 )   152 - 153   2006

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  • 野菜及び果物中のD-アミノ酸の定量分析と植物におけるD-アミノ酸の生合成機構

    郷上佳孝, 伊藤克佳, 老川典夫

    微量栄養素研究   第23集, 1-4   2006

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  • NGF-induced phosphatidylinositol 3-kinase signaling pathway prevents thapsigargin-triggered ER stress-mediated apoptosis in PC12 cells

    K Shimoke, S Kishi, T Utsumi, Y Shimamura, H Sasaya, T Oikawa, S Uesato, T Ikeuchi

    NEUROSCIENCE LETTERS   389 ( 3 )   124 - 128   2005.12

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    Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC 12 cells. We also found that LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), reduced the survival of cells treated with NGF for 24 It in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal. (C) 2005 Elsevier Ireland Ltd. All rights reserved.

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  • NGF-induced phosphatidylinositol 3-kinase signaling pathway prevents thapsigargin-triggered ER stress-mediated apoptosis in PC12 cells

    K Shimoke, S Kishi, T Utsumi, Y Shimamura, H Sasaya, T Oikawa, S Uesato, T Ikeuchi

    NEUROSCIENCE LETTERS   389 ( 3 )   124 - 128   2005.12

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    Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC 12 cells. We also found that LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), reduced the survival of cells treated with NGF for 24 It in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal. (C) 2005 Elsevier Ireland Ltd. All rights reserved.

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  • Purification, characterization, and overexpression of psychrophilic and thermolabile malate dehydrogenase of a novel antarctic psychrotolerant, Flavobacterium frigidimaris KUC-1

    T Oikawa, N Yamamoto, K Shimoke, S Uesato, T Ikeuchi, T Fujioka

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   69 ( 11 )   2146 - 2154   2005.11

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    We purified the psychrophilic and thermolabile malate dehydrogenase to homogeneity from a novel psychrotolerant, Flavobacterium frigidimaris KUC-1, isolated from Antarctic seawater. The enzyme was a homotetramer with a molecular weight of about 123k and that of the subunit was about 32 k. The enzyme required NAD(P)(+) as a coenzyme and catalyzed the oxidation Of L-malate and the reduction of oxalacetate specifically. The reaction proceeded through an ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment, and the half-life time at 40 degrees C was estimated to be 3.0 min. The k(cat)/K-m (mu m(-1).s(-1)) values for L-malate and NAD(+) at 30 degrees C were 289 and 2,790, respectively. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of the coenzyme. The enzyme contained 311 amino acid residues and much lower numbers of proline and arginine residues than other malate dehydrogenases.

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  • Purification, characterization, and overexpression of psychrophilic and thermolabile malate dehydrogenase of a novel antarctic psychrotolerant, Flavobacterium frigidimaris KUC-1

    T Oikawa, N Yamamoto, K Shimoke, S Uesato, T Ikeuchi, T Fujioka

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   69 ( 11 )   2146 - 2154   2005.11

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    We purified the psychrophilic and thermolabile malate dehydrogenase to homogeneity from a novel psychrotolerant, Flavobacterium frigidimaris KUC-1, isolated from Antarctic seawater. The enzyme was a homotetramer with a molecular weight of about 123k and that of the subunit was about 32 k. The enzyme required NAD(P)(+) as a coenzyme and catalyzed the oxidation Of L-malate and the reduction of oxalacetate specifically. The reaction proceeded through an ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment, and the half-life time at 40 degrees C was estimated to be 3.0 min. The k(cat)/K-m (mu m(-1).s(-1)) values for L-malate and NAD(+) at 30 degrees C were 289 and 2,790, respectively. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of the coenzyme. The enzyme contained 311 amino acid residues and much lower numbers of proline and arginine residues than other malate dehydrogenases.

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  • Flavobacterium frigidimaris sp nov., isolated from Antarctic seawater

    Y Nogi, K Soda, T Oikawa

    SYSTEMATIC AND APPLIED MICROBIOLOGY   28 ( 4 )   310 - 315   2005.6

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    We described the polyphasic characterization of the psychrotolerant isolated from Antarctic seawater. The strain was closely related to Flarobacterium hydatis, F. pectinororum, and F. saccharophilum on the basis of the 16S rDNA sequence analysis. However, DNA-DNA hybridization experiments showed that the DNA-similarities between strain KUC-1(T) and the reference strains of Flavobacterium were less than 30%. Therefore, we can definite a new species of Flavobacterium phylogenetically, and strain KUC-1(T) can be considered to be a new species of Flavobacterium. i.e. F. frigidimaris (KUC-1(T): JCM 12218(T) and DSM 15937(T): mol% G + C or DNA or the type strain is 34.5 mol%), Useful phenotypical features for discrimination of F. frigidimaris from other Flavobacterium species, such as a resistance to NaCl, optimum growth temperature. and cellular fatty acid composition, were also determined. (c) 2005 Elsevier GmbH. All rights reserved.

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  • Flavobacterium frigidimaris sp nov., isolated from Antarctic seawater

    Y Nogi, K Soda, T Oikawa

    SYSTEMATIC AND APPLIED MICROBIOLOGY   28 ( 4 )   310 - 315   2005.6

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    We described the polyphasic characterization of the psychrotolerant isolated from Antarctic seawater. The strain was closely related to Flarobacterium hydatis, F. pectinororum, and F. saccharophilum on the basis of the 16S rDNA sequence analysis. However, DNA-DNA hybridization experiments showed that the DNA-similarities between strain KUC-1(T) and the reference strains of Flavobacterium were less than 30%. Therefore, we can definite a new species of Flavobacterium phylogenetically, and strain KUC-1(T) can be considered to be a new species of Flavobacterium. i.e. F. frigidimaris (KUC-1(T): JCM 12218(T) and DSM 15937(T): mol% G + C or DNA or the type strain is 34.5 mol%), Useful phenotypical features for discrimination of F. frigidimaris from other Flavobacterium species, such as a resistance to NaCl, optimum growth temperature. and cellular fatty acid composition, were also determined. (c) 2005 Elsevier GmbH. All rights reserved.

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  • 好冷微生物の生産する好冷性酵素の開発と高生産系の構築

    老川典夫, 栗原達夫, 江崎芳信

    日本応用酵素協会誌   39巻   2005.3

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  • 大腸菌のコールドショック発現系を用いる亜鉛含有型金属酵素の高発現系構築法 Reviewed

    郷上佳孝, 老川典夫

    Trace Nutrients Research   22: 39-44   2005

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  • カイワレダイコン種子およびスプラウトのセレンの蓄積 Reviewed

    岡田敏英, 福永健治, 吉田宗弘, 老川典夫

    微量栄養素研究   21巻37頁   2004.12

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  • Thermophilic, reversible gamma-resorcylate decarboxylase from Rhizobium sp strain MTP-10005: Purification, molecular characterization, and expression

    M Yoshida, N Fukuhara, T Oikawa

    JOURNAL OF BACTERIOLOGY   186 ( 20 )   6855 - 6863   2004.10

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    We found the occurrence of thermophilic reversible gamma-resorcylate decarboxylase (gamma-RDC) in the cell extract of a bacterium isolated from natural water, Rhizobium sp. strain MTP-10005, and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be about 151 kDa by gel filtration, and that of the subunit was 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; in other words, the enzyme was a homotetramer. The enzyme was induced specifically by the addition of gamma-resorcylate to the medium. The enzyme required no coenzyme and did not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. It was relatively thermostable to heat treatment, and its half-life at 50degreesC was estimated to be 122 min; furthermore, it catalyzed the reverse carboxylation of resorcinol. The values of k(cat)/K-m (mM(-1) s(-1)) for gamma-resorcylate and resorcinol at 30degreesC and pH 7 were 13.4 and 0.098, respectively. The enzyme contains 327 amino acid residues, and sequence identities were found with those of hypothetical protein AGR C 4595p from Agrobacterium tumefaciens strain C58 (96% identity), 5-carboxyvanillate decarboxylase from Sphingomonas paucimobilis (32%), and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylases from Bacillus cereus ATCC 10987 (26%), Rattus norvegicus (26%), and Homo sapiens (25%). The genes (graA [1,230 bp], graB [888 bp], and graC [1,056 bp]) that are homologous to those in the resorcinol pathway also exist upstream and downstream of the gamma-RDC gene. Judging from these results, the resorcinol pathway also exists in Rhizobium sp. strain MTP-10005, and gamma-RDC probably catalyzes a reaction just before the hydroxylase in it does.

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  • Acyl-CoA carboxylases (accD2 and accD3), together with a unique polyketide synthase (Cg-pks), are key to mycolic acid biosynthesis in corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis

    R Gande, KJC Gibson, AK Brown, K Krumbach, LG Dover, H Sahm, S Shioyama, T Oikawa, GS Besra, L Eggeling

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 43 )   44847 - 44857   2004.10

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    The Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis possess several unique and structurally diverse lipids, including the genus-specific mycolic acids. Although the function of a number of genes involved in fatty acid and mycolic acid biosynthesis is known, information relevant to the initial steps within these biosynthetic pathways is relatively sparse. Interestingly, the genomes of Corynebacterianeae possess a high number of accD genes, whose gene products resemble the beta-subunit of the acetyl-CoA carboxylase of Escherichia coli, providing the activated intermediate for fatty acid synthesis. We present here our studies on four putative accD genes found in C. glutamicum. Although growth of the accD4 mutant remained unchanged, growth of the accD1 mutant was strongly impaired and partially recovered by the addition of exogenous oleic acid. Overexpression of accD1 and accBC, encoding the carboxylase alpha-subunit, resulted in an 8-fold increase in malonyl-CoA formation from acetyl-CoA in cell lysates, providing evidence that accD1 encodes a carboxyltransferase involved in the biosynthesis of malonyl-CoA. Interestingly, fatty acid profiles remained unchanged in both our accD2 and accD3 mutants, but a complete loss of mycolic acids, either as organic extractable trehalose and glucose mycolates or as cell wall-bound mycolates, was observed. These two carboxyltransferases are also retained in all Corynebacterianeae, including Mycobacterium leprae, constituting two distinct groups of orthologs. Furthermore, carboxyl fixation assays, as well as a study of a Cg-pks deletion mutant, led us to conclude that accD2 and accD3 are key to mycolic acid biosynthesis, thus providing a carboxylated intermediate during condensation of the mero-chain and alpha-branch directed by the pks-encoded polyketide synthase. This study illustrates that the high number of accD paralogs have evolved to represent specific variations on the well known basic theme of providing carboxylated intermediates in lipid biosynthesis.

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  • Acyl-CoA carboxylases (accD2 and accD3), together with a unique polyketide synthase (Cg-pks), are key to mycolic acid biosynthesis in corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis

    R Gande, KJC Gibson, AK Brown, K Krumbach, LG Dover, H Sahm, S Shioyama, T Oikawa, GS Besra, L Eggeling

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 43 )   44847 - 44857   2004.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis possess several unique and structurally diverse lipids, including the genus-specific mycolic acids. Although the function of a number of genes involved in fatty acid and mycolic acid biosynthesis is known, information relevant to the initial steps within these biosynthetic pathways is relatively sparse. Interestingly, the genomes of Corynebacterianeae possess a high number of accD genes, whose gene products resemble the beta-subunit of the acetyl-CoA carboxylase of Escherichia coli, providing the activated intermediate for fatty acid synthesis. We present here our studies on four putative accD genes found in C. glutamicum. Although growth of the accD4 mutant remained unchanged, growth of the accD1 mutant was strongly impaired and partially recovered by the addition of exogenous oleic acid. Overexpression of accD1 and accBC, encoding the carboxylase alpha-subunit, resulted in an 8-fold increase in malonyl-CoA formation from acetyl-CoA in cell lysates, providing evidence that accD1 encodes a carboxyltransferase involved in the biosynthesis of malonyl-CoA. Interestingly, fatty acid profiles remained unchanged in both our accD2 and accD3 mutants, but a complete loss of mycolic acids, either as organic extractable trehalose and glucose mycolates or as cell wall-bound mycolates, was observed. These two carboxyltransferases are also retained in all Corynebacterianeae, including Mycobacterium leprae, constituting two distinct groups of orthologs. Furthermore, carboxyl fixation assays, as well as a study of a Cg-pks deletion mutant, led us to conclude that accD2 and accD3 are key to mycolic acid biosynthesis, thus providing a carboxylated intermediate during condensation of the mero-chain and alpha-branch directed by the pks-encoded polyketide synthase. This study illustrates that the high number of accD paralogs have evolved to represent specific variations on the well known basic theme of providing carboxylated intermediates in lipid biosynthesis.

    DOI: 10.1074/jbc.M408648200

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  • Paradoxical thermostable enzymes from psychrophile: molecular characterization and potentiality for biotechnological application

    T Oikawa, T Kazuoka, K Soda

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   23 ( 2-6 )   65 - 70   2003.9

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    NAD(P)(+)-dependent aldehyde dehydrogenase (EC 1.2.1.5) and aspartase (EC 4.3.1.1) in the cells of an atypical psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, were paradoxically thermostable, although they derived from a psychrophile. Both enzymes showed the highest activity at about 55 degreesC, and also active even under cold conditions. The enzymes contained more lie residues than the enzymes from mesophiles. The Ile/Ile + Val + Len ratio of the Cytophaga thermostable enzymes was much higher than that of the enzymes from mesophiles. As compared with the enzymes from other microorganisms, the Cytophaga thermostable enzymes have the structural differences in the C-terminal region of the enzymes. Therefore, the C-terminal region might be important for the paradoxical thermostability of the enzymes. The psychrophilic microorganism produces not only psychrophilic enzyme, but thermostable enzyme with psychrophilicity. Therefore, the psychrophilic microorganism is one of the candidates for isolation of novel biocatalysts, which have potential for various industrial applications. (C) 2003 Elsevier B.V. All rights reserved.

    DOI: 10.1016/S1381-1177(03)00073-0

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  • Novel psychrophilic and thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp strain KUC-1

    T Kazuoka, S Takigawa, N Arakawa, Y Hizukuri, Muraoka, I, T Oikawa, K Soda

    JOURNAL OF BACTERIOLOGY   185 ( 15 )   4483 - 4489   2003.8

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    A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD+-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD(+) and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45 degreesC. The kinetic parameters of the enzyme are highly influenced by temperatures. The K-m for L-threonine was lowest at 20 degreesC. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD(+) binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 by encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.

