start-ver=1.4 cd-journal=joma no-vol=95 cd-vols= no-issue=1 article-no= start-page=1 end-page=5 dt-received= dt-revised= dt-accepted= dt-pub-year=2006 dt-pub=200602 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Analysis of Decolorization Mechanism by Azo Dyes Decolizing Microorganism kn-title=染料脱色微生物による染料脱色機構の解析 en-subtitle= kn-subtitle= en-abstract= kn-abstract=The mehcanism of azo dyes decolorization by Candida sp. MK-1, Aeromonas sp. B-5 and Actinobacillus sp. B-11 were analyzed. The maximal decolorization activity was observed at pH 7.5 and 30℃ on Candida sp. MK-1, at alkaline and at 35℃ on Aeromonas sp. B-5 and Actinobacillus sp. B-11. The HPLC analysis of the supernatant of the Acid Red 27 detected in the blank. The retention time of this peak matched that of a reference standard compound of 4-amino-1-naphthalenesulfonate, produced by reductive cleavage of Acid Red 27. The decolorization of azo dyes with cell free extract of Candida sp. MK-a was promoted by the addition of several coenzymes or lawsone. The remarkable promotion of decolorization was observed by the addition of glutamate dehydrogenase with α-ketoglutarate and NH4+. Therefore, it was suggested that Candida sp. MK-1 azoreductase catalyzed decolorization of azo dye by NADPH dependent reductive cleavage. en-copyright= kn-copyright= en-aut-name=BabaNaoko en-aut-sei=Baba en-aut-mei=Naoko kn-aut-name=馬場直子 kn-aut-sei=馬場 kn-aut-mei=直子 aut-affil-num=1 ORCID= en-aut-name=SakaguchiHiromichi en-aut-sei=Sakaguchi en-aut-mei=Hiromichi kn-aut-name=坂口博脩 kn-aut-sei=坂口 kn-aut-mei=博脩 aut-affil-num=2 ORCID= en-aut-name=TaguchiTakaaki en-aut-sei=Taguchi en-aut-mei=Takaaki kn-aut-name=田口隆章 kn-aut-sei=田口 kn-aut-mei=隆章 aut-affil-num=3 ORCID= en-aut-name=HayaseNobuki en-aut-sei=Hayase en-aut-mei=Nobuki kn-aut-name=早瀬伸樹 kn-aut-sei=早瀬 kn-aut-mei=伸樹 aut-affil-num=4 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=5 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=有限会社タグチ affil-num=4 en-affil= kn-affil=新居浜工業高専 affil-num=5 en-affil= kn-affil=岡山大学 affil-num=6 en-affil= kn-affil=岡山大学 en-keyword=azo dyes kn-keyword=azo dyes en-keyword=decolorizing microorganism kn-keyword=decolorizing microorganism en-keyword=azoreductase kn-keyword=azoreductase END start-ver=1.4 cd-journal=joma no-vol=96 cd-vols= no-issue=1 article-no= start-page=1 end-page=5 dt-received= dt-revised= dt-accepted= dt-pub-year=2007 dt-pub=200702 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=キイロショウジョウバエ由来のチオレドキシン還元酵素のC未端テトラペプチド配列は、ヒト肺由来のチオレドキシン還元酵素では酸化還元活性を示さない kn-title=The C-Terminal Tetrapeptide Sequence of Drosophila Thioredoxin Reductase Does not Function as a Redox-active Motif in the Human Lung Counterpart en-subtitle= kn-subtitle= en-abstract=ほ乳類チオレドキシン還元酵素はＣ末端配列-Gly-Cys-SeCys-Gly（end）の後ろから２番目にセレノシステイン（SeCys）残基を持つ．SeCys をシステインに変換すると酵素の活性は大きく低下するので，SeCys 残基が触媒活性に必須であることが分かる．これに対してキイロショウジョウバエのチオレドキシン還元酵素（Dm-TrxR）のＣ末端配列にはセレンが含まれず，システイン残基の対が２つのセリンに挟まれた配列-Ser-Cys-Cys-Ser (end）を持つ．それでも Dm-TrxR はほ乳類のセレン含有酵素と同程度の触媒能を示す．われわれはヒト肺チオレドキシン還元酵素に Dm-TrxR のＣ末端テトラペプチド配列を導入してその効果を調べた．しかし，酵素活性はまったく上昇せず，Dm-TrxR のＣ末端のテトラペプチド配列-Ser-Cys-Cys-Ser だけでは Cys 残基のチオール基を活性化する効果はなかった．そこで，分子軌道計算 MOPAC を用いて酸化還元機能を担うためのＣ末端配列モチーフを探索した．その結果，テトラペプチドにさらに２つ先のプロリンまでを含めた Pro-X-Ser-Cys-Cys-Ser（end）により初めて酸化還元モチーフとして機能する可能性が示唆された．Pro を含むこの配列モチーフはミツバチや蚊などほかの昆虫の TrxR でも保存されていた kn-abstract=The isozymes of mammalian thioredoxin reductase (TrxR) contain the penultimate selenocysteineresidue (SeCys) in the redox-active C-terminal tetrapeptide, -Gly-Cys-SeCys-Gly (end). Amutant form of the mammalian enzyme TrxR-X498C in which SeCys is replaced with Cys showsa dramatically decreased catalytic activity, suggesting that SeCys residue plays an integral role inthe catalysis. In contrast, TrxR of the fruit fly, Drosophila melanogaster, has no selenium in the corresponding C-terminal redox sequence, which instead of SeCys has flanking serine residues in the terminal sequence, -Ser-Cys-Cys-Ser (end). Because the catalytic activity of Dm-TrxR is comparable to that of the mammalian selenoenzyme, we introduced the serine residues at the corresponding positions of the recombinant TrxR-X498C and mimicked the redox center of the fruit fly TrxR. However, the catalysis remained as low as the Cys mutant of the selenoenzyme, suggesting that the additional structural features are still required for the tetrapeptide to function as a redox center. MOPAC calculation suggested that the complete motif might involve the hexapeptide sequence, which includes a proline residue, -Pro-X-Ser-Cys-Cys-Ser (end). The proline-containing motif is conserved among other insect TrxRs such as those of honeybee and fruit fly. en-copyright= kn-copyright= en-aut-name=KuwaharaMitsuhiko en-aut-sei=Kuwahara en-aut-mei=Mitsuhiko kn-aut-name=桑原光彦 kn-aut-sei=桑原 kn-aut-mei=光彦 aut-affil-num=1 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=2 ORCID= en-aut-name=KawamuraKentaro en-aut-sei=Kawamura en-aut-mei=Kentaro kn-aut-name=河村健太郎 kn-aut-sei=河村 kn-aut-mei=健太郎 aut-affil-num=3 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=4 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 en-keyword=?Thioredoxin reductase kn-keyword=?Thioredoxin reductase en-keyword=Drosophila melanogaster kn-keyword=Drosophila melanogaster en-keyword=MOPAC kn-keyword=MOPAC END start-ver=1.4 cd-journal=joma no-vol=93 cd-vols= no-issue=1 article-no= start-page=13 end-page=18 dt-received= dt-revised= dt-accepted= dt-pub-year=2004 dt-pub=200402 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Saccharomyces cerevisiae 由来 D-アミノ酸アセチルトランスフェラーゼの精製と性質 en-subtitle= kn-subtitle= en-abstract= kn-abstract=Some properties of D-amino scid acetyltransferase purified from Saccharomyces cerevisiae were investigated. The enzyme was purified to homogeneity by ammonium sulfate fractionation, column chromatographies on DEAE-Toyopearl 650M, Sephacryl S-200, QAE-Toyopearl 550C and affinity chromatography with D-glutamate as a ligand. The molecular weight was estimated to be about 53,000 by gel filtration. Relative molecular mass studies indicated that the enzyme was a monomer structure. The purified enzyme had an optimum pH of 8.4 and an optimum temperature of 40C. The Km values of the purified enzyme determined with tryptophan and acetyl-CoA were 4.5 * 10 -3M, respectively. The 20 residues of N-terminal amino acid sequence were analyzed. en-copyright= kn-copyright= en-aut-name=Tashiro (Yamada)Yuriko en-aut-sei=Tashiro (Yamada) en-aut-mei=Yuriko kn-aut-name=田代 (山田)百合子 kn-aut-sei=田代 (山田) kn-aut-mei=百合子 aut-affil-num=1 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=2 ORCID= en-aut-name=TanakaHidehiko en-aut-sei=Tanaka en-aut-mei=Hidehiko kn-aut-name=田中英彦 kn-aut-sei=田中 kn-aut-mei=英彦 aut-affil-num=3 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=4 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 en-keyword=D-amino acid acetyltransferase kn-keyword=D-amino acid acetyltransferase en-keyword=Saccharomyces cerevisiae kn-keyword=Saccharomyces cerevisiae en-keyword=acyl donor kn-keyword=acyl donor en-keyword=affinity chromatography kn-keyword=affinity chromatography END start-ver=1.4 cd-journal=joma no-vol=92 cd-vols= no-issue=1 article-no= start-page=9 end-page=15 dt-received= dt-revised= dt-accepted= dt-pub-year=2003 dt-pub=200302 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Cloning and Nucleotide Sequenceof the Genes Encoding Restriction-Modification System from Acidophilic Bacterium Acidocella facilis 22M kn-title=好酸性細菌Acidocella facilis 22Mの制限修飾系遺伝子のクローニングと塩基配列決定 en-subtitle= kn-subtitle= en-abstract=CGATCGを認識する Afa22MI 制限修飾系遺伝子を好酸性細菌 Acidocella facilis 22M より、クローニングし、塩基配列を決定した。その結果、C5-シトシンメチラーゼに特徴的なモチーフが保存された。M.Afa22MI 遺伝子、その上流に、M.Afa22MI 遺伝子とは逆方向に、 very short patch repair endonuclease 様タンパク質遺伝子（Afa22MI vsr）、制限酵素（R.Afa22MI）遺伝子と推定されるオープンリーディングフレームが見出された。M.Afa22MI の推定アミノ酸配列は、Xanthomonas oryzae pv. oryzae 由来 M.XorII の配列と全体で約63%、veriable region 内で約53%の高い配列類似性を示した。また、Afa22MI usr の推定アミノ酸配列も、M.XorIIに付随する XorII vsr の配列と約66%の高い類似性を示した。 kn-abstract=The gene encoding the Afa22MI restriction-modification system recognizing the sequence 5'-CGATCG-3' was cloned from Acidocella facilis 22M and sequenced. The cloned DNA fragment contained three genes encoding the Afa22MI methylase (M.Afa22MI) , the putative restriction endonuclease Afa22MI (R.Afa22MI) and a very short patch repair endonuclease (Afa22MI vsr) . M. Afa22MI gene has the conserved motifs of C5-cytosine methyltransferase. Afa22MI vsr gene was localized upstream of M. Afa22MI gene in opposite orientation, and an open reading frame of R. Afa22MI has about 53% sequence similarity to the amino acid sequence for the variable region of M.XorU. Afa22MI vsr has about 66% sequence similarity to the amino acid sequence of XorII vsr which was associated M. XorII. en-copyright= kn-copyright= en-aut-name=YamaokaSeiji en-aut-sei=Yamaoka en-aut-mei=Seiji kn-aut-name=山岡誠司 kn-aut-sei=山岡 kn-aut-mei=誠司 aut-affil-num=1 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=2 ORCID= en-aut-name=TakenobuHisanori en-aut-sei=Takenobu