    DOI: 10.1128/JB.185.15.4483-4489.2003

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  • D-arginase of Arthrobacter sp KUJ 8602: Characterization and its identity with Zn2+-guanidinobutyrase

    N Arakawa, M Igarashi, T Kazuoka, T Oikawa, K Soda

    JOURNAL OF BIOCHEMISTRY   133 ( 1 )   33 - 42   2003.1

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    D-Arginase activity was found in the cells of an isolate, Arthrobacter sp. KUJ 8602, grown in the L-arginine medium, and the enzyme was purified and characterized. Its molecular weight was estimated to be about 232,000 by gel filtration, and that of the subunit was approximately 40,000 by SDS-PAGE, suggesting that the enzyme is a homohexamer. The enzyme acted on not only D-arginine but also 4-guanidinobutyrate, 3-guanidinopropionate and even L-arginine. The V-max/K-m values for 4-guanidinobutyrate and D-arginine were determined to be 87 and 0.81 mumol/min/mg/mM, respectively. Accordingly, the enzyme is regarded as a kind of guanidinobutyrase [EC 3.5.3.7]. The pH optima for 4-guanidinobutyrate and D-arginine were 9.0 and 9.5, respectively. The enzyme was inhibited competitively by 5-aminovalerate, and thiol carboxylates such as mercaptoacetate served as strong mixed-type inhibitors. The enzyme contained about 1 g-atom of firmly bound Zn2+ per mol of subunit, and removal of the metal ions by incubation with 1,10-phenanthroline resulted in loss of activity. The inactivated enzyme was reactivated markedly by incubation with either Zn2+ or Co2+, and slightly by incubation with Mn2+. The nucleotide sequence of enzyme contains an open reading frame that encodes a polypeptide of 353 amino acid residues (M,: 37,933). The predicted amino acid sequence contains sequences involved in the binding of metal ions and the guanidino group of the substrate, which show a high homology with corresponding sequences of Mn2+-dependent amidinohydrolases such as agmatinase from Escherichia coli and L-arginase from rat liver, though the homology of their entire sequences is relatively low (24-43%).

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  • Thermostable aspartase from a marine psychrophile, Cytophaga sp KUC-1: Molecular characterization and primary structure

    T Kazuoka, Y Masuda, T Oikawa, K Soda

    JOURNAL OF BIOCHEMISTRY   133 ( 1 )   51 - 58   2003.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:JAPANESE BIOCHEMICAL SOC  

    We found that a psychrophilic bacterium isolated from Antarctic seawater, Cytophaga sp. KUC-1, abundantly produces aspartase [EC4.3.1.1], and the enzyme was purified to homogeneity. The molecular weight of the enzyme was estimated to be 192,000, and that of the subunit was determined to be 51,000: the enzyme is a homotetramer. L-Aspartate was the exclusive substrate. The optimum pH in the absence and presence of magnesium ions was determined to be pH 7.5 and 8.5, respectively. The enzyme was activated cooperatively by the presence of L-aspartate and by magnesium ions at neutral and alkaline pHs. In the deamination reaction, the K-m value for L-aspartate was 1.09 mM at pH 7.0, and the S-1/2 value was 2.13 mM at pH 8.5. The V-max value were 99.2 U/ mg at pH 7.0 and 326 U/mg at pH 8.5. In the deamination reaction, the Km values for fumarate and ammonium were 0.797 and 25.2 mM, respectively, and V-max was 604 U/ mg. The optimum temperature of the enzyme was 55degreesC. The enzyme showed higher pH and thermal stabilities than that from mesophile: the enzyme was stable in the pH range of 4.5-10.5, and about 80% of its activity remained after incubation at 50degreesC for 60 min. The gene encoding the enzyme was cloned into Escherichia coli, and its nucleotides were sequenced. The gene consisted of an open reading frame of 1,410-bp encoding a protein of 469 amino acid residues. The amino acid sequence of the enzyme showed a high degree of identity to those of other aspartases, although these enzymes show different thermostabilities.

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  • マグロ血合肉中に含まれるセレンの化学種の同定 Reviewed

    吉田宗弘, 杉原悟, 千原優子, 近藤真理子, 老川典夫

    微量栄養素研究   20巻117-120頁   2003

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    食品中セレンの栄養有効性は種々の要因によって変動するため、食品中セレンの化学分析値と栄養有効性の間にはしばしば齟齬が生ずる。魚肉は日本人の主要なセレン供給源であるが、含有されるセレンの栄養有効性は他の食品由来のセレンに比較して低いと信じられている。魚肉中セレンの低栄養有効性の原因として、しばしば共存する水銀の影響が指摘されている。しかし、水銀含有量が低い魚肉製品でも、含有セレンが低栄養有効性を示すと報告されており、魚肉中セレンの一部が栄養有効性の低い化学種である可能性も否定できない。 従来、食品中の微量セレンの化学種の同定には、化学試薬との反応性を検討するなどの間接的な手法が多く用いられてきたため、食品中セレンを化学種ごとに分別定量することは困難であった。近年、高速液体クロマトグラフィー(HPLC)で分離したセレン化合物を誘導結合プラズマ質量分析器(ICPMS)で特異的に検出する手法が開発・普及しており、種々の食品に含有されるセレンの化学種の同定が可能となりつつある。 本研究では、魚肉の中でもセレン含有量がきわめて高いマグロの血合肉について、その化学種の同定をHPLC-ICPMSを用いて試みた。

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  • 第22番目のアミノ酸,ピロリシン

    老川 典夫

    日本生物工学会誌   81巻23頁   2003

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  • 食品中のD-アミノ酸:定量的解析と微量栄養素としての可能性 Reviewed

    老川典夫, 山田高史, 杉原祐貴, 吉田宗弘, 左右田健次

    微量栄養素研究   20巻121-124頁   2003

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    D-アミノ酸は、非天然型と見なされ生体内に存在するアミノ酸はすべてL-アミノ酸であると考えられてきた。しかし近年、ヒトを含む哺乳動物の特定の部位に高濃度の遊離型D-アミノ酸が存在することが報告され、それらの由来や生理機能に関心が寄せられている。本研究では、従来不分明の状態にある食品、とりわけ飲料中の各種D-アミノ酸に焦点を当て、食品中に含まれるD-アミノ酸の量的分布を明らかにするとともに、D-アミノ酸の生成機構や生理機能を解明する。

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  • 低温菌Cytophagasp.KUC-1の亜鉛含有耐熱・低基質特異性アルコールデヒドロゲナーゼとアルデヒドデヒドロゲナーゼ:酵素科学的性質とアルコール定量への応用 Reviewed

    数岡孝幸, 村岡郁夫, 吉田雅博, 神澤範行, 荒川憲昭, 老川典夫, 左右田健次

    微量栄養素研究   19巻83頁   2002.12

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    アルコールデヒドロゲナーゼ(AlcDH)とアルデヒドデヒドロゲナーゼ(AldDH)は共にアルコールとアルデヒド代謝に中心的位置を占め、前者は各基質の可逆的,後者は不可逆的脱水素反応を触媒するNAD(P)要求性酵素である。馬肝臓のAlcDHはZnを含み、哺乳類、常温性微生物由来の両酵素に関する研究は多いが極限微生物起源のこれらの酵素については不分明なままである。我々は先に南極海水由来の低温菌Cytophagasp.KUC-1株が著量のAlcDH及びAldDHを生産することを見いだした。本研究ではこの両酵素の酵素科学的諸性質を明らかにするとともに高発現系を構築し、その精製酵素を用いる高感度のアルコール定量法を開発した。

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  • Thermostable aldehyde dehydrogenase from psychrophile, Cytophaga sp. KUC-1: Enzymological characteristics and functional properties

    Yuko Yamanaka, Takayuki Kazuoka, Masahiro Yoshida, Kazuya Yamanaka, Tadao Oikawa, Kenji Soda

    Biochemical and Biophysical Research Communications   298 ( 5 )   632 - 637   2002

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    We found the occurrence of NAD(P)--dependent aldehyde dehydrogenase (EC1.2.1.5) in the cells of a psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, and purified to homogeneity. About 50% of the enzyme activity remained even after heating at 50°C for 65 min and the highest activity was observed in the range of 55-60°C. The enzyme was thermostable and thermophilic, although it was derived from a psychrophile. The circular dichroism at 222 nm of the enzyme showed a peak at 32°C. This temperature was closely similar to the transition temperature in the Arrhenius plots. The stereospecificity for the hydride transfer at C4-site of nicotinamide moiety of NADH was pro-R. The gene encoding the enzyme consisted of an open reading frame of 1506-bp encoding a protein of 501 amino acid residues. The significant sequence identity (61%) was found between the Cytophaga and the Pseudomonas aeruginosa enzymes, although their thermostabilities are completely different. © 2002 Elsevier Science (USA). All rights reserved.

    DOI: 10.1016/S0006-291X(02)02523-8

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  • 微生物のグアニジノブチラーゼ:結合2価金属による基質特異性の変化 Reviewed

    荒川憲昭, 老川典夫, 左右田健次

    微量栄養素研究   18巻77頁   2001.12

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    グアニジノ化合物を尿素とアミノ化合物へ加水分解する反応を触媒するアミジン加水分解酵素は、主にMn2+で活性化することが知られており,中でもL-アルギナーゼ(L-arginine amidinohydrolase, EC 3.5.3.1)は自然界に広く存在する。これらの中で種々のL-アルギナーゼやアグマチナーゼ(agmatine ureohydrolase, EC 3.5.3.11)はすでにクローニングされ、そのほとんどがMn2+要求性アミジン加水分解酵素であり、これらの推定アミノ酸配列は各々部分的に高い相同性を示す。このため、これらの酵素は共通の起源から分岐し,異なる基質特異性を持って進化したと考えられている。現在、L-アルギナーゼ分子と反応速度論的性質に関する研究が報告され,さらにラット肝臓L-アルギナーゼの3次元結晶構造が明らかになり,Mn2+が関与したL-アルギニン加水分解の反応機構が予測された。 一方、グアニジノブチラーゼ(4-guanidinobutyrate amidinohydrolase, EC 3.5.3.7)についてはPseudomonas属細菌やコリネ型細菌での存在が知られており,爬虫類や鳥類の肝臓においてもその活性が認められている。これらの酵素もまた,コリネ型細菌のBrevibacterium由来グアニジノブチラーゼが活性にZn2+を必要とすることを除いて、Mn2+要求性酵素である。しかし,グアニジノブチラーゼの金属イオンや一次構造については不分明なままである。 我々が土壌より単離したD-アルギニン資化性菌Arthrobacter sp. KUJ 6802から精製したグアニジノブチラーゼはMn2+を要求しなかった。本研究では本酵素の一次構造を明らかにし、本酵素の触媒過程における金属イオンの役割を解明することを目的としている。

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  • ホモシステインスルフィン酸、ホモシステインスルフォン酸、ホモシステインスルフォンアミドの合成とアミノ酸代謝酵素の反応性 Reviewed

    数岡孝幸, 前田彰久, 老川典夫, 左右田健次

    微量栄養素研究   18巻155頁   2001.12

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    酵素は一般的に基質特異性が高いが性質や構造が類似する基質アナログに対しても,しばしば作用する。酵素反応では基質の-COOHと-SOOHは類似する反応性を示す。例えばアスパラギン酸のアナログであるシステインスルフィン酸は,アスパラギン酸アミノトランスフェラーゼやアスパラギン酸デカルボキシラーゼに対して高い反応性を示す。しかしグルタミン酸のアナログであるホモシステインスルフィン酸(HCS)などについては不分明な状態にある。本研究では,グルタミン酸のアナログとしてHCSやホモシステインスルフォン酸(HCA)を,またグルタミンのアナログとしてホモシステインスルフォンアミド(HCN)を取り上げ,これらを科学的に合成し,様々なグルタミン酸及びグルタミン代謝関連酵素に対する反応性を調べた。

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  • Relationship between cytocidal activity and glutathione-S-transferase inhibition using doxorubicin coupled to stereoisomers of glutathione with different substrate specificity

    Y Hashizume, T Asakura, T Oikawa, T Yamauchi, K Soda, K Ohkawa

    ANTI-CANCER DRUGS   12 ( 6 )   549 - 554   2001.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:LIPPINCOTT WILLIAMS & WILKINS  

    To determine the cytotoxic mode of action of a glutathione (GSH)-doxorubicin (DXR) conjugate, which exhibited point cytotoxicity against various multidrug-resistant as well as DXR-sensitive cell lines, the molecular interaction between covalent GSH-DXR conjugates and glutathione-S-transferase (GST), a possible molecular target of the conjugates, was investigated. The following four GSH molecules with stereoisomeric forms were prepared: L-Glu-L-Cys-Gly (LL-GSH), D-Glu-L-Cys-Gly (DL-GSH), L-Glu-D-Cys-Gly (LD-GSH) anal D-Glu-D-Cys-Gly (DD-GSH). The enzymic activity of GST against each GSH stereoisomer was 88, 38, 8 and 4 nmol/ mg/min, respectively, suggesting that the L-form cysteine residue in the molecule was an important substrate of GST. Addition of DXR conjugated with each isomer (10 muM) to a GSH-containing GST assay mixture inhibited the GST activity to 32% for LL-GSH-DXR, 16% for DL-GSH-DXR and 61% for LD-GSH-DXR as compared with the solvent control. Moreover, IC,, values for these conjugates were 30, 20 and 250 nM, respectively. The cytocidal activity of each conjugate corresponded to the substrate specificity of GST activity for the GSH isomer. These conjugates bound to the GST molecule, and the binding ability was 0.746, 0.627 and 0.462 mol/mol of GST for LL-GSH-DXR, DL-GSH-DXR and LD-GSH-DXR, respectively. These findings suggested that GSH-DXR interacted with the substrate-binding site of the GST molecule and inhibition of GST activity exhibited potent cytotoxicity. [(C) 2001 Lippincott Williams & Wilkins.].

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  • Increased Transglycosylation Activity of Rhodotorula glutinisEndo-beta-Glucanase in MediaContaining Organic Solvent, Reviewed

    Biosci. Biotechnol.Biochem.   Vol.65 pp.1889-1892   2001.5

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    The transglycosylation of p-nitrophenyl-β-D-cellotrioside to cellotetraose catalyzed by endo-1,4-β-glucanase (cellulase, EC 3.2.1.4) from a psychrotrophic yeast, Phodotorula glutinis KUJ 2731, was increased by addition of a miscible organic solvent in the reaction mixture.Among various organic solvents tested, acetone was most effective.The transglycosylation activity increased with an increase in acetone concentrations, while hydrolysis activity was suppressed .The transglycosylation preferably occurred at acidic pH with the optimum pH at 2 in 10mΜ Gly-HCl buffer. The optimum temperature of transglycosylation was found to be 50℃ in the presence of 40℃ acetone.

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  • One-pot chemo-enzymatic enantiomerization of racemates , Reviewed

    Journal of Molecular Catalysis,   Vol.11 pp . 149-153   2001.4

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    A new one-pot chemo-enzymatic procedure was developed for enantiomerization of racemates based on enzymatic enantiospecific oxidation of a substrate and chemical non-enantiospecific reduction of the product. The principle is shown as follows for the L-proline production.L-Pro ←ゥゥゥゥゥゥゥゥゥゥゥゥゥ・ ・ ・ ゥゥゥゥゥゥゥゥゥゥゥゥゥゥゥゥ・ ↓ ・D-Pro―D-Amino acid oxidase→△1-Pyrroline-2-carboxylic acidL-Proline and L-pipecolate were produced from racemic proline and pipecolate by means of D-amino acid oxidase and sodium borohydride in high yield in this reaction system [J. W. Huh, K. Yokoigama, N. Esaki, K. Soda, Biosci., Biotechnol., Biochem. 56 (1992) 2081].DL-and L-Lactate were DL-enantiomerized in a one-pot reaction systems containing L-lactate oxidase and sodium borohydride in the similar mannar [S. Mukoyama, K. Yamanaka, T. Oikawa, K. Soda, Nippon Nogei Kagaku Kaishi 73 (1999) 62].Pyruvate was also converted to an equimolar amount of D-lactate in the same system. D-α-Hydroxybutyrate can be produced from the DL- and L-isomers, and α-ketobutyrate in the same manner though slowly.This method is applicable to production of other chiral compounds from the corresponding racemates. Ⓒ2001 Elsevier Science B. V. All right reserved.

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  • Chemo-Enzymatic D-Enantiomerization of DL-Lactate, Reviewed

    Biotechnology and Bioengineering,   Vol . 73 pp . 80-82   2001.3

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    Abstract:┏B/┓ We investigated the total conversion of racemic lactate, L-lactate, and pyruvate into D-lactate, which is very useful as a starting material for the synthesis of chiral compounds and much more valuable than the L-enantiomer by means of coupling of L-specific oxidation of the recemate with L-lactate oxidase and non-enantiospecific reduction of pyruvate to DL-lactate with sodiumborohydride.In this one-pot system, L-lactate was enantiospecifically oxidized to an achiral product, pyruvate, which was chemically reduced to DL-lactate leading to a turnover.Consequently, either DL-lactate, L-lactate, or pyruvate was fully converted to the D-enantiomer.We optimized the reaction conditions: DL-lactate was converted to D-lactate in 99% of the theoretical yield and with more than 99% enantiomeric excess.DL-α-Hydroxybutyrate and α- ketobutyrate were converted also to D-α- hydroxybutyrate in the same way, though slowly.Ⓒ2001

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  • Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC-1. Purification, molecular characterization and expression

    Tadao Oikawa, Kazuya Yamanaka, Takayuki Kazuoka, Noriyuki Kanzawa, Kenji Soda

    European Journal of Biochemistry   268 ( 16 )   4375 - 4383   2001

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    Language:English   Publishing type:Research paper (scientific journal)  

    We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and puriffied the enzyme to homogeneity. The molecular mass of the enzyme was determined to be ≈ 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD+ as a coenzyme, and catalyzed the oxidative deamination of L-valine, L-isoleucine, L-leucine and the reductive amination of α-ketoisovalerate, α-ketovalerate, α-ketoisocaproate, and α-ketocaproate. The reaction proceeds through an isoordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 °C was estimated to be 2.4 min. The kcat/Km (μ-1·s-1) values for L-valine and NAD+ at 20 °C were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.

    DOI: 10.1046/j.1432-1327.2001.02353.x

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  • Fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus: Its construction and reconstitution of active fragmentary enzyme

    Tadao Oikawa, Kunishige Kataoka, Yui Jin, Shinnichiro Suzuki, Kenji Soda

    Biochemical and Biophysical Research Communications   280 ( 4 )   1177 - 1182   2001

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    X-ray crystallographic studies revealed that various amino acid dehydrogenases fold into two domains in each subunit, a substrate-binding domain and an NAD(P)+-binding domain (Baker, P. J., Turnbull, A. P., Sedelnikova, S. E., Stillman, T. J., and Rice, D. W. (1995) Structure 3, 693-705). To elucidate the function and folding process of these two domains, we have genetically constructed a fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus consisting of an N-terminal polypeptide fragment corresponding to the substrate-binding domain including an N-terminus, and a C-terminal fragment corresponding to the NAD+-binding domain. The two peptide fragments were expressed in separate host cells and purified. When both fragments were mixed, the leucine dehydrogenase activity with a specific activity of 1.4% of that of the wild-type enzyme appeared. This suggests that both peptide fragments mutually recognize each other, associate and fold correctly to be catalytically active, although the activity is low. However, the fragmentary form of enzyme produced catalyzed the oxidative deamination of L-leucine, L-isoleucine, and L-valine with broad substrate specificity compared to that of the wild-type enzyme. The fragmentary enzyme retained more than 75% of the initial activity after heating at 50°C for 60 min. The fragmentary enzyme was more stable on heating than separate peptide fragments. These results suggest that the two domains of leucine dehydrogenase probably fold independently, and the two peptide fragments interact and associate with each other to form a functional active site. © 2001 Academic Press.

    DOI: 10.1006/bbrc.2001.4252

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  • 低温微生物と好冷性酵素:新しい生体触媒としての可能性

    老川典夫

    日本生物工学会誌   78巻138頁   2000

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  • Production of D-glutamate from L-glutamate with glutamate racemase and L-glutamate oxidase

    T Oikawa, M Watanabe, H Makiura, H Kusakabe, K Yamade, K Soda

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   63 ( 12 )   2168 - 2173   1999.12

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    We studied production of D-glutamate from L-glutamate using a bioreactor consisting of two columns of sequentially connected immobilized glutamate racemase (EC 5.1.1.3, from Bacillus subtilis no 3336) and L-glutamate oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6): L-glutamate was racemized by the glutamate racemase column, and then L-glutamate was oxidized by the L-glutamate oxidase column. Consequently only D-glutamate remained, and was easily separated from the alpha-ketoglutarate formed by anion-exchange chromatography. Both enzymes were highly stabilized by immobilization. The pH and temperature optima of immobilized glutamate racemase (pH 8, 40 degrees C) were similar to those of immobilized L-glutamate oxidase (pH 7, 50 degrees C). Accordingly, we connected the two columns tandemly to do both enzyme reactions under the same conditions. Actually 4.5 mu mol of D-glutamate was produced and isolated from 10 mu mol of L-glutamate, about 90% of the theoretical yield.

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  • Stereoisomers of glutathione: Preparation and enzymatic reactivities

    T Oikawa, T Yamauchi, H Kumagai, K Soda

    JOURNAL OF NUTRITIONAL SCIENCE AND VITAMINOLOGY   45 ( 2 )   223 - 229   1999.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:CENTER ACADEMIC PUBL JAPAN  

    We synthesized a series of stereoisomers of glutathione (GSH) and glutathione disulfide (GSSG) by the solid-phase method. These peptides were used to examine their reactivities with enzymes acting on glutathione. The glutathione reductase of yeast acted only on LL-GSSG. Glutathione S-transferase catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with LL-GSH and DL-GSH (Km (mM): for LL-GSH, 0.035; and for DL-GSH, 0.62), but the DD- and LD-diastereomers were inert, gamma-Glutamyl transpeptidase catalyzed the transfer of gamma-glutamyl moiety of LL-GSH and DL-GSH to taurine forming gamma-glutamyl taurine and cysteinyl taurine (Km (mM): for LL-GSH, 0.336; and for DL-GSH, 0.628), but the other diastereomers were not the substrates. The occurrence of L-cysteinyl residue in the tripeptides is required for the glutathione analogue to be a substrate of the enzymes.

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  • Vitamin B-6 enzymes participating in selenium amino acid metabolism

    K Soda, T Oikawa, N Esaki

    BIOFACTORS   10 ( 2-3 )   257 - 262   1999

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    Various vitamin B-6 enzymes play important roles in mammalian and microbial metabolism of selenium amino acids. Selenocysteine is synthesized from selenohomocysteine by catalysis of cystathionine beta-synthase and cystathionine gamma-lyase, which both require pyridoxal phosphate. Selenocysteine beta-lyase, a new B-6-enzyme, exclusively catalyzes beta-elimination of selenocysteine, and occurs in mammalian systems and bacteria. Methionine gamma-lyase, cysteine desulfurase, cysteine sulfinate desulfinase, and D-selenocystine alpha, beta-lyase, which are B-6-enzymes, act on cysteine, cysteine sulfinate, D-cystine, and their derivatives, and their selenium counterparts indiscriminately. Their reaction mechanisms are comparatively described.

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  • Endo-beta-glucanase secreted by a psychrotrophic yeast: Purification and characterization

    T Oikawa, Y Tsukagawa, K Soda

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   62 ( 9 )   1751 - 1756   1998.9

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    A psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, isolated from soil, effectively produced an extracellular endo-beta-glucanase (EC 3.2.1.4). The enzyme was monomeric, and the molecular mass was about 40,000 Da. The N-terminal amino acid sequence was H-Ser-Leu-Pro-Lys-Leu-Gly-Gly-Val-Asp-Leu-Ala-Gly-Leu-Asp-Ile-Gly-Lys-Asp-Lys-Asn-. alpha-Helix content was calculated to be about 32.6%. The isoelectric point was 8.57. The activation energy was 20.9 kJ/mol, which was much smaller than that of mesophilic enzymes. The enzyme was active at temperatures from 0 to 70 degrees C, with a highest initial velocity at 50 degrees C similar to other psychrotrophic enzymes. The enzyme was inhibited by Hg2+. The enzyme catalyzed hydrolysis of carboxymethyl cellulose with an apparent K-m of 1.1% and V-max of 556 mu mol/min/mg. Products from the enzymatic hydrolysis of carboxymethyl cellulose by the enzyme were glucose, cellobiose, and cellotriose. The enzyme also catalyzed the transglycosylation of p-nitrophenyl-beta-cellotrioside to cellotetraose.

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  • A novel type of D-mannitol dehydrogenase from Acetobacter xylinum: Occurrence, purification, and basic properties

    T Oikawa, J Nakai, Y Tsukagawa, K Soda

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   61 ( 10 )   1778 - 1782   1997.10

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    We purified a novel type of D-mannitol dehydrogenase, which contains a c-type cytochrome and an unknown chromophore in the soluble fraction of an acetic acid bacterium, Acetobacter xylinum KU-1, to homogeneity. The enzyme showed the maximum activity at pH 5 and 40 degrees C. It was stable up to 60 degrees C at pH 6, and was inhibited by Hg2+ and p-quinone (K-i = 0.18 mM). The molecular weight of the enzyme was about 140,000, and those of the subunits were 69,000, 51,000, and 20,000; the enzyme is hetero-trimeric and contained 8 g-atoms of Fe per mole. The alpha-helix was estimated to be about 52.9%. The enzyme catalyzed phenazine methosulfate dependent oxidation of D-mannitol with an apparent K-m of 98 mu M (for D-mannitol) and V-max of 213 mu mol/min/mg. The reduced form of the enzyme showed the absorption maxima at 386, 416, 480, 518, 550, and 586 nm, which are attributable to a c-type cytochrome in the enzyme.

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  • Endo-beta-glucanase from Acetobacter xylinum: Purification and characterization

    T Oikawa, T Kamatani, T Kaimura, M Ameyama, K Soda

    CURRENT MICROBIOLOGY   34 ( 5 )   309 - 313   1997.5

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    A cellulose-producing acetic acid bacterium, Acetobacter xylinum KU-1, abundantly produces an extracellular endo-beta-glucanase (EC 3.2.1.4) in the culture broth. The enzyme was purified to homogeneity by DEAE- and CM- Toyopearl 650M ion-exchange chromatography, Butyl-Toyopearl 650M hydrophobic chromatography, and Toyopearl HW-50 gel filtration. The purified enzyme showed the maximum activity at pH 5 and 50 degrees C: it was stable up to 50 degrees C at pH 5, activated by Co2+, and competitively inhibited by Hg2+; the apparent K-i was 7 mu m. The molecular weight of the enzyme was determined to be about 39,000 by sodium dodesyl sulfate/polyacrylamide gel electrophoresis, and about 41,000 by Toyopearl HW-50 gel filtration; the enzyme is monomeric. The enzyme hydrolyzed carboxy-methylcellulose with an apparent K-m of 30 mg/ml and V-max of 1.2 mu M/min. It hydrolyzed cellohexaose to cellobiose, cellotriose and cellotetraose, and also cellopentaose to cellobiose and cellotriose, but did not act on cellobiose, cellotriose, or cellotetraose.

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  • 酢酸菌のバクテリアセルロース生産とその代謝関連酵素の生化学的研究

    老川典夫

    (財)サッポロ生物科学振興財団第11回助成研究報告書   11-18頁   1997

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  • セレン含有アミノ酸,ペプチド,タンパク質,工学と技術

    老川典夫

    11巻2号79-84頁   1996

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  • PRODUCTION OF CELLULOSE FROM D-ARABITOL BY ACETOBACTER-XYLINUM KU-1

    T OIKAWA, T MORINO, M AMEYAMA

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   59 ( 8 )   1564 - 1565   1995.8

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    We found that Acetobacter xylinum KU-1 produced cellulose from D-arabitol. The maximum cellulose production was obtained when it was grown in a medium containing 2.0% D-arabitol, 1.0% tryptone, and 1.0% yeast extract (pH 5) at 30 degrees C for 96h statically. The productivity was more than 6 times as much as that of D-glucose [productivity (mg/ml-medium): from D-arabitol, 12.4; from D-glucose, 2.0].