en-aut-mei=Hisanori kn-aut-name=竹信尚典 kn-aut-sei=竹信 kn-aut-mei=尚典 aut-affil-num=3 ORCID= en-aut-name=KojoToshiyuki en-aut-sei=Kojo en-aut-mei=Toshiyuki kn-aut-name=古城俊之 kn-aut-sei=古城 kn-aut-mei=俊之 aut-affil-num=4 ORCID= en-aut-name=TanakaHidehiko en-aut-sei=Tanaka en-aut-mei=Hidehiko kn-aut-name=田中英彦 kn-aut-sei=田中 kn-aut-mei=英彦 aut-affil-num=5 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 affil-num=5 en-affil= kn-affil=岡山大学 affil-num=6 en-affil= kn-affil=岡山大学 en-keyword=restriction-modification system kn-keyword=restriction-modification system en-keyword=restriction endonuclease kn-keyword=restriction endonuclease en-keyword=C5-cytosine methyltransferase kn-keyword=C5-cytosine methyltransferase en-keyword=very short patch repair endonuclease kn-keyword=very short patch repair endonuclease END start-ver=1.4 cd-journal=joma no-vol=92 cd-vols= no-issue=1 article-no= start-page=17 end-page=20 dt-received= dt-revised= dt-accepted= dt-pub-year=2003 dt-pub=200302 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=ヒト由来チオキドキシン還元酵素のカルボキシル末端配列の酸化還元状態の半経験的分子軌道計算 kn-title=Semiempirical Molecular Orbital Calculation for the Redox Property of C-Terminal Active Site　Sequence of Human Thioredoxin Reductase en-subtitle= kn-subtitle= en-abstract=ヒトのチオレドキシン還元酵素（TrxR）のカルボキシル末端配列の酸化還元に伴うエンタルピー変化を計算した。半経験的分子軌道計算 WinMOPAC 3.5Pro を用いてモデルペプチド、N-Ac-Ser-Ile-Leu-Gln-Ala-Gly-X1-X2-Gly の生成熱を算出した。X1,X2のアミノ酸配列は-SeCys-Cys、 -Cys-SeCys-、 -SeCys-SeCys-、 -Cys-Cys- のいずれかで、それぞれ酸化状態と還元状態を計算し、そのエンタルピー差を求めた。ハミルトニアン AM1 と PM3は同じ傾向の計算結果を示し、セレノスルフィドまたはジセレニドを形成するペプチドは酸化状態でより安定化するのに対して-Cys-Cys-の配列を持つペプチドは還元型の方が安定的であることを示した。ほ乳類の TrxR の SeCys498 を Cys に置換すると酵素活性が1%程度にまで低下することが報告されている。これは、変異酵素が -Cys497-Cys498- 間で酸化的架橋を形成しにくいからと考察されていたが、今回の分子軌道計算の結果は、この仮説を支持している。 kn-abstract=Semiempirical molecular orbital calculation (MOPAC) was used to estimate the enthalpy difference (ΔH) between the reduce and oxidized states of the C-terminal rebox center of human thioredoxin reductase. Heat of formation was computed by WinMOPAC 3.5Pro for the model peptides, N-Acetyl-Ser-Ile-Leu-Gly-X1-X2-Gly, whose-X1-X2-sequence was-Cys SeCys-(natural sequence), -SeCys-Cys-(reverse sequence), -Cys-Cys, and-SeCys-SeCys-. Calculation by Hamiltonian AM1 and PM3 agreed that the oxidized state with selenosulfide bonds and a diselenide bond were more favoralbe than their reduced states. Only the peptide that contained-Cys-Cys-sequence was shown to have lower enthalpy when the two Cys were in the reduced form. It has been reported that substitution of SeCys498 to Cys results in the mujtant TrxRs retaining only about 1% of the enzyme activity. The results of computational estimation supported the experimental hypothesis that the inactivation by SeCys498Cys mutation was due to the unfavorable formation of disulfide bond between Cys497-Cys498. en-copyright= kn-copyright= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=1 ORCID= en-aut-name=TanakaHidehiko en-aut-sei=Tanaka en-aut-mei=Hidehiko kn-aut-name=田中英彦 kn-aut-sei=田中 kn-aut-mei=英彦 aut-affil-num=2 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=3 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 en-keyword=MOPAC kn-keyword=MOPAC en-keyword=thioredoxin reductase kn-keyword=thioredoxin reductase en-keyword=selenocysteine kn-keyword=selenocysteine en-keyword=enthalpy kn-keyword=enthalpy END start-ver=1.4 cd-journal=joma no-vol=91 cd-vols= no-issue=1 article-no= start-page=1 end-page=5 dt-received= dt-revised= dt-accepted= dt-pub-year=2002 dt-pub=200202 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=ＵＶ照射とプロトプラスト再生法によるStreptomyces incarnatusのリファンビシン耐性とシネフンギン生産性の向上 kn-title=Improvement of Sinefungin-Producing Strain of Streptomyces incarnatus by Conferring Rifampicin-Resistance through Ultraviolet Light Irradiation and Protoplast Regeneration en-subtitle= kn-subtitle= en-abstract=グラム陽性菌の二次代謝は，貧栄養条件などの緊縮応答として，その生合成遺伝子の転写段階が厳密な制御を受けている．そこで，これらのバクテリアのRNAポリメラーゼの制御部位を突然変異導入により破壊すれば，二次代謝産物の生産性向上が期待できる．本研究ではシネフンギンを生産するStreptomycesincarnatusNRRL8057プロトプラストに紫外線を照射して，RNAポリメラーゼ阻害剤であるリファンピシンに耐性を獲得した変異株を取得した．突然変異操作を三回行なった結果，異なるリファンピシン耐性を持つ突然変異株10株を得た．この中で最も高い耐性を示した突然変異株が最も高いシネフンギン生産能を示し，その生産性（0.45±0.11μg/ml）は野生株（0.19±0.07μg/ml）の約2.4倍あった．リァンピシン耐性を指標とする菌株の改良は，ランダムなスクリーニングを行なうよりも効率よく選抜を行うことができるという点で有効であるといえる kn-abstract=Secondary metabolite production by gram-positive bacteria is strictly regulated at the transcription of the biosynthetic genes to mRNA in response to certain stringent conditions. Therefore, some mutational disruption of regulatory domains of the bacterial RNA polymerase might increase the production of the antibiotics. In this study, we have attempted to improve the sinefungin-producing strain of Streptomyces incarnatus NRRL 8057 by irradiating ultraviolet light on the protoplast, and selecting mutants that acquired the resistance to rifampicin, the antibiotic which specifically binds to the β-subunit of bacterial RNA polymerase. After three rounds of mutation, 10 strains were obtained with varied resistance to rifampicin. A mutant which showed the highest resistance was found to have the highest sinefungin production, which was 2.4 times higher(0.45±0.11μg/ml)than the wild type strain (0.19±0.07μg/ml). The breeding approach by rifampicin-resistance may be advantageous over the classical random screening since it requires much smaller number of candidates to be examined. en-copyright= kn-copyright= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=1 ORCID= en-aut-name=LiYin Shu en-aut-sei=Li en-aut-mei=Yin Shu kn-aut-name=李銀淑 kn-aut-sei=李 kn-aut-mei=銀淑 aut-affil-num=2 ORCID= en-aut-name=TanakaHidehiko en-aut-sei=Tanaka en-aut-mei=Hidehiko kn-aut-name=田中英彦 kn-aut-sei=田中 kn-aut-mei=英彦 aut-affil-num=3 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=4 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 en-keyword=Sinefungin kn-keyword=Sinefungin en-keyword=Protoplast regeneration kn-keyword=Protoplast regeneration en-keyword=Rifampicin kn-keyword=Rifampicin en-keyword=RNA polymerase kn-keyword=RNA polymerase END start-ver=1.4 cd-journal=joma no-vol=91 cd-vols= no-issue=1 article-no= start-page=7 end-page=13 dt-received= dt-revised= dt-accepted= dt-pub-year=2002 dt-pub=200202 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Cloning of the Extracellular Acid Esterase Gene from Acidophilic Bacterium, Acidocella facilis kn-title=好酸性細菌Acidocella Facilis由来菌体外酸性エステラーゼ遺伝子のクローニング en-subtitle= kn-subtitle= en-abstract=好酸性従属栄養細菌AcidocellafacilisAIU409株は，基質としてソルビタンモノエステルであるTweenを含む培地中で培養された時に，菌体外に熱安定性の高い酸性エステラーゼを生産する．エステラーゼをコードする遺伝子（estA）をA.facilisAIU409株のゲノムライブラリーから単離し，大腸菌MV1184にクローニングし，その遺伝子の全塩基配列を決定した．その結果，estAの構造遺伝子が，1881塩基対であることが明らかになった．酸性エステラーゼ遺伝子のオープンリーディングフレーム（ORF）は，627アミノ酸残基（計算された分子量は64,140ダルトン）をコードしていた．ロー因子非依存性の転写終結シグナルが終止コドンであるTGAのすぐ下流に存在していた．そのＮ末端予想アミノ酸配列より，酸性エステラーゼの前駆体は，Ｎ末端に23個のアミノ酸残基から成るシグナルペプチドを有していた．また，リパーゼのコンセンサス配列であるG-X-S-X-Sの配列が存在することが明らかとなった．酸性エステラーゼの予想アミノ酸配列から計算された分子量は61,486であり，これはSDS-PAGEによって予想されていた分子量より，やや低い値であった．また，Acidocellafacilis酸性エステラーゼの予想アミノ酸配列は，Aeromonashydrophila由来のacyltrans-feraseやXenorhabdusluminescens由来のリパーゼと高い相同性を示した． kn-abstract=An acidophilic heterotrophic bacterium, Acidocella facilis sp. AIU409 produces an extracellular acid esterase when grown in a medium containing sorbitan mono ester, Tween 80. The estA gene encoding for the acid esterase was isolated from the genomic library of A. facilis AIU409, cloned into Escherichia coli MV1184, and the nucleotide sequence of the gene was determined. The structural gene of estA was found to be 1881bp. The open reading frame of estA encodes 627 amino acid residues (calculated molecular mass, 64,140 daltons). A rho-independent terminator was present just downstream of the termination codon, TGA. The deduced N-terminal amino acid showed that the presursor of the acid esterase had a signal peptide composed of 23 amino acids and a consensus sequence of lipase, G-X-S-X-S. The molecular mass excluding the signal peptide calculated from the deduced amino acid sequence if the acid esterase is 61,846. This is lower than the molecular mass, 64kDa estimated by gel electrophoresis. The predicated amino acid sequence of the acid esterase has high similarity to the acyltransferase from Aeromonas hydrophila and the lipase from Xenorhadbus luminescens. en-copyright= kn-copyright= en-aut-name=TakahashiTatsuya en-aut-sei=Takahashi en-aut-mei=Tatsuya kn-aut-name=高橋竜也 kn-aut-sei=高橋 kn-aut-mei=竜也 aut-affil-num=1 ORCID= en-aut-name=TanakaNoboru en-aut-sei=Tanaka en-aut-mei=Noboru kn-aut-name=田中登 kn-aut-sei=田中 kn-aut-mei=登 aut-affil-num=2 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=3 ORCID= en-aut-name=MukaiChika en-aut-sei=Mukai en-aut-mei=Chika kn-aut-name=向井千佳 kn-aut-sei=向井 kn-aut-mei=千佳 aut-affil-num=4 ORCID= en-aut-name=IsobeKimiyasu en-aut-sei=Isobe en-aut-mei=Kimiyasu kn-aut-name=磯部公安 kn-aut-sei=磯部 kn-aut-mei=公安 aut-affil-num=5 ORCID= en-aut-name=WakaoNorio en-aut-sei=Wakao en-aut-mei=Norio kn-aut-name=若尾紀夫 kn-aut-sei=若尾 kn-aut-mei=紀夫 aut-affil-num=6 ORCID= en-aut-name=TanakaHidehiko en-aut-sei=Tanaka en-aut-mei=Hidehiko