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  • PRODUCTION OF CELLULOSE FROM D-MANNITOL BY ACETOBACTER-XYLINUM KU-1

    T OIKAWA, T OHTORI, M AMEYAMA

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   59 ( 2 )   331 - 332   1995.2

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    We found that Acetobacter xylinum KU-1 produced cellulose from D-mannitol. The optimum culture conditions for cellulose production were 1.5% D-mannitol, 0.5% Polypeptone, 2.0% yeast extract, pH 5, 30 degrees C, and 48 h. The amount of cellulose from D-mannitol was more than 3 times as much as that from D-glucose under the same culture conditions [productivity (mg/ml-medium): from D-mannitol, 4.6; from D-glucose, 1.2].

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  • DETECTION OF CARBOXYMETHYL CELLULASE ACTIVITY IN ACETOBACTER-XYLINUM KU-1

    T OIKAWA, M TAKAGI, M AMEYAMA

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   58 ( 11 )   2102 - 2103   1994.11

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    We detected carboxymethyl cellulase activity in a crude extract of Acetobacter xylinum KU-1. The enzyme activity was detected when glycerol, D-fructose, D-mannitol, D-glucose, D-arabitol, D-sorbitol, or carboxynmethyl cellulose was used as a carbon source. The optimum pH was found to be 4.0, while the optimum temperature was 50 degrees C. The enzyme activity was inhibited characteristically by the addition of Hg2+.

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  • DETECTION OF DYE-LINKED D-MANNITOL DEHYDROGENASE-ACTIVITY IN ACETOBACTER-XYLINUM KU-1

    T OIKAWA, M AMEYAMA

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   57 ( 9 )   1580 - 1581   1993.9

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    We detected dye-linked D-mannitol dehydrogenase activity in the crude extract of Acetobacter xylinum KU-1. The enzyme activity was specific for D-mannitol, and not pyridine nucleotide (NAD+, NADP+)-dependent. The optimal pH was found to be 5.0, while the optimal temperature was at 50-degrees-C. The enzyme activity was inhibited by p-quinone noncompetitively.

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  • SYNTHESIS AND CHARACTERIZATION OF THE SELENIUM ANALOG OF GLUTATHIONE DISULFIDE

    T TAMURA, T OIKAWA, A OHTAKA, N FUJII, N ESAKI, K SODA

    ANALYTICAL BIOCHEMISTRY   208 ( 1 )   151 - 154   1993.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

    Grant-in-Aid for Scientific Research 199204-199304

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  • A Selenium Analogue of Metallothionein Chemical Synthesis and Characterization

    OIKAWA Tadao

    Toyobo Biotechnology Fundation Record of Activities   pp. 196-197   1993

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  • セレンを含むアミノ酸,ペプチド,酵素,バイオサイエンスとインダストリ_

    老川典夫, 左右田健次

    50巻12号1197-1201頁   1992

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  • グルタチオンペルオキシダ_ゼとその酵素モデル

    老川典夫, 左右田健次

    日本農芸化学会誌   66巻10号1501-1504頁   1992

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  • PURIFICATION AND PROPERTIES OF DYE-LINKED ALDEHYDE DEHYDROGENASE IN RHODOPSEUDOMONAS-ACIDOPHILA M402

    K YAMANAKA, H IINO, T OIKAWA

    AGRICULTURAL AND BIOLOGICAL CHEMISTRY   55 ( 4 )   989 - 996   1991.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

    Rhodopseudomonas acidophila M402 grown under aerobic-dark condition shows two band of dye-linked aldehyde dehydrogenase activity by polyacrylamide gel electrophoresis. The slower-migrating band coincided with the dye-linked aromatic alcohol dehydrogenase that was active on aromatic and aliphatic alcohols and aldehydes. The faster-migrating band was a new dye-linked aldehyde dehydrogenase. The latter enzyme was purified 125-fold by ultracentrifugation and column chromatographies on DEAE-cellulose, Bio-Gel HTP, and Sepharose CL-6B. The enzyme has a relative molecular mass of 70,000 daltons, and a subunit size of 35,000 dalton indicates the dehydrogenase has a dimeric structure. The isoelectric point was pH 4.74. NAD+ and NADP+ do not act as electron acceptors. The enzyme was active specifically on straight chain aldehydes (C3-C10) rather than on benzaldehyde and its substitutes. Aromatic and aliphatic alcohols were inert for this enzyme. The Michaelis constant (mM) were 1.6, 0.6, 0.9, 3.6, 0.5, 0.3, 0.1, 0.9, 0.6, 0.3, and 0.1 for benzaldehyde, m-hydroxybenzaldehyde, m-anisaldehyde, vanillin, propionaldehyde, butyraldehyde, hexaldehyde, heptaldehyde, octaldehyde, nonaldehyde, and decaldehyde, respectively.

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  • METALLOSELENONEIN, THE SELENIUM ANALOG OF METALLOTHIONEIN - SYNTHESIS AND CHARACTERIZATION OF ITS COMPLEX WITH COPPER IONS

    T OIKAWA, N ESAKI, H TANAKA, K SODA

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   88 ( 8 )   3057 - 3059   1991.4

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    We used an automated peptide synthesizer to produce a peptide, metalloselenonein, that contains selenocysteine residues substituted for all cysteine residues in Neurospora crassa copper metallothionein. Metalloselenonein binds 3 mol of Cu(I) per mol. This adduct shows a broad absorption band between 230 and 400 nm and a fluorescence band at 395 nm, which can be attributed to copper-selenolate coordination. The circular dichroism spectrum of the copper-metalloselenonein complex shows a positive band around 245 nm attributable to asymmetry in metal coordination.

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  • セレノシステイン含有ペプチド(メタロセレノネイン,グルタセレノン):その化学合成と機能

    老川典夫

    技苑   69巻27-32頁   1991

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  • 含セレンペプチド,グルタセレノンのグルタチオンぺルオキシダ_ゼ活性 Reviewed

    老川典夫, 江崎信芳, 芦田裕之, 左右田健次

    微量栄養素研究   第8集75-80頁   1991

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  • グルタチオンのセレノシステインアナログの合成と性質 Reviewed

    江崎信芳, 老川典夫, 田中英彦, 左右田健次

    Biomed Res Trace Elem   1 ( 2 )   261 - 262   1990.12

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    J-GLOBAL

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  • 新しいセレノシステイン含有ペプチドの合成と性質 Reviewed

    老川典夫, 江崎信芳, 田中英彦, 左右田健次

    微量栄養素研究   第7集97-101頁   1990

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  • セレン置換メタロチオネインの合成と性質 Reviewed

    老川典夫, 杉本学, 江崎信芳, 田中英彦, 左右田健次

    微量栄養素研究   第5集75-79頁   1988

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  • SYNTHESIS OF BIOLOGICALLY-ACTIVE SELENIUM-CONTAINING AMINO-ACIDS AND PEPTIDES

    H TANAKA, N ESAKI, M SUGIMOTO, T OIKAWA, P CHOCAT, K SODA

    PHOSPHORUS SULFUR AND SILICON AND THE RELATED ELEMENTS   38 ( 1-2 )   19 - 24   1988

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  • Chemical synthesis and expression of copper metallothionein gene of Neurospora crassa

    Manabu Sugimoto, Tadao Oikawa, Nobuyoshi Esaki, Hidehiko Tanaka, Kenji Soda

    Journal of Biochemistry   104 ( 6 )   924 - 926   1988

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press  

    The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene. The MT-β-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the β-galactosidase through methionine residues. An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation. The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs. © 1988 COPYRIGHT 1988 BY THE JOURNAL OF BIOCHEMISTRY.

    DOI: 10.1093/oxfordjournals.jbchem.a122584

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  • 再び注目を浴びるメタロチオネイン

    老川典夫, 左右田健次

    化学   44巻6号418-419頁   1988

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  • エッセンシャル タンパク質工学

    老川 典夫, 大島 敏久, 保川 清, 三原 久明, 宮原 郁子

    講談社サイエンティフィク  2018.2 

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  • D-Amino Acids Physiology, Metabolism, and Application Reviewed

    老川 典夫( Role: Contributor)

    Springer  2016.10 

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  • 日本酒の新たな呈味性成分「D-アミノ酸」

    老川 典夫( Role: Sole author)

    日本醸造協会誌  2015 

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  • X線構造解析による代謝酵素の反応機構解明

    老川典夫( Role: Sole author)

    京都大学化学研究所共同利用・共同研究拠点平成23年成果報告書  2012.6 

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  • 環境アポトジェンを含む環境汚染科学物質の作用動態解析と化学生態学的防除法の開発研究プロジェクト

    土戸哲明, 池内俊彦, 上里新一, 下家浩二, 吉田宗弘, 福永健治, 安原裕紀, 長谷川喜衛, 老川典夫, 松村吉信, 岩木宏明

    技苑  2012.3 

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  • D-アミノ酸を利用した旨み増強日本酒の製造方法

    老川典夫( Role: Sole author)

    関西大学新技術説明会資料集  2011.11 

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  • X線構造解析による代謝酵素の反応機構解明

    老川典夫( Role: Sole author)

    京都大学化学研究所共同利用・共同研究拠点平成22年成果報告書  2011.6 

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  • Amino Acid Biosynthesis-pathways, Regulation and Metabolic Engineering

    OIKAWA Tadao

    Springer, Berlin Heidelberg New York  2007 

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  • Thermotorelant D-Amino acid transferase:Characterization and Reaction Mechanism

    Kenji Soda, Tadao Oikawa, Nobuyoshi Esaki, Tohru Yoshimura

    1998.3 

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  • Trace elements and Euzymes:Reaction Mechanism of Enzyme Activation

    Nobuyoshi Esaki, Tadao Oikawa, Kenji Soda

    1998.1 

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  • 生化学辞典

    老川 典夫( Role: Contributor)

    東京化学同人  1998 

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  • 新生化学実験講座 13・バイオテクノロジー

    老川 典夫( Role: Contributor)

    東京化学同人  1993 

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Presentations

  • 乳酸菌Leuconostoc mesenteroides LK-151のセレノシステインβ-リアーゼ:酵素科学的性質と触媒機能発現機構の解明

    岡島 浩平, 山中 一也, 加藤 志郎, 老川 典夫

    2020.2 

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  • X-ray Structural Studies on Reaction Mechanism of Maleylacetate Reductase

    老川 典夫

    2020 

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  • ポリ-D-ジアミノブタン酸生産放線菌Streptoalloteichus hindustannusに見出した新規D-ペプチド分解酵素の同定

    宮脇 大輝, 福本 響, 濱野 吉十, 老川 典夫, 山中 一也

    第71回日本生物工学会大会  2019.9 

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    Event date: 2019.9

    Venue:岡山  

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  • 放線菌Streptomyces hindustanusに見出した新規PLP非依存型Diaminobutyric acid ラセマーゼの機能解析

    尾崎 僚, 福本 響, 濱野 吉十, 老川 典夫, 山中 一也

    第71回日本生物工学会大会  2019.9 

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    Event date: 2019.9

    Venue:岡山  

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  • 放線菌Streptoalloteichus hindustanusが生産する新規カチオン性ホモポリアミノ酸の構造決定及びその抗菌活性評価

    福本 響, 戸口 梨那, 荒川 賢治, 濱野 吉十, 老川 典夫, 山中 一也

    第71回日本生物工学会大会  2019.9 

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    Venue:岡山  

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  • A NOVEL PYRIDOXAL 5'-PHOSPHATE-DEPENDENT HISTIDINE RACEMASE FROM Leuconostoc mesenteroides SUBSP. SAKE NBRC 102480: DISCOVERY AND STRUCTURAL CHARACTERIZATION

    老川 典夫

    The 4th International Conference of D-Amino Acid Research (IDAR-2019)  2019.9 

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    Event date: 2019.9

    Venue:Tokyo  

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  • D-アラニンおよびD-ロイシンの給餌がマウスおよびラットの血清生化学検査値に及ぼす影響

    清水 栄人, 中川 航希, 老川 典夫, 細見 亮太, 福永 健治, 吉田 宗弘

    第36回日本微量栄養素学会学術集会  2019.6 

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    Venue:茨木  

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  • ヒトD-アミノ酸酸化酵素発現微生物株の構築と評価

    加藤 志郎, 老川 典夫

    第36回日本微量栄養素学会学術集会  2019.6 

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    Venue:茨木  

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  • ε-poly-L-lysine合成酵素ホモログにおける基質特異性予測手法の確立と新規生理活性ホモポリアミノ酸探索への応用

    木村ほのか, 福本 響, 濱野 吉十, 老川 典夫, 山中 一也

    日本農芸化学会2019年度大会  2019.3 

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    Event date: 2019.3

    Venue:京都  

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  • Lactobacillus sakei LK-145のアスパラギナーゼ ホモログ遺伝子の大腸菌を宿主とする高発現系の構築と遺伝子産物の機能解析

    田村 和也, 山中 一也, 老川 典夫

    日本農芸化学会2019年度大会  2019.3 

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    Event date: 2019.3

    Venue:京都  

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  • 放線菌Streptomyces albulusの全ゲノム及び網羅的遺伝子発現解析により見出した新奇ペプチド合成酵素遺伝子群の機能解析

    友杉 玲那, 藤田 彩香, 宮本 昴, 濱野 吉十, 老川 典夫, 山中 一也

    日本農芸化学会2019年度大会  2019.3 

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    Venue:京都  

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  • 乳酸菌Leuconostoc mesenteroides LK-151 のセレン耐性能の評価と セレノシステインβ-リアーゼホモログの機能解析

    岡島 浩平, 大塚 政志, 細見 亮太, 吉田 宗弘, 山中 一也, 老川 典夫

    第35回日本微量栄養素学会学術集会  2018.6 

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    Venue:京都  

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  • 亜セレン酸ナトリウムおよびセレノ -L- メチオニン曝露におけるシロイヌナズナ (Arabidopsis thaliana)の遺伝子発現量の網羅的解析

    大塚 政志, 細見 亮太, 老川 典夫, 福永 健治, 吉田 宗弘

    第35回日本微量栄養素学会学術集会  2018.6 

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    Venue:京都  

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  • ゲノムマイニングにより見出した新規ポリアミノ酸の構造及び生合成機構の解析