kn-aut-name=田中英彦 kn-aut-sei=田中 kn-aut-mei=英彦 aut-affil-num=7 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=8 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 affil-num=5 en-affil= kn-affil=岩手大学農学部 affil-num=6 en-affil= kn-affil=岩手大学農学部 affil-num=7 en-affil= kn-affil=岡山大学 affil-num=8 en-affil= kn-affil=岡山大学 en-keyword=acid esterase kn-keyword=acid esterase en-keyword=extracellular enzyme kn-keyword=extracellular enzyme en-keyword=Acidocella facilis kn-keyword=Acidocella facilis en-keyword=Acidophiles kn-keyword=Acidophiles END start-ver=1.4 cd-journal=joma no-vol=91 cd-vols= no-issue=1 article-no= start-page=15 end-page=22 dt-received= dt-revised= dt-accepted= dt-pub-year=2002 dt-pub=200202 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Glutamate Snsor Using L-Glutamate Oxidase and Its Application for Sensing GOT/GPT Activity kn-title=Ｌ−グルタミン酸オキシダーゼを用いたグルタミン酸センサーの開発及びＧＯＴ/ＧＰＴセンシングへの応用 en-subtitle= kn-subtitle= en-abstract= kn-abstract=L-Glutamate measurement and GOT/GPT assay was successful by H2O2 measurement using the L-glutamate oxidase with 4-aminoantipyrine / phenol method. But, in examination of oxigen electrode, immobilized L-glutamate oxidase at the cellulose to L-glutamate and GOT-GPT sensor, Lpglutamate measurement was used for the amperometric determination with non-fixed enzyme. On examination of electron mediator, response for L-glutamate was observed with each of the compounds ferricyane, ferrocene-COOH, ferrocene-MeOH, and benzoquinone. L-Glutamate was measured by carbon printed tip electrode the L-glutamate oxidase and ferricyane based on the principle of chronoamperometry. A linear calibration graph was obtained between 1mM and 30mM. These results suggest that L-glutamate oxidase is able to utilize to L-glutamate sensor, and that there is a strong possibility to put this sensor to sensing for GOT/GPT activity. en-copyright= kn-copyright= en-aut-name=ArimaJiro en-aut-sei=Arima en-aut-mei=Jiro kn-aut-name=有馬二朗 kn-aut-sei=有馬 kn-aut-mei=二朗 aut-affil-num=1 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=2 ORCID= en-aut-name=ShinoharaHiroaki en-aut-sei=Shinohara en-aut-mei=Hiroaki kn-aut-name=篠原寛明 kn-aut-sei=篠原 kn-aut-mei=寛明 aut-affil-num=3 ORCID= en-aut-name=KusakabeHitoshi en-aut-sei=Kusakabe en-aut-mei=Hitoshi kn-aut-name=日下部均 kn-aut-sei=日下部 kn-aut-mei=均 aut-affil-num=4 ORCID= en-aut-name=TanakaHidehiko en-aut-sei=Tanaka en-aut-mei=Hidehiko kn-aut-name=田中英彦 kn-aut-sei=田中 kn-aut-mei=英彦 aut-affil-num=5 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=ヤマサ醤油株式会社 affil-num=5 en-affil= kn-affil=岡山大学 affil-num=6 en-affil= kn-affil=岡山大学 en-keyword=L-glutamate ocidase kn-keyword=L-glutamate ocidase en-keyword=L-glutamate sensor kn-keyword=L-glutamate sensor en-keyword=GOT/GPT sensor kn-keyword=GOT/GPT sensor END start-ver=1.4 cd-journal=joma no-vol=87 cd-vols= no-issue=1 article-no= start-page=47 end-page=51 dt-received= dt-revised= dt-accepted= dt-pub-year=1998 dt-pub=199802 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Screening nad lsolation of Azo Dyes Decolorizing Microorganisms and Analysis of Their Mechanism kn-title=染料脱色微生物の検索，単離及び脱色機構の解析 en-subtitle= kn-subtitle= en-abstract=アゾ染科を脱色する微生物の検索を行い、岡山市旭西浄化センターの活性汚泥サンプルから染科脱色菌1株を単離した。単離菌MK-1株はCandida属に分類される酵母であり、実験に用いた5種のアゾ染科のうちColor Index(C.I.)Reactive orange 16, C.I.Reactive red 21の2種類の染科を効率的に脱色した。脱色機構を検討したところCandida sp.MK-1株による脱色はグルコースんい依存していることが判明した。さらに菌体を用いた染色生体の脱色も可能であった。これらの結果は、今回単離したCandida sp. MK-1株が、抜染法等に応用可能であり、更に染科を多量に含んだ有色廃水の処理等のバイオレメディエーションにも応用できる可能性を示した。 kn-abstract=A yeast, Candida sp. MK-1, newly isolated from activated sludge as a dye decolorizing microorganism, decolorized Color lndex (C.l.) Reactive orange 16 and C.l. Reactive red 21 on the solid medium. Both azo dyes were also decolorized even in the liquid medium. The decolorizing activity by Candida sp. MK-1 depends on glucose concentration. Textile stained by Reactive orange 16 or Reactive red 21 was decolorized by Candida sp. MK-1. There results suggest that Candida sp. MK-1 has potential applications for the decolorization of textile and for the bioremediation of dye-contaminated wastewater. en-copyright= kn-copyright= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=1 ORCID= en-aut-name=KawaguchiMasakazu en-aut-sei=Kawaguchi en-aut-mei=Masakazu kn-aut-name=川口将和 kn-aut-sei=川口 kn-aut-mei=将和 aut-affil-num=2 ORCID= en-aut-name=TaguchiTakaaki en-aut-sei=Taguchi en-aut-mei=Takaaki kn-aut-name=田口隆章 kn-aut-sei=田口 kn-aut-mei=隆章 aut-affil-num=3 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=4 