    福本 響, 濱野 吉十, 老川 典夫, 山中 一也

    日本農芸化学会2018年度大会  2018.3 

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    Venue:名古屋  

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  • 直鎖状ホモポリアミノ酸を合成する膜結合型NRPS様ペプチド合成酵素

    山中 一也, 老川 典夫, 濱野 吉十

    日本農芸化学会2018年度大会  2018.3 

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    Venue:名古屋  

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  • 黒酢醪由来 D-アミノ酸高生産乳酸菌 Pediococcus pentosaceus KTCC12 の D-デヒドラターゼの機能解析

    岩田 音々, 山中 一也, 老川 典夫

    日本農芸化学会関西支部第502回講演会  2018.2 

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    Venue:京都  

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  • X線結晶構造解析によるL-アスパラギナーゼ の耐熱性及び基質特異性の研究

    老川 典夫

    京都大学化学研究所共同利用・共同研究拠点化学関連分野の深化・連携を基軸とする先端・学際研究拠点平成29年成果報告書  2018 

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  • D-アミノ酸高生産乳酸菌Lactobacillus casei M10-8由来新奇二機能グルタミン酸ラセマーゼのC末端側ドメインの機能解析

    宮本 将崇, 山中 一也, 老川 典夫

    ConBio2017  2017.12 

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    Event date: 2017.12

    Venue:神戸  

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  • クエン酸による乳酸菌のD-アミノ酸生産量の変化とゲノム情報の統合的解析

    加藤 志郎, 老川 典夫

    第34回日本微量栄養素学会学術集会  2017.6 

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    Venue:京都  

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  • 食品中のD-アスパラギン酸定量への応用を目的としたD-アスパラギン酸の酵素定量法の開発

    鷲尾 翼, 老川 典夫

    第34回日本微量栄養素学会学術集会  2017.6 

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    Venue:京都  

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  • シロイヌナズナ由来ホモシステインS-メチルトランスフェラーゼ3遺伝子の大腸菌での発現系の構築とin vitroでの機能解析

    寺田 俊輝, 山中 一也, 吉田 宗弘, 老川 典夫

    第34回日本微量栄養素学会学術集会  2017.6 

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    Venue:京都  

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  • 微生物二次代謝産物生合成研究をアシストする高効率TAR直接クローンング法

    安田 真央, 老川 典夫, 山中 一也

    日本農芸化学会2017年度大会  2017.3 

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    Venue:京都  

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  • epsilon-Poly-L-lysine合成酵素(Pls)における縮合ドメインの機能解析

    阿部 修平, 吉村 友宏, 老川 典夫, 山中 一也

    日本農芸化学会2017年度大会  2017.3 

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    Venue:京都  

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  • レゾルシノール代謝に関わるフラビンレダクターゼのX線構造生物学研究

    老川 典夫

    京都大学化学研究所共同利用・共同研究拠点化学関連分野の深化・連携を基軸とする先端・学際研究拠点平成28年成果報告書  2017 

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  • Lactobacillus sakei由来新規二機能性アミノ酸ラセマーゼ, MalYの機能解析

    加藤志郎, 老川 典夫

    第12回D-アミノ酸研究会学術講演会  2016.9 

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    Event date: 2016.9

    Venue:高知  

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  • Thermococcus litoralis DSM 5473由来アスパラギン酸ラセマーゼとL-アスパラギン酸オキシダーゼを用いたD-およびL-Aspの新規定量法の開発

    鷲尾 翼, 老川 典夫

    第12回D-アミノ酸研究会学術講演会  2016.9 

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    Venue:高知  

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  • シロイヌナズナにおける微量D-アミノ酸の吸収および生育阻害解析

    加藤 志郎, 安原 裕紀, 老川 典夫

    第33回日本微量栄養素学会学術集会  2016.6 

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    Venue:京都  

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  • レゾルシノールモノオキシゲナーゼの構造ー機能に関するX線構造解析

    老川 典夫

    京都大学化学研究所 共同利用・共同研究拠点 化学関連分野の深化・連携を基軸とする先端・学際研究拠点 平成27年成果報告書  2016 

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  • AMINO ACID ANALYSIS OF MOUSE MACROPHAGE

    加藤 志郎, 増田 有紀, 小西 守周, 老川 典夫

    The 2nd International Conference of D-Amino Acid Research (IDAR-2014)  2015.9 

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    Event date: 2015.9

    Venue:Utsunomiya, Japan  

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  • 乳酸菌Lactobacillus sakei由来シスタチオニンβ-リアーゼの機能解析

    加藤 志郎, 老川 典夫

    第11回D-アミノ酸学会学術講演会  2015.8 

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    Event date: 2015.8

    Venue:新潟  

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  • Thermococcus litoralis DSM 5473の耐熱性L-アスパラギン酸オキシダーゼの分子特性の解析

    鷲尾 翼, 老川 典夫

    日本ビタミン学会第67回大会  2015.6 

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    Venue:奈良  

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  • 乳酸菌Lactobacillus sakei LK-145のD-アミノ酸生産に及ぼす培地成分と培養条件の影響

    森田 朱香, 老川 典夫

    第32回日本微量栄養素学術集会  2015.5 

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    Event date: 2015.5

    Venue:京都  

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  • D-アミノ酸高生産乳酸球菌のゲノム解析

    加藤 志郎, 高橋 俊成, 老川 典夫

    第32回日本微量栄養素学術集会  2015.5 

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    Venue:京都  

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  • D-アミノ酸高生産乳酸菌のドラフトゲノム解析

    加藤 志郎, 高橋 俊成, 老川 典夫

    第62回日本生化学会近畿支部例会  2015.5 

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    Event date: 2015.5

    Venue:滋賀  

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  • ゲノム解析から見た乳酸菌のD-アミノ酸高生産機構

    加藤志郎, 老川 典夫

    日本農芸化学会2015年度大会  2015.3 

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    Event date: 2015.3

    Venue:岡山  

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  • 超好熱アーキアThermococcus litoralis DSM 5473のL-アスパラギン酸オキシダーゼ:性質と反応速度論的解析

    鷲尾 翼, 老川 典夫

    日本農芸化学会2015年度大会  2015.3 

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    Venue:岡山  

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  • Flavobacterium indicum DSM 177447の好熱性スレオニンデヒドロゲナーゼ:性質と反応速度論的解析

    井上 淳, 老川 典夫

    日本農芸化学会2015年度大会  2015.3 

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    Venue:岡山  

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  • レゾルシノールモノオキシゲナーゼの構造ー機能に関するX線構造解析

    老川 典夫

    都大学化学研究所 共同利用・共同研究拠点 化学関連分野の深化・連携を基軸とする先端・学際研究拠点 平成26年成果報告書  2015 

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  • 次世代ベンチトップ型シーケンサーによるゲノム・エピゲノム解析に基づく統合的健康生命研究

    老川 典夫, 吉田 宗弘, 池内 俊彦, 下家 浩二, 土戸 哲明, 松村 吉信

    平成26(2014)年度研究成果報告書  2015 

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  • Thermococcus litoralis DSM 5473のL-アスパラギン酸オキシダーゼホモログ遺伝子のクローニングとその遺伝子産物の精製と酵素科学的性質の解明

    鷲尾 翼, 老川 典夫

    第87回日本生化学大会  2014.10 

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  • Arabidopsis thaliana芽生えの生育と外因性アミノ酸取り込みとの関連

    加藤 志郎, 老川 典夫

    第87回日本生化学大会  2014.10 

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    Venue:京都  

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  • Flavobacterium indicum DSM 177447のスレオニンデヒドロゲナーゼホモログ遺伝子のクローニングとその遺伝子産物の酵素科学的性質の解明

    井 上淳, 老川 典夫

    第87回日本生化学大会  2014.10 

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    Venue:京都  

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  • D-AMINO ACID IN SAKE: DISTRIBUTION, PRODUCTION MECHNISM, AND FUNCTION

    老川 典夫

    The 2nd International Conference of D-Amino Acid Research (IDAR-2014)  2014.9 

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    Event date: 2014.9

    Venue:Utsunomiya, Japan  

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  • 超好熱アーキアThermococcus litoralis DSM 5473のアスパラギナーゼホモログ遺伝子のクローニングとその遺伝子産物の酵素科学的性質の解明

    山崎 遼, 老川典夫

    日本農芸化学会2014年度大会  2014.3 

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    Event date: 2014.3

    Venue:神奈川  

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  • X線構造解析によるレゾルシノールヒドロキシラーゼの反応機構研究

    老川 典夫

    京都大学化学研究所 共同利用・共同研究拠点 化学関連分野の深化・連携を基軸とする先端・学際研究拠点 平成25年成果報告書  2014 

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  • 日本酒中のD-アミノ酸の定量的解析に基づく生成機構及び呈味機能の解明とD-アミノ酸に着目した新商品開発への展望

    老川典夫

    新アミノ酸分析研究会第3回学術講演会  2013.12 

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    Event date: 2013.12

    Venue:東京  

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  • 食品中のD-アミノ酸とその機能

    老川典夫

    日本ペプチド学会市民フォーラム2013  2013.11 

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    Event date: 2013.11

    Venue:大阪  

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  • 食品中のD-アミノ酸の定量的解析とD-アミノ酸強化新規機能性食品の開発

    老川典夫

    第86回日本生化学会大会  2013.9 

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    Event date: 2013.9

    Venue:横浜  

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  • 乳酸菌を用いるD-アミノ酸強化福山黒酢生産方法の開発

    老川典夫, 竹下義隆

    第9回D-アミノ酸研究会学術講演会  2013.9 

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    Venue:大阪  

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  • 乳酸菌Lactobacillus casei M10-8のアミノ酸ラセマーゼホモログ遺伝子の網羅的発現と遺伝子産物の酵素化学的性質の解明

    齊藤瑠実, 老川典夫

    第9回D-アミノ酸研究会学術講演会  2013.9 

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    Venue:大阪  

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  • D-アラニンを利用した旨み増強日本酒の製造方法

    老川典夫

    第99回清酒製造技術セミナー  2013.4 

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    Event date: 2013.4

    Venue:東京  

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  • X線構造解析によるレゾルシノールヒドロキシラーゼの反応機構研究

    老川典夫

    京都大学化学研究所 共同利用・共同研究拠点化学関連分野の深化・連携を基軸とする先端・学際研究拠点平成24年成果報告書  2013 

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  • 生酛由来乳酸菌Lactobacillus sakei NBRC 15983及びLeuconostoc mesenteroides NBRC 102480のアミノ酸ラセマーゼホモログ遺伝子の網羅的発現と新規ヒスチジンラセマーゼの部位特異的変異導入による触媒機能発現機構の解明

    郷上佳孝, 老川典夫

    2012年度酵素補酵素研究会  2012.7 

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    Event date: 2012.7

    Venue:名古屋  

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  • 日本酒中のD-アミノ酸の生成機構の解明

    郷上佳孝, 岡田かおり, 森山昌和(菊正宗酒造), 溝口晴彦(菊正宗酒造), 老川典夫

    第29回日本微量栄養素学術集会  2012.6 

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    Venue:京都  

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  • 黒酢及び食酢中のD-アミノ酸の定量的解析

    岡田かおり, 郷上佳孝, 竹下義隆(福山黒酢), 老川典夫

    第29回日本微量栄養素学術集会  2012.6 

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    Venue:京都  

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  • レゾルシノールヒドロキシラーゼの構造-機能解析

    山内貴恵(京都大学), 小林一隆(京都大学), 藤井知実(京都大学), 吉田雅博, 老川典夫, 畑安雄(京都大学)

    第59回日本生化学会近畿支部例会  2012.5 

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    Venue:京都  

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  • 生もと由来Lactbacillus sakaiのグルタミン酸ラセマーゼ遺伝子導入Corynebacterium glutamicumを用いるD-グルタミン酸生産

    矢野正博(D), 老川典夫

    日本農芸化学会2012年度大会  2012.3 

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    Venue:京都  

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  • Rhizobium sp. Strain MTP-10005由来フラビン還元酵素のX線結晶構造解析

    山内貴恵(京都大学), 藤井知実(京都大学), 吉田雅博, 老川典夫, 畑安雄(京都大学)

    日本農芸化学会2012年度大会  2012.3 

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    Venue:京都  

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  • 生もと由来乳酸菌Leuconostoc mesenteroides NBRC 102481のヒスチジンラセマーゼ:部位特異的変異導入による変異型酵素の調製とその酵素科学的性質

    郷上佳孝, 老川典夫

    日本農芸化学会2012年度大会  2012.3 

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    Venue:京都  

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  • D-アスパラギン酸高含有日本酒の醸造工程におけるD-アミノ酸の定量的解析と生成機構の解明

    斉藤瑠実(B), 岡田かおり, 郷上佳孝, 重藤憲史(若戎酒造), 老川典夫

    日本農芸化学会2012年度大会  2012.3 

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    Venue:京都  

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  • 食品中のD-アミノ酸:存在,生成機構, 機能

    老川典夫

    第37回生命の起源および進化学会学術講演会  2012.3 

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    Venue:大阪  

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  • X線構造解析による代謝酵素の反応機構解明

    老川典夫

    京都大学化学研究所共同利用・共同研究拠点化学関連分野の深化・連携を基軸とする先端・学際研究拠点平成23年成果報告書  2012 

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    Event date: 2012

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  • 好冷細菌 Flavobacterium frigidimaris KUC-1由来マレートデヒドロゲナーゼの部位特異的変異導入及び分子動力学計算による低温適応機構の解析

    郷上佳孝, 茨木 将(D), 藤井知実(京都大学), 畑 安雄(京都大学), 老川典夫

    第2回近畿地区ビタミン懇話会  2011.11 

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    Event date: 2011.11

    Venue:神戸  

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  • 生酛作りは日本酒中のD-アミノ酸含有量を高める

    郷上佳孝, 岡田かおり, 森山昌和(菊正宗), 溝口晴彦(菊正宗), 老川典夫

    第63回日本生物工学会  2011.9 

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    Event date: 2011.9

    Venue:東京  

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  • 生もと由来乳酸菌Leuconostoc mesenteroides NBRC 102480の新規ヒスチジンラセマーゼ:クローニングと特性解明

    郷上佳孝, 老川典夫

    第84回日本生化学大会  2011.9 

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    Event date: 2011.9

    Venue:京都  

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  • 超好熱アーキアThermococcus litoralis DSM 5473のアスパラギン酸アミノトランスフェラーゼの大腸菌を用いた発現系の構築と性質

    内田悠喜, 老川典夫

    第84回日本生化学大会  2011.9 

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    Event date: 2011.9

    Venue:京都  

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  • 生酛由来乳酸菌のグルタミン酸ラセマーゼ遺伝子導入 Corynebacterium glutamicumのD-グルタミン酸合成と分泌

    矢野正博, 老川典夫

    第84回日本生化学大会  2011.9 

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    Event date: 2011.9

    Venue:京都  

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  • 食品中のD-アミノ酸とその呈味性:D-アミノ酸には日本酒の味や総合評価を高める効果がある

    岡田かおり, 郷上佳孝, 老川典夫

    第7回D-アミノ酸研究会  2011.9 

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    Event date: 2011.9

    Venue:東京  

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  • 生酛由来乳酸菌のアミノ酸ラセマーゼホモログ遺伝子の網羅的発現と新規ヒスチジンラセマーゼの発見

    郷上佳孝, 岡田かおり, 老川典夫

    第7回D-アミノ酸研究会  2011.9 

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    Event date: 2011.9

    Venue:東京  

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  • D-Amino acid productin and amino acid racemase of Lactobacillus sakei NBRC 15893 isolated from kimoto, starter culture of sake

    Yoshitaka Gogami, Kaori Okada, Masahiro Yano(D), Tadao oikawa

    XIII International Congress of Bacteriology and Applied Microbiology/ XIII International Congress of Mycology  2011.9 

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    Event date: 2011.9

    Venue:Sapporo  

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  • 日本酒中のD-アミノ酸の定量と生成機構の解析

    岡田かおり, 郷上佳孝, 老川典夫

    第28回日本微量栄養素学会  2011.6 

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    Event date: 2011.6

    Venue:京都  

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  • 日本酒原料中のD-アミノ酸含量の地域差と局在性

    郷上佳孝, 岡田かおり, 老川典夫

    第28回日本微量栄養素学会  2011.6 

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    Event date: 2011.6

    Venue:京都  

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  • 生もと由来Lactobacillus sakei NBRC 15893のグルタミン酸ラセマーゼ:酵素反応速度論的解析

    矢野正博D, 松井大亮D, 郷上佳孝D, 老川典夫

    日本農芸化学会  2011.3 

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    Event date: 2011.3

    Venue:京都  

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  • 根粒菌由来フラビン還元酵素のX線結晶構造解析

    山内貴恵(京都大学), 藤井知実(京都大学), 吉田雅博, 老川典夫, 畑安雄(京都大学)

    日本農芸化学会  2011.3 

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    Event date: 2011.3

    Venue:京都  

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  • 超好熱アーキアのアスパラギン酸アミノトランスフェラーゼ:クローニングと酵素科学的性質

    内田悠喜D, 老川典夫

    日本農芸化学会  2011.3 

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    Event date: 2011.3

    Venue:京都  

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  • Corynebacterium glutamicum ATCC 13032の芳香族アルデヒドデヒドロゲナーゼの酵素科学的特性と機能の解明

    山田菜美子D, 老川典夫

    日本農芸化学会  2011.3 

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    Event date: 2011.3

    Venue:京都  

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  • アルギニンラセマーゼ組み換えCorynebacterium glutamicum ATCC13032のL-アミノ酸生産変異株を用いたDArg及びD-Lys生産

    松井大亮D, 老川典夫

    日本農芸化学会  2011.3 

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    Event date: 2011.3

    Venue:京都  

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  • Flavobacterium細菌における耐熱性アルデヒドデヒドロゲナーゼの分布とその特性解明

    下浦正朝B, 老川典夫

    日本農芸化学会  2011.3 

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    Event date: 2011.3

    Venue:京都  

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  • 日本酒原料米中のD-アミノ酸の定量的解析と日本酒中のD-アミノ酸含有量に及ぼす影響

    郷上佳孝D, 岡田かおり, 老川典夫

    日本農芸化学会  2011.3 

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    Event date: 2011.3

    Venue:京都  

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  • 日本酒醸造方法の日本酒中のD-アミノ酸含有量に及ぼす影響

    岡田かおり, 郷上佳孝D, 森山昌和(菊正宗), 溝口晴彦(菊正宗), 老川典夫

    日本農芸化学会  2011.3 

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    Event date: 2011.3

    Venue:京都  

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  • ビタミンB6酵素, 超好熱アーキアの分岐鎖アミノ酸アミノトランスフェラーゼ:酵素科学的特性と応用

    内田悠喜D, 林 秀行, 老川典夫

    1回近畿地区ビタミン懇話会  2011.1 

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    Event date: 2011.1

    Venue:兵庫  

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  • コリネ型細菌のNADP+要求性ベンズアルデヒドデヒドロゲナーゼ:酵素科学的性質の解明

    山田菜美子D, 老川典夫

    1回近畿地区ビタミン懇話会  2011.1 

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    Event date: 2011.1

    Venue:兵庫  

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  • 生命科学における鏡の中の世界:食品とD-アミノ酸

    老川典夫

    第27回関西地区ペプチドセミナー  2010.12 

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    Event date: 2010.12

    Venue:大阪  

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  • 超好熱アーキアThermococcus litoralis DSM 5473の低基質特異性分岐鎖アミノ酸アミノトランスフェラーゼ:ストップトフロー分光法による基質特異性の解析

    内田悠喜D, 林 秀行, 老川典夫

    第33回日本分子生物学会年会・第83回日本生化学会大会 合同大会  2010.12 

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    Event date: 2010.12

    Venue:兵庫  

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  • Corynebacterium glutamicum ATCC 13032のNADP+_芳香族アルデヒドデヒドロゲナーゼの酵素科学的性質の解明,

    山田菜美子D, 老川典夫

    第33回日本分子生物学会年会・第83回日本生化学会大会 合同大会  2010.12 

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    Event date: 2010.12

    Venue:兵庫  

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  • Lactobacillus sakei NBRC15893由来、murl遺伝子の大腸菌における発現とその遺伝子産物の機能解明

    矢野正博D, 横路菜緒B, 松井大亮D, 郷上佳孝D, 老川典夫

    第33回日本分子生物学会年会・第83回日本生化学会大会 合同大会  2010.12 

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    Event date: 2010.12

    Venue:兵庫  

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  • 超好熱アーキアのアミノトランスフェラーゼの非天然アミノ酸合成への応用

    内田悠喜D, 林 秀行, 老川典夫

    第5回産業用酵素シンポジウム  2010.11 

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    Event date: 2010.11

    Venue:滋賀  

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  • 日本酒中のD-アミノ酸の存在と生成機構

    老川典夫

    第62回日本生物工学会  2010.10 

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    Event date: 2010.10

    Venue:宮崎  

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  • Corynebacterium glutamicum ATCC 13032のベンズアルデヒドデヒドロゲナーゼ: クローニングと酵素科学的特性解明,

    山田菜美子D, 老川典夫

    第62回日本生物工学会  2010.10 

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    Event date: 2010.10

    Venue:宮崎  

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  • 生もと由来Lactobacillus sakeiNBRC15893のアスパラギン酸ラセマーゼ:クローニングと酵素科学的性質の解明

    郷上佳孝D, 松井大亮D, 老川典夫

    第62回日本生物工学会  2010.10 

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    Event date: 2010.10

    Venue:宮崎  

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  • 生もと由来Lactobacillus sakei NBRC15893のグルタミン酸ラセマーゼ:クローニングと特性解明

    矢野正博D, 横路菜緒B, 松井大亮D, 郷上佳孝D, 老川典夫

    第62回日本生物工学会  2010.10 

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    Event date: 2010.10

    Venue:宮崎  

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  • 生もと由来乳酸菌のD-アミノ酸生産とアミノ酸ラセマーゼ

    松井大亮D, 郷上佳孝D, 岡田かおり, 矢野正博D, 老川典夫

    第6回D-アミノ酸研究会学術講演会  2010.9 

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    Event date: 2010.9

    Venue:富山  

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  • 食品中のD-アミノ酸:定量的解析とその生産を担う生体触媒

    老川典夫

    関西大学技術交流セミナー2010  2010.6 

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    Event date: 2010.6

    Venue:東京  

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  • PLP-要求性アルギニンラセマーゼ遺伝子導入Corynebacterium glutamicumによるD-アルギニン及びD-リシンの分泌生産

    松井大亮D, 老川典夫

    日本ビタミン学会第62回大会  2010.6 

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    Event date: 2010.6

    Venue:岩手  

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  • 微生物がD-リシンを利用するのに必須な酵素,アルギニンラセマーゼの構造特性

    松井大亮D, 老川典夫

    第27回日本微量栄養素学会2010年度  2010.6 

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    Event date: 2010.6

    Venue:京都  

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  • Pseudomonas taetrolens NBRC3460のアルギニンラセマーゼ:構造と機能の解明

    松井大亮D, 老川典夫

    第57回日本生化学会近畿支部例会  2010.5 

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    Event date: 2010.5

    Venue:奈良  

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  • マレイル酢酸還元酵素の活性部構造探索

    畑 安雄, 藤井知実, 吉田雅博, 老川典夫

    日本農芸化学会2010年度大会  2010.3 

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    Event date: 2010.3

    Venue:東京  

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  • 日本酒中のD-アミノ酸の定量的解析

    岡田かおり, 郷上佳孝D, 松井大亮D, 老川典夫

    日本農芸化学会2010年度大会  2010.3 

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    Event date: 2010.3

    Venue:東京  

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  • 生もと由来Lactobacillus sakei NBRC15893:Dアミノ酸生産の発見とアラニンラセマーゼのクローニングと特性解明

    松島由貴D, 松井大亮D, 郷上佳孝D, 老川典夫

    日本農芸化学会2010年度大会  2010.3 

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    Event date: 2010.3

    Venue:東京  

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  • 日本酒原料米中のD-アミノ酸の定量的解析

    保井美穂B, 郷上佳孝D, 松井大亮D, 老川典夫

    日本農芸化学会2010年度大会  2010.3 

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    Event date: 2010.3

    Venue:東京  

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  • 低温菌Flavobacterium frigidimarisKUC-1由来L-スレオニン脱水素酵素の分子特性

    米田一成, 櫻庭春彦, 村岡郁夫, 老川典夫, 荒木朋洋, 河村俊介, 大島敏久

    日本農芸化学会2010年度大会  2010.3 

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    Event date: 2010.3

    Venue:東京  

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  • マレイル酢酸還元酵素GraCの結晶構造

    畑 安雄, 藤井知実, 吉田雅博, 老川典夫

    日本農芸化学会2009年度  2009.3 

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    Event date: 2009.3

    Venue:博多  

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  • 低温菌Flavobacterium frigidimarisKUC-1由来L-スレオニン脱水素酵素の構造解析

    米田一成, 櫻庭春彦, 大島敏久, 河村俊介, 荒木朋洋, 村岡郁夫, 老川典夫

    日本農芸化学会2009年度  2009.3 

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    Event date: 2009.3

    Venue:博多  

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  • 低温菌 Flavobacterium frigidimaris KUC-1由来L-スレオニン脱水素酵素の構造解析

    米田一成, 櫻庭春彦, 大島敏久, 河村俊介, 荒木朋洋, 村岡郁夫, 老川典夫

    日本農芸化学会2009年度  2009.3 

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    Event date: 2009.3

    Venue:博多  

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  • イネのセリンラセマーゼのE219A/D225A変異型酵素:マグネシウムイオン (Ⅱ) の活性及び構造に及ぼす影響

    郷上佳孝, 老川典夫

    日本農芸化学会2009年度  2009.3 

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    Event date: 2009.3

    Venue:博多  

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  • PLP依存性アルギニンラセマーゼの輸送及び機能の解明

    松井大亮, 老川典夫

    日本農芸化学会2009年度  2009.3 

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    Event date: 2009.3

    Venue:博多  

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  • マレイル酢酸還元酵素GraCの結晶構造

    畑 安雄, 藤井知実, 吉田雅博, 老川典夫

    日本農芸化学会2009年度  2009.3 

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    Event date: 2009.3

    Venue:博多  

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  • 日本酒中のD-アミノ酸の定量的解析

    老川典夫, 野田まり恵, 松島由貴

    日本農芸化学会2009年度  2009.3 

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    Event date: 2009.3

    Venue:博多  

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  • Pseudomonas taetrolens NBRC3460のアルギニンラセマーゼの機能解析

    松井大亮, 老川典夫

    第4回D-アミノ酸研究会学術講演会  2008.9 

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    Event date: 2008.9

    Venue:名古屋  

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  • 食品中のD-アミノ酸:現状と展望

    老川典夫

    第4回D-アミノ酸研究会学術講演会  2008.9 

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    Event date: 2008.9

    Venue:名古屋  

    特別講演

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  • イネセリンラセマーゼ:2つの酵素活性に及ぼすMg2+,Na+の影響とその速度論解析

    郷上佳孝, 老川典夫

    第4回D-アミノ酸研究会学術講演会  2008.9 

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    Event date: 2008.9

    Venue:名古屋  

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  • コーヒーチェリー付着微生物の単離・同定とそのコーヒー生豆中のGABA含量の影響

    南塚博司, 右山由美香, 紙谷雄志, 大西いづみ, 福永泰司, 老川 典夫

    第60回日本生物工学会大会講演  2008.8 

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    Event date: 2008.8

    Venue:仙台  

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  • イネのセリンラセマーゼ:Mg2+による構造変化と2酵素活性の制御

    郷上佳孝, 松島由貴, 池内俊彦, 老川典夫

    第25回微量栄養素研究会シンポジウム講演  2008.5 

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    Event date: 2008.5

    Venue:京都  

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  • イネのセリンラセマーゼ:金属イオンによる酵素活性と構造変化

    郷上佳孝, 伊藤克佳, 松島由貴, 老川典夫

    日本農芸化学会2008年度大会  2008.3 

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    Event date: 2008.3

    Venue:名古屋  

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  • Thermococcus litoralisのアラニンアミノトランスフェラーゼ:遺伝子破壊株の調製と特性

    毛笠秀昭, 松見理恵, 跡見晴幸, 今中忠行, 老川典夫

    日本農芸化学会2008年度大会  2008.3 

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    Event date: 2008.3

    Venue:名古屋  

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  • 植物中のD-アミノ酸とアミノ酸ラセマーゼの生化学