ORCID= en-aut-name=TanakaHidehiko en-aut-sei=Tanaka en-aut-mei=Hidehiko kn-aut-name=田中英彦 kn-aut-sei=田中 kn-aut-mei=英彦 aut-affil-num=5 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=有限会社タグチ affil-num=4 en-affil= kn-affil=岡山大学 affil-num=5 en-affil= kn-affil=岡山大学 en-keyword=decolorization kn-keyword=decolorization en-keyword=azo dyes kn-keyword=azo dyes en-keyword=Candida sp kn-keyword=Candida sp en-keyword=screening kn-keyword=screening en-keyword=bioremediation kn-keyword=bioremediation END start-ver=1.4 cd-journal=joma no-vol=87 cd-vols= no-issue=1 article-no= start-page=53 end-page=58 dt-received= dt-revised= dt-accepted= dt-pub-year=1998 dt-pub=199802 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Structural Analysis of the mde Operon lnvolved in L-Methionine Degradative Metabolism of Pseudomonas putida kn-title=Pseudomonas putida のＬ−メチオニン分解系オペロンの解析 en-subtitle= kn-subtitle= en-abstract=mdeオペロン及びその上流調節遺伝子(mdeR)がPseudomonas putida染色体DNAからクローニングされ、これらの遺伝子の塩基配列が決定された。mdeオペロンは、L-メチオニン分解代謝に伴われる2つの構造遺伝子mdeA (L-メチオニン　γ-リアーゼ遺伝子)及び、mdeB(ピルビン酸デヒドロゲナーゼ複合体のE1成分に相同性のあるタンパク質をコードする遺伝子)を含んでいた。ロー因子非依存型のターミネーター構造がmdeBのすぐ下流に存在し、α-ケト酸デヒドロゲナーゼ複合体の他の成分に相当するオープンリーディングフレームは見られなかった。mdeB産物が大腸菌中で過剰発現されたとき、その細胞抽出液は、ピルビン酸よりもむしろα-ケト酪酸に対し高い特異性を持つE1活性を示した。すなわちmdeB遺伝子は新規のE1成分であるα-ケト酪酸デヒドロゲナーゼE1成分をコードしており、L-メチオニンからL-メチオニンγ-リアーゼによって生産されたα-ケト酪酸の代謝に、mdeBが重要な役割を果たすことが示唆された。さらに我々は、mdeA遺伝子の翻訳開始点から127bp上流反対方向にコードされるmdeR遺伝子を見い出した。mdeR産物は、ロイシン応答調節タンパク質(Lrp)ファミリーの一つとして同定され、mdeABオペロンの発現に必須な正の調整因子として作用することが明らかとなった。 kn-abstract=The mde operon and an upstream regulatory gene (mdeR) have been cloned and sequenced from Pseudomonas putida chromosomal DNA. The mde operon contains two structural genes involved in L-methionine degradative metabolism, which are mdeA (L-methionine γ-lyase gene) and mdeB (a gene encoding a homologous protein to the E1 component of pyruvate dehydrogenase complex). A rho-independent terminator was present just downstream of mdeB and open reading frames corresponding to other components of α-keto acid dehydrogenase complex were not found. When the mdeB gene product was overproduced in Escherichia coli, the E1 activity of the cell extract showed high specificity for α-ketobutyrate rather than pyruvate. these results suggest that mdeB encodes a novel E1 component, α-ketobutyrate dehydrogenase E1 component, and plays an important role in the metabolism of α-ketobutyrate produced by L-methionine γ-lyase from L-methionine. In addition,we found that mdeR gene was located on the opposite strand and began at 127 bp from the translational start site of mdeA. The mdeR gene product has been idetified as a member of the leucine responsive regulatory protein (Lrp) family and revealed to act as an essential positive regulator allowing the expression of the mdeAB operon. en-copyright= kn-copyright= en-aut-name=InoueHiroyuki en-aut-sei=Inoue en-aut-mei=Hiroyuki kn-aut-name=井上浩之 kn-aut-sei=井上 kn-aut-mei=浩之 aut-affil-num=1 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=2 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=3 ORCID= en-aut-name=TanakaHidehiko en-aut-sei=Tanaka en-aut-mei=Hidehiko kn-aut-name=田中英彦 kn-aut-sei=田中 kn-aut-mei=英彦 aut-affil-num=4 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 en-keyword=L-methionine kn-keyword=L-methionine en-keyword=α-ketobutyrate kn-keyword=α-ketobutyrate en-keyword=mde operon kn-keyword=mde operon en-keyword=pyruvate dehydrogenase E1 component kn-keyword=pyruvate dehydrogenase E1 component en-keyword=leucine responsive regulatory protein kn-keyword=leucine responsive regulatory protein END start-ver=1.4 cd-journal=joma no-vol=86 cd-vols= no-issue=1 article-no= start-page=13 end-page=19 dt-received= dt-revised= dt-accepted= dt-pub-year=1997 dt-pub=199702 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=好酸性細菌 Acidiphilium organovorum 由来のアラニンセマーゼ kn-title=Alanine Racemase from an Acidophilic Bacterium, Acidiphilium organovorum en-subtitle= kn-subtitle= en-abstract=Acidiphilium属好酸性細菌中のアラニンラセマーゼ活性を検索した。Acidiphilium organovorum 13Hが最も高い活性を示したので、好酸性細菌由来の代表的なアラニンラセマーゼとして研究対象とした。本酵素の局在性を調べたところ、細胞質に存在することが明らかとなった。また本酵素は同一のサブユニット（約34ｋDa）からなる二重構造体をとることが示された。至適反応条件は50-60℃、pH9であった。また65℃で30分のインキュベーション後も活性が失われず、熱さに対して比較的安定な酵素であった。Hydroxylamineによる透析で活性が失われ、これをpyriodoxal 5'-phosphate を含む緩衝液により透析をした結果、活性が部分的に回復したことから、本酵素はpyridoxial 5'-phosphateを補酵素として要求することが示唆された。 