    老川典夫

    第30回日本分子生物学会年会第80回日本生化学会大会合同大会  2007.12 

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    Event date: 2007.12

    Venue:横浜  

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  • イネのセリンデヒドラターゼ/ラセマーゼ:Mg2+による酵素反応の制御機構

    郷上佳孝, 伊藤克佳, 松島由貴, 老川典夫

    第59回日本生物工学会大会  2007.9 

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    Event date: 2007.9

    Venue:広島  

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  • カフェイン分解微生物のスクリーニングと微生物を用いるコーヒー抽出物からのクロロゲン酸の残存とカフェインの選択的分解方法の検討

    老川典夫, 白江純子, 磯田真代, 岩井和也, 福永泰司

    第59回日本生物工学会大会  2007.9 

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    Event date: 2007.9

    Venue:広島  

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  • イネセリンデヒドラターゼ/ラセマーゼ:酵素科学的特性とマグネシウムイオンによるバイファンクショナル酵素反応の調節機構

    郷上佳孝, 伊藤克佳, 松島由貴, 老川典夫

    第3回D-アミノ酸研究会学術講演会  2007.9 

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    Event date: 2007.9

    Venue:徳島  

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  • イネのセリンデヒドラターゼ/ラセマーゼ:微量元素Mgによる酵素反応の制御機構の発見

    郷上佳孝, 伊藤克佳, 老川典夫

    第24回微量栄養素研究会シンポジウム講演  2007.6 

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    Event date: 2007.6

    Venue:京都  

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  • イネセリンラセマーゼ/デヒドラターゼのクローニングと酵素科学的性質の解明

    伊藤克佳, 郷上佳孝, 岸本勝也, 老川典夫

    日本農芸化学会2007年度(平成19年度)大会  2007.3 

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    Event date: 2007.3

    Venue:東京  

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  • 新規γーレゾルシン酸(2, 6-ジヒドロキシ安息香酸)代謝経路の発見とその特性解明

    吉田雅博, 老川典夫, 阿部勝正, 三原久明, 江崎信芳

    日本農芸化学会2007年度(平成19年度)大会  2007.3 

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    Event date: 2007.3

    Venue:東京  

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  • イネOryza sativaのセリンラセマーゼホモログ:クローニングと酵素科学的性質の解明

    伊藤克佳, 郷上佳孝, 老川典夫

    第2回D-アミノ酸研究会学術講演会  2006.9 

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    Event date: 2006.9

    Venue:京都  

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  • アルファルファ芽生えのアラニンラセマーゼ:発見と酵素科学的性質の解明

    郷上佳孝, 老川典夫, 小野和利, 左右田健次

    第2回D-アミノ酸研究会学術講演会  2006.9 

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    Event date: 2006.9

    Venue:京都  

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  • Pseudomonas taetrolens NBRC 3460の2種のアミノ酸ラセマーゼのin vivoにおける機能

    松井大亮, 老川典夫

    第2回D-アミノ酸研究会学術講演会  2006.9 

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    Event date: 2006.9

    Venue:京都  

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  • 野菜及び果物中のD-アミノ酸の定量的解析と植物におけるD-アミノ酸の生合成機構

    郷上佳孝, 伊藤克佳, 老川典夫

    第23回微量栄養素研究会シンポジウム講演  2006.6 

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    Event date: 2006.6

    Venue:京都  

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  • 植物におけるD-アミノ酸の存在とアミノ酸ラセマーゼ

    紙谷雄志, 郷上佳孝, 吉田雅博, 左右田健次, 老川典夫

    日本農芸化学会2006年度 (平成18年度) 大会  2006.3 

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    Event date: 2006.3

    Venue:京都  

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  • 耐熱性低温活性型アルコールデヒドロゲナーゼの低温適応戦略:Asn265Val変異酵素の調製と機能解析

    村岡郁夫, 土戸哲明, 老川典夫

    日本農芸化学会2006年度 (平成18年度) 大会  2006.3 

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    Event date: 2006.3

    Venue:京都  

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  • 微生物の新規γ-レゾルシン酸分解経路の発見と関連酵素遺伝子群の同定

    吉田雅博, 小幡 斉, 老川典夫

    日本農芸化学会2006年度 (平成18年度) 大会  2006.3 

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    Event date: 2006.3

    Venue:京都  

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  • Pseudomonas taetrolens NBRC 3460のアラニンラセマーゼ:クローニングとin vitroにおける機能の解明

    老川典夫, 大角真太郎, 松井大亮, 紙谷雄志

    日本ビタミン学会第58回大会  2006.3 

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    Event date: 2006.3

    Venue:徳島  

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  • イネOryza sativaのセリンラセマーゼホモログ:クローニングと酵素科学的性質の解明

    伊藤克佳, 紙谷雄志, 郷上佳孝, 吉田雅博, 老川典夫

    日本ビタミン学会第58回大会  2006.3 

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    Event date: 2006.3

    Venue:徳島  

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  • Amino acids racemase with low substrate specificity of Peudomonas putida IFO 12996: Its localization and export

    Kamitani, D.Matsui, T. Oikawa

    2005.10 

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    Event date: 2005.10

    Venue:Kobe  

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  • The enzymological studies on recombinant L-lysine:2-oxoglutarate 6-aminotransferase

    Y. Kashiwabara, Y. Uchida, K. Soda, T.Fujii, T. Narita, H. Agematsu, N. Agata, K. Isshiki, T. Oikawa

    2005.10 

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    Event date: 2005.10

    Venue:Kobe  

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  • Genetical analysisi of γ-resorcylate degradation pathway in Rhizobium sp. MTP-10005

    M. Yoshida, T. Oikawa

    2005.10 

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    Event date: 2005.10

    Venue:Kobe  

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  • Structural analysisi of thermostable and cold-active alcohol dehydrogenase from Flavobacterium frigidimaris KUC-1

    I. Muraoka, A. Kashima, S. Sugio, T. Oikawa

    2005.10 

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    Event date: 2005.10

    Venue:Kobe  

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  • Pseudomonas putida IFO12996の低基質特異性アミノ酸ラセマーゼ:局在性と輸送機構の解明

    松井大亮, 紙谷雄志, 老川典夫

    第1回D-アミノ酸学術講演会研究会講演  2005.9 

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    Event date: 2005.9

    Venue:東京  

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  • Pseudomonas taetrolens IFO 3460のアルギニンラセマーゼ:酵素科学的特性と機能解析

    松井大亮, 荒川憲昭, 左右田健次, 老川典夫

    日本ビタミン学会第57回大会  2005.5 

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    Event date: 2005.5

    Venue:三重  

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  • 大腸菌のコールドショック発現系を用いる亜鉛含有型金属酵素における高発現系構築法

    郷上佳孝, 村岡郁夫, 老川典夫

    第22回微量栄養素研究会シンポジウム講演  2005.5 

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    Event date: 2005.5

    Venue:京都  

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  • γ―レゾルシン酸デカルボキシラーゼ,高発現系の構築と一次構造の解明

    吉田雅博, 有井輝夫, 老川典夫

    2005年度(平成17年度)日本農芸化学会大会  2005 

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    Event date: 2005

    Venue:北海道  

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  • 特異な一次構造を有する好冷性L―スレオニンデヒドロゲナーゼ,高発現系の構築と精製

    村岡郁夫, 左右田健次, 老川典夫

    2005年度(平成17年度)日本農芸化学会大会  2005 

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    Event date: 2005

    Venue:北海道  

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  • 微生物によるレゾルシンからのγ―レゾルシン酸の合成

    柴本寛子, 有井輝夫, 吉田雅博, 老川典夫

    化学工学会第69年会講演  2004 

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    Event date: 2004

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  • Zn2+‐Guanidinobutyrase of Arthrobacter sp.KUJ8602:construction of high expression system in Escherichia coli

    Y. Gougami, I. Muraoka, K. Soda, T. Oikawa

    Biochemistry  2004 

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    Event date: 2004

    Venue:Yokohama  

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  • Thermophilic, Reversible γ ‐Resorcylate Decarboxylase from Rhizobium sp. MTP‐10005. Purification, Molecular Characterization, and Primary Structure

    M. Yoshida, N. Fukuhara, T. Oikawa

    Biochemistry  2004 

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    Event date: 2004

    Venue:Yokohama  

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  • D-乳酸生産に用いる乳酸菌宿主の開発と高生産系の構築

    左右田健次, 老川典夫

    平成16年度地域新生コンソーシアム研究開発事業成果報告書  2004 

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    Event date: 2004

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  • Pseudomonas putida IFO12996の低基質特異性アミノ酸ラセマーゼ:bsrc 遺伝子破壊株の調製と機能の解明

    紙谷雄志, Andrea Tauch, 左右田健次, 老川典夫

    日本ビタミン学会第56回大会  2004 

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    Event date: 2004

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  • 耐熱性及び有機溶媒耐性菌株の育種

    老川典夫, 左右田健次

    平成15年度地域新生コンソーシアム研究開発事業成果報告書  2003 

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    Event date: 2003

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  • Ultrathermostable Alanine Aminotrausterases of Hyperthermophilic Archaeron, Pyrococcus furiosus

    Kenji Soda, Satoru Sakaguchi, Tadao Oikawa

    1999.10 

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    Event date: 1999.10

    We found the occurrence of alanine aminotransferase (AlaAT, EC 2.6.1.2) isozymesⅠand Ⅱin a hyperthermophilic archaeron, Pyrococcus furiosus DSM 3638 grown on pyruvate as a sole carbon source, and purified to characterize it. The enzyme was homotetrameric, and the molecular mass was about 168,000 Da. The enzyme showed the maximum activity at pH 9, and Km for L-alanine and 2-oxoglutarate were 2.3 and 2.4 mM, respectively. The enzyme catalyzes the pyridoxal phosphate-dependent transamination of alanine, 2-amino-butyrate, norvaline, and norleucine with α-ketoglutarate, whereas phenylalanine, tyrosine, and tryotophane are insert as an amino donor. The inhibition experiment showed that both pyridoxal phosphate and sulfhydryl group were directly involved in catalysis. The arrhenius plots of from P. furisus: activation energy, 57 kJ/mol. The consensus sequence (YAVR of alanine aminotransferase Ⅱwas found as 1XKASKRAMSIXYAIRNVVLP-in the N-terminal region.

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  • Bacterial Guanidino butyrase acting on D-Arginine

    Noriaki Arakawa, Tadao Oikawa, kenji Soda

    1999.10 

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    Event date: 1999.10

    We found the occurrence of D-arginase in cells of Acinetobacter haemolyticus KUJ 8661 grown in the L-arginine medium, purified and characterized the enzyme. The molecular weight and subunit of the enzyme was estimated to be about 232,000 and 40,000, respectively, suggesting that the enzyme is a homohexamer. The enzyme does not act on only D-arginine, but 4-guanidinobutrate, 3-guanidinopropionate and L-arginine. The Km values for 4-guanidinobutyrate and D-arginine were determined 3.9 and 22 mM, respectively. Accoringly, it is more pertinent to name the enzyme guanidinobutyrase (guanidinobutyrate amidinohydrorase, Ec.3.5.3.7). The optimum pH was 9.0. Incubation of the enzyme in part. The activity of the inactivated enzyme was substantially restored by incubation with Co2+.

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  • Alanine Aminotransferase Ⅱ from Hyperthermophilic Archaeron, Pyrococcus furiosus : Primary structure

    Satoru Sakaguchi, Tadao Oikawa, Shun-ichi Kuroda, Katsuyuki Tanizawa, Kenji Soda

    1999 

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    Event date: 1999

    Alanine aminotransferase isozyme Ⅱ (AlaAT Ⅱ) purified from Pyrococcus furiosus DSM 3638 was digested with lysylendopeptidase, and the peptides were purified by reversed phase high performance liquid chromatography. The amino acid sequences of the peptides were analyzed by protein sequencer. N-Terminal and internal amino acid sequences of the enzyme were highly homologous to those of thermostable aspartate aminotransferase of P. horikoshii OT3. Based on these peptides sequences, the primers for polymerase chain reaction (PCR) were designed and AlaAT Ⅱ gene in chromosomal DNA of P. furiosus DSM 3638 was amplified by PCR. The DNA purified from agarose gel was ligated with pT7 Blue T-vector and tansformed into NovaBlue. The plasmid prepared by alkali miniprep method was purified by polyethylene glycol, and the insert (1.0 kb) was analyzed by DNA sequencer. The partial primary structure of AlaAT Ⅱ determined by DNA sequence agreed with the internal amino acid sequences, and the total DNA of AlaAT Ⅱ was obtained by genome-walking PCR.

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  • Ultrathermostable Alanine Aminotransferase Ⅱ of Hyperthermophilic Archaeron, Pyrococcus furiosus : Purification and characterization

    Tadao Oikawa, Satoru Sakaguchi, Kenji Soda

    1999 

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    Event date: 1999

    Alanine aminotransferase (EC 2.6.1.2) isozyme Ⅱ (AlaAT Ⅱ) was purified to homogeneity from hyperthermophilic archaeron Pyrococcus furiosus DSM 3638 grown on pyruvate as a sole carbon source. The enzyme was homotetrameric, and the molecular mass was about 168,000 Da. The enzyme showed the maximum activity at pH 9, and K m for L-alanine and 2-oxoglutarate were 2.3 and 2.4 mM, respectively. The enzyme catalyzes the pyridoxal5'-phosphate-dependent transamination of L-alanine, 2-amino-L-butyric acid, L-norvaline, and L-norleucine with α-ketoglutarate, whereas L-phenylalanine, L-tyrosine, and L-tryotophane are inert as an amino donor. The inhibition experiment showed that both pyridoxal 5'-phosphate and sulfhydryl groups were directly involved in catalysis. The arrhenius plots of the enzyme showed a single phase not similar to those of other enzymes from P. furiosus: activation energy, 57 kJ/mol. The consensus sequence (YAVR) of alanine aminotransferase was found as 1XKASKRAMSIXYAIRNVVLP-in N-terminal region.

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  • 納豆菌由来グルタミン酸ラセマーゼのD-グルタミン酸生産への応用に関する研究

    老川典夫

    タカノ農芸化学研究助成財団,平成10年度助成研究報告書  1998 

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    Event date: 1998

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  • 乳酸菌Weissella viridescens JCM1174の新規2ドメイン型アミノ酸ラセマーゼの発見と基礎的性質

    大島 尚哉, 安達 基泰, 加藤 志郎, 山中 一也, 老川 典夫

    2021.9 

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  • D-アミノ酸を新たな生物系素材とする新規機能性食品開発拠点の形成

    老川 典夫, 松村 吉信, 細見 亮太, 加藤 志郎

    2021.3 

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  • 大腸菌由来D-システインデスルフィドラーゼ:高発現系および変異型酵素の構築とD-システイン定量への応用

    長光 佑介, 山中 一也, 老川 典夫

    2021.2 

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  • Crystal structure analysis of GraE protein from root-nodule-forming bacterium,

    老川 典夫

    2021 

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  • 次世代ベンチトップ型シーケンサーによるゲノム・エピゲノム解析に基づく統合的健康生命研究

    老川 典夫, 吉田宗弘, 池内俊彦, 下家浩二, 土戸哲明, 松村吉信

    2014 

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  • 好冷菌ADHの低温環境適応分子機構

    畑 安雄, 藤井知実, 老川典夫, 村岡郁夫, 左右田健次

    日本農芸化学会2007年度(平成19年度)大会  2007.3 

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    Venue:東京  

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  • 好冷菌ADHの低温環境適応分子機構

    村岡郁夫, 老川典夫, 鹿島亜希子, 杉尾成俊

    日本農芸化学会2007年度(平成19年度)大会  2007.3 

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    Venue:東京  

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Industrial property rights

  • アルコール脱水素酵素及びその製造方法

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    Application no:H10-250931  Date applied:1998.9

    Announcement no:2000-078969  Date announced:2000.3

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Awards

  • 学の実化賞

    2016.5   関西大学科学技術振興会  

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Research Projects

  • Development for quantum-theoretical approach by observing hydrogen atom positions in drug target proteins

    Grant number:20H03377  2020.4 - 2023.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

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    Grant amount:\17680000 ( Direct Cost: \13600000 、 Indirect Cost:\4080000 )

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  • Crystal structure analysis for intermediate in enzymatic reaction by using neutron beam

    Grant number:16KT0063  2016.7 - 2020.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    Adachi Motoyasu

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    Grant amount:\18460000 ( Direct Cost: \14200000 、 Indirect Cost:\4260000 )

    In this study, we aimed to realize neutron structural analysis including orientation of hydrogen atoms and water molecules in reaction intermediates for controlling transition states through three-dimensional structural analysis of enzyme. For the histidine racemase, which is one of the amino acid racemases, we succeeded in X-ray crystallography for the first time in the world. Furthermore, by examining crystallization conditions and continuing optimization, we succeeded in X-ray crystal structure analysis to a high resolution of 1.0 angstrom, and in producing large crystals with a volume of above 1 mm cubed. For CK2, we succeeded in neutron structure analysis for the first time in the world, and showed a hydrogen bond network that traverses the molecule and its functionality.

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  • Elucidation of protection strategy for toxicity of D-amino acid in plant

    Grant number:24580151  2012.4 - 2015.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    OIKAWA Tadao

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    Grant amount:\5330000 ( Direct Cost: \4100000 、 Indirect Cost:\1230000 )

    We succeeded to construct the serine racemase gene-disruption mutant and overexpressed mutant of Arabidopsis thaliana. By using these mutants and wild-type strain of Arabidopsis thaliana, we evaluated the effects of various D- and L- amino acids on germination and growth of these strains. In case of D-serine, the serine racemase gene overexpressed mutant showed higher tolerant than other strains. This probably attributed to the D-serine degradation activity of serine rasemase. In case of L-serine, both serine racemase gene-disruption mutant and overexpressed mutant showed higher tolerant than wild-type strain.

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  • Strategy for cold adaptation of enzyme : studies on structure and molecular evolution of cold active alcohol dehydrogenase

    Grant number:16550150  2004 - 2005

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    OIKAWA Tadao

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    Grant amount:\2700000 ( Direct Cost: \2700000 )

    An NAD^+-dependent alcohol dehydrogenase from the Antarctic psychrotolerant, Flavobacterium frigidimaris KUC-1 was purified to be homogeneity with an overall yield of about 20% and characterized enzymologically. The native enzyme had an apparent molecular mass of 160 kDa and consisted of four identical 40 kDa subunits. The pI of the enzyme was determined to be 6.7 and its optimum pH for oxidation reaction was 7.0. The enzyme contained 2 zinc atoms/subunit. The enzyme exclusively required NAD^+ as a coenzyme and showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. F.frigidimaris KUC-1 alcohol dehydrogenase showed a remarkable thermal stability similar to its thermophilic counterparts and in contrast to other microbial alcohol dehydrogenases. The enzyme was active in the temperature range of 0 to over 85℃ and the most active at 70℃. The half-life time and k_<cat> at 60℃ were calculated to be 50 min and 27,370 (min^<-1>), respectively. The enzyme also showed high catalytic efficiency at low temperatures (0-20℃) (kcat/K_m at 20℃ ; 25,500 mM^<-1> min^<-1>) similar to its psychrophilic counterpart. The alcohol dehydrogenase gene was composed of 1,035 bp and coded 344 amino acid residues with an estimated molecular mass of 36,823 Da. The sequence identities were found with the amino acid sequences of Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%), and Geobacillus stearothermophilus LLD-R (56%) alcohol dehydrogenases. To our knowledge, this is the first example of a cold-active and thermostable alcohol dehydrogenase.

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  • 好冷性酵素の構造と低温での特性に関する研究

    Grant number:12878112  2000 - 2002

    日本学術振興会  科学研究費助成事業  萌芽研究

    左右田 健次, 白岩 正, 小幡 斉, 老川 典夫, 河原 秀久

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    Grant amount:\2300000 ( Direct Cost: \2300000 )

    本研究では、南極海水から単離した低温菌Cytophaga sp.KUC-1の酵素を対象として、酵素科学的,物理化学的性質を解明するとともに、好冷性酵素の構造と低温での特性を解明することを目的としている。本菌由来のアルデヒドデヒドロゲナーゼは、低温菌由来の酵素であるにもかかわらず、60℃付近に最適反応温度を示し、中温菌酵素と比較してもかなり高い耐熱性を示した。アレニウスプロットの結果、32℃に遷移温度を持つ二相性が認められ、低温域での活性化エネルギーは中温菌からの同酵素の示す値より低い。すなわち本酵素は耐熱性を示すと共に機能的にも低温条件に適応していると考えられる。円二色性の測定結果も32℃付近での大きな変化を示し、熱による構造面での変化が触媒作用の促進と良く相関していた。本酵素の構成アミノ酸残基中のIle残基含量が高く、本酵素がより強固な疎水性コアを形成しており、高い耐熱性発現の一因となっていることが示唆される。本菌由来のアスパルターゼも耐熱性と好冷性の双方を示し、大腸菌やBacillus属細菌の同酵素のアミノ酸配列と比較した結果、分岐鎖アミノ酸残基数に対するIleの数の比率が好熱菌Bacillus属酵素の値に近似することを示した。本酵素のC-末端のαヘリックス形成能が高いことや、コア形成能が高いことが本酵素の耐熱性発現に寄与していると考えられる。またアルコール代謝において最も重要な役割を果たしているアルコールデヒドロゲナーゼはK_m値が20℃で最大値を示すのに、最適反応温度が70℃にあり、50℃での活性半減期が200分以上の高い熱安定性を示した。Znを必須金属として含む事実に基づいて、本酵素の触媒機構を解明し、本酵素と上記のAldDHの両反応の共役により種々の基質アルコールを特異的に微量定量する方法を確立した。

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  • Structural and functional analysis of bacterial D-amino acid metabolic enzymes to develop their inhibitors

    Grant number:08456052  1996 - 1997

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    SODA Kenji, ESAKI Nobuyoshi, OIKAWA Tadao

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    Grant amount:\8000000 ( Direct Cost: \8000000 )

    D-Amino acid aminotransferase (D-AAT), alanine racemase (AlaR), amino acid racemase of broad substrate specificities (AAR), and glutamate racemase (GluR) participate in the metabolism of D-amino acids, some of which are indispensable for bacteria as components of the peptidoglycan layr of cell walls. Physiological roles of these D-amino acids in mammals are not known, thus these enzymes have been regarded as a target for the development of novel antibacterial agents serving, for example, as suicide substrates. To develop the mechanism-based inhibitors, we studied the structure-function relationship of these enzymes. We studied the role of Arg98 of D-AAT,the binding site for the alpha-carboxyl group of substrates. This is presumably crucial for the unique stereospecificity of the enzyme. Replacement of Arg98 by replacement by methionine and lysine, resulted in decreases in the kmax values and increases in the Kd values for both amino donors and amino acceptors. The introduction of another mutation, replacement of Tyr88 being located near Arg98 by arginine, in addition to the above Arg98 mutation, resulted in increases in the kmax values but little change in the Kd values. We cloned the glutamate racemase gene (murl) of Bacillus pumilus cells into E.coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B.pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG,which is probably the reason for the low expression efficiency of the gene. We also studied the half and overall transaminations catalyzed by AAR,and found that the enzyme catalizes the non-stereospecific transaminations.

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  • バクテリアセルロース生産菌のセルラーゼの構造と機能解析

    Grant number:08760105  1996

    日本学術振興会  科学研究費助成事業  奨励研究(A)

    老川 典夫

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    Grant amount:\900000 ( Direct Cost: \900000 )

    本研究ではAcetobacterxylinum KU-1が菌体外に生産するカルボキシメチルセルラーゼ(CMCase)を収率14.2%で電気泳動的に均一状態に精製した。本酵素は、60℃まで安定であり、既報の好アルカリ性菌Bacillus sp.KSM-635のCMCaseより安定であった。また本酵素は酸性条件下で非常に安定であり50mM Gly-HCl buffer(pH2)で30℃、60分間インキュベート後でさえ最大活性の75%の活性が検出された。本酵素は1mMコバルトイオンなどによって活性化され、0.1mM水銀イオンによって完全に失活した。本酵素の分子量は、SDS-PAGEで39,000、ゲル濾過で41,000であった。したがって本酵素はモノマーである。この値は、他の微生物由来のCMCaseと類似している:Aspergillus aculeatus(25,000);Aspergillus niger(31,000);Trichodermaressei(48,000)。本酵素のCMCに対する見かけのKm値は3.00%であり、既報のAspergillus niger(0.0086%)やTrichodermaviride(0.054%)と比較して13-60倍大きい。N末端アミノ酸配列相同性検索の結果、本酵素のN末端アミノ酸配列は、既報のAcetobacter pasturianusのβ-glucanase(Val-27〜Lys-37)およびPseudomonas aeruginosaの熱ショックタンパク質(Ala-60〜Pro-67,Ile-98〜Asn-107)と高い相同性を示し、その配列相同性はそれぞれ72,87,60%であった。
    これまでに種々の微生物由来のさまざまなタイプのセルラーゼの研究が行われてきた。私はAcetobacter xylinum KU-1が培養液中にCMCaseを生産することを見いだし、Acetobacter属の細菌から初めて均一状態に精製することに成功した。最近Bacillus subtilis由来のCMCaseをAcetobacterxylinumの培地に添加するとBC生産が促進されるという現象が報告されている。したがって、Acetobacterxylinum由来のCMCaseは本菌のBC生産への関与が強く示唆される。

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  • ANALYSIS OF OXIDATIVE FERMENTATION, BIOCHEMICAL AND GENE-TECHNOLOGICAL RECONSTITUTION OF CN-INSENSITIVE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA

    Grant number:03660118  1991 - 1992

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for General Scientific Research (C)

    AMEYAMA Minoru, MATSUSHITA Kazunobu, OIKAWA Tadao

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    Grant amount:\2400000 ( Direct Cost: \2400000 )

    1) Sugar oxidizing dehydrogenases have been investigated widely onstrains of acetic acid bacteria, genus Pseudomanas, and methylotrophic bacteria. These enzymes have been purified and characterized to having as the prosthetic groups PQQ or covalently bound flavin.
    2) The ethanol oxidase respiratoruy chain of Gluconobacter suboxydans was characterized by using G.suboxydans subsp. alpha , a variant species of G.suboxydans incapable of oxidizing ethanol. The organism is known to be defective in the second subunit of allochol dehydrogenase (ADH) so that the membranes of the strain exhibit neither ADH nor ethanol oxidase activity. The enzyme activities have been shown to be restored by reconstituting the second subunit to the membranes. The second subunit of ADH has been shown to be a cytochrome c-553(CO).
    3) Membrane-bound, pyrroloquinoline quinone-dependent, alcohol dehydrogenase functions as the primary dehydrogenase in the respiratory chain of acetic acid bacteria. In this study, an ability of the enzyme to derectly react with ubiquinone was investigated in alcohol dehuydrogenases purified from both A.aceti and G.suboxydans. It has been shown that alcohol dehydrogenase of acetic acid bacteria donates electrons directly to ubiquinone in the cytoplasmic membranes and thus the ethanol oxidase respiratory chain of acetic acid bacteria is constituted of only three membranous respiratory component, alcohol dehydrogenase, ubiquinone, and terminal ubiquinol oxidase.
    4) A plasmid, pGEACl, carrying the cytochrome c-553(CO) gene was introduced in G.suboxydans subsp. alpha and could increase dehydrogenase activities for D-glucose, D-sorbitol and glycerol in the membranes. Oxidase activities for glucose, D-sorbitol and glycerol were also increasedin both the membrane and the cells of G.suboxydans subsp.alpha haboring pGEACl. Furthermore, pGEACl could restore azide insensitivity in the respiratory chain of G.suboxydans subsp.alpha which lacks insensitivity against azide or cyanide. Thazide insensitivity was observed not only in the membranes but also in the intact cells of the organism(pGEACl). Thus, cytochrome c-553 (CO) seems to be indispensable in the pathway of the azide-insensitive respriratory chain bypass of G.suboxydans. In addition, the increase of oxidative ability in the practical oxidative fermentation was observed in the intact cells of G.suboxydans subsp.alpha (PGEACl) which increased the azide insensitivity in the respiratoy chain.

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  • ドイツアーヘン工科大学から交流研究生の受け入れ

    2005.3 - 2005.9

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