kn-abstract=Alanine racemase(EC 5.1.1.1)was screened from several acidophilic bacteria.Acidiphilium organovorum 13H showed the highest activity and was chosen as the representative source to study alaine racemase from acidphlic bacteria.The enzyme was found to be localised in the cytoplasm of the bacteria.Relative molecular mass syudies indicated that it had a dimeric native structure with identical subunits of about 34 kDa each.Maximum activity was observed between 50 and 60℃and at pH9.There was no loss of enzyme activity even after incubation at 65℃.The loss of activity upon dialysis against pyridoxal 5'-phosphate-free buffer containing hydroxylamine,and its partial recovery upon subsequent dialysis against buffer containing phyridoxal 5'-phosphate suggested that the enzyme required piridoxal 5'-phosphate as a co-factor for its catalytic activity. en-copyright= kn-copyright= en-aut-name=SeowTeck Keong en-aut-sei=Seow en-aut-mei=Teck Keong kn-aut-name=スィアオテッキョン kn-aut-sei=スィアオ kn-aut-mei=テッキョン aut-affil-num=1 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=2 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=3 ORCID= en-aut-name=TanakaHidehiko en-aut-sei=Tanaka en-aut-mei=Hidehiko kn-aut-name=田中英彦 kn-aut-sei=田中 kn-aut-mei=英彦 aut-affil-num=4 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 en-keyword=Alanine racemase kn-keyword=Alanine racemase en-keyword=Acidophile kn-keyword=Acidophile en-keyword=Acidiphilium organovorum kn-keyword=Acidiphilium organovorum en-keyword=Phyridoxal 5'-phosphate kn-keyword=Phyridoxal 5'-phosphate END start-ver=1.4 cd-journal=joma no-vol=84 cd-vols= no-issue=1 article-no= start-page=69 end-page=72 dt-received= dt-revised= dt-accepted= dt-pub-year=1995 dt-pub=19950201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Synthesis and Biochemical Studies of Selenocysteine-Containing Peptides kn-title=セレノシステイン含有ペプチドの合成と生化学的機能の研究 en-subtitle= kn-subtitle= en-abstract=セレンは2-、0、2+、4+、6+と多彩な酸化還元状態をとるという点で金属的な性質を持つ。また有機セレン化合物として求核的・求電子的な反応性に関与できるという点で酸素や硫黄などの典型元素のような振る舞いをする。生命は進化の過程で、セレンのユニークな化学的特性をうまく利用し、グルタチオンペルオキシダーゼなどのセレン酵素を獲得してきたと考えられる。セレン酵素の研究に触発されて、セレン酵素化合物を医薬品として利用する研究が進められており注目を集めている。このような背景のもと、グルタセレノンとテトラペプチドを合成した。とくにテトラペプチドはグルタセレノンの反応機構研究から生まれたペプチドである。今後さらに高い活性を持つ人工酵素がデザインされ、合成されるだろう。その時、鋳型として蛋白質や核酸、オリゴ糖などある特定のコンフォメーションをとる生体物質が利用されると思われる。 kn-abstract=Selenium belongs to the Yb group of the periodic table,and possesses both metallic and nonmetallic characteristics. Physicochemical properties of selenium resemble more or less those of sulfur,and selenium may be indiscriminately incorporated in place of sulfur in cellular constituents and disturb metabolism. Alkali disease and blind stagger disease of livestock are caused by selenium-polluted grass. Carcinogenic effect is also one of the marked biological properties of selenium. In spite of the toxic and carcinogenic effects,selenium is actually an essential trace element for bacteria,fish,and mammals. Life has exploied the high reactivity and unique characteristics of organoselenium compounds,especially in the form of selenoenzymes. Mammalian glutathione peroxidase(EC 1.11.1.9) has selenocysteine residue at the active site, and the enzyme plays a central role in the biological defense system against oxidative challenge by activated oxygens and radicals. The author has studied the low redox potential and high reactivity of selenium-containing compounds, and developed glutathione peroxidase mimics. Glutaselenone, a selenium analog of glutaheione, catalyzes a glutathione peroxidase like reaction in vitro. Studies on the mechanisum of glutaselenone-catalyzed reaction revealed that glutaselenone is converted to a selenosulfide conjugate with glutathione in its catalysis. Thioredoxins contain a conserved sequence,Cys-Gly-Pro-Cys, which is known to form intramolecular disulfide bond with consecutive β-turn conformations. THe peptide is expected to serve as an template for intramolecular selenosulfide bond formation when either the cysteine is replaced by selenocystein. A tetrapeptide,Secys-Gly-Pro-Cys, was synthesized chemically. It showed glutathione peroxidase-like activity three times as high as glutaselenone. The high catalytic activity is ascribed to an intramolecular selenosulfide bond formation in the catalytic reaction. en-copyright= kn-copyright= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=1 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 en-keyword=Selenium kn-keyword=Selenium en-keyword=Glutathione Peroxidase kn-keyword=Glutathione Peroxidase en-keyword=Selenocysteine kn-keyword=Selenocysteine en-keyword=Thioredoxin kn-keyword=Thioredoxin END start-ver=1.4 cd-journal=joma no-vol=99 cd-vols= no-issue=1 article-no= start-page=1 end-page=5 dt-received= dt-revised= dt-accepted= dt-pub-year=2010 dt-pub=20100201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Identification of Ornithine-lactam Converted from Arginine in Streptomyces incarnatus NRRL8089 kn-title=シネフンギン生産菌Streptomyces incarnatus NRRL 8089由来アルギニン変換化合物「オルニチンラクタム」の同定 en-subtitle= kn-subtitle= en-abstract=シネフンギンは抗真菌，抗マラリア活性を有する核酸系抗生物質であり，放線菌 S. incarnatus により生合成される．シネフンギンはアデノシンとオルニチンがＣﾝＣ結合した構造であり，無細胞抽出液での取り込み実験からＬﾝアルギニンと ATP から生合成されると推測される．Ｌﾝアルギニン，Ｌﾝオルニチンを S. incarnatus の休止菌体反応系への投与を行いシネフンギン中間体の探索を行った．その結果50ﾋアルギニンは24時間以内に低極性化合物へと変換された．一方50ﾋオルニチンは変換されず反応液中に残存した．HPLC で化合物を精製し，1HﾝNMR，FABﾝMS での分析の結果オルニチン環状モノペプチド，「オルニチンラクタム」（分子量114）であることを明らかにした．この結果は S. incarnatus がアルギニンからオルニチンラクタムへの変換酵素を有する事を示唆する．このような酵素の報告例はこれまでになく，ニ次代謝酵素であることが示唆され，シネフンギン生合成との関連性に興味が持たれる． kn-abstract=Sinefungin is a nucleoside antibiotic, in which a molecule of L-ornithine is linked to the 5' end of adenosine through a C-C bond. The antibiotic was isolated from the culture broth of Streptomyces incarnatus. For the purpose of detecting intermediate of sinefungin biosynthesis, resting cell suspensions were incubated with supplemental L-arginine, and L-ornithine. 50mM Arginine was converted to a compound X that has low polarity. 50mM ornithine was not converted and remained in reaction solution. Compound X was purified using HPLC, and analyzed using (1)H-NMR and FAB-MS. These analyses showed that a compound X is "ornithine-lactam" (Mw＝114), which has a structure of circularized ornithine. These results indicated that S. incarnatus has an enzyme that converts arginine to ornithine-lactam. Such an enzyme has never been reported, and suggested that it may be relevant to sinefungin biosynthesis. en-copyright= kn-copyright= en-aut-name=FukudaKoji en-aut-sei=Fukuda en-aut-mei=Koji kn-aut-name=福田康二 kn-aut-sei=福田 kn-aut-mei=康二 aut-affil-num=1 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=2 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=3 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 en-keyword=sinefungin kn-keyword=sinefungin en-keyword=arginine kn-keyword=arginine en-keyword=ornithine kn-keyword=ornithine en-keyword=Streptomyces kn-keyword=Streptomyces END start-ver=1.4 cd-journal=joma no-vol=99 cd-vols= no-issue=1 article-no= start-page=7 end-page=12 dt-received= dt-revised= dt-accepted= dt-pub-year=2010 dt-pub=20100201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Purification and Characterization of Cystathionine γ-Synthase from Thermoacidophilic Archaea Sulfolobus tokodaii kn-title=好熱好酸性アーキア Sulfolobus tokodaii 由来シスタチオニン γ-シンターゼの精製及び性質検討 en-subtitle= kn-subtitle= en-abstract=好熱好酸性アーキア Sulfolobus tokodaii 由来シスタチオニンγﾝシンターゼ（stCGS）遺伝子を pET-11a に組み込み pET-stCGS を構築した．このベクターでE. coli Rosettaﾝgami（DE3）を形質転換し，本遺伝子を発現させ，精製及び性質検討を行った．大腸菌で発現したシスタチオニンγﾝシンターゼの活性が無細胞抽出液で確認できた．S. tokodaii シスタチオニンγﾝシンターゼを70℃熱処理 DEAEﾝトヨパールイオン交換カラム等により単一精製した．精製酵素の最適温度は100℃以上であり，熱安定性は60分間処理で70℃までほぼ100ｵの残存活性を示した．また，最適pHについてはリン酸緩衝液やブリトンﾝロビンソン広域緩衝液の場合はpH7.0の時が最も活性が高く，トリス塩酸緩衝液の場合はpH9.0が最適であった．pH安定性についてはpH5.0〜9.0において安定であった．O-ホスホ-l-ホモセリンに対するKm，Vmaxは，それぞれ0.82mM，2.42U/rであった．アポ酵素のホロ化実験により，本酵素活性がPLP に依存していることが明らかとなった．更に本酵素の脱離反応での基質特異性の検討を行った．変異酵素を用いた実験により，stCGSの基質特異性には，活性中心に存在するPhe97を含む領域が深く関わっていることが示唆された． kn-abstract=The gene encoding a cystathionine γ-synthase from Sulfolobus tokodaii was cloned and expressed in Escherihia coli Rosetta-gami (DE3). Cystathionine γ-synthase ［EC 2. 5. 1. 48］ from Sulfolobus tokodaii (stCGS) was purified by heat treatment, DEAE- Toyopearl 650M and Sephacryl S-300 column chromatographies from E. coli transformants. stCGS shows optimum activity at pH 7.0, and is stable between pH5.0 and pH9.0. The optimum temperature of stCGS is above 100℃, and the enzyme showed the remaining activity of almost 100% up to 70℃. The K(m) and V(max) with O-phospho-L- homoserine as a substrate are 0.82 mM and 2.42 U/mg. To analyze the role of Phe 97 in the active site of stCGS, we constructed F97Y, R99C, and F97Y-R99C mutant enzymes. Although native stCGS has no activity toward l-methionine, F97Y mutant enzyme gained the elimination activity toward L-methionine. en-copyright= kn-copyright= en-aut-name=ShinozakiMai en-aut-sei=Shinozaki en-aut-mei=Mai kn-aut-name=篠崎舞 kn-aut-sei=篠崎 kn-aut-mei=舞 aut-affil-num=1 ORCID= en-aut-name=YanagitaniMasahiko en-aut-sei=Yanagitani en-aut-mei=Masahiko kn-aut-name=柳谷昌彦 kn-aut-sei=柳谷 kn-aut-mei=昌彦 aut-affil-num=2 ORCID= en-aut-name=KanedaShouichirou en-aut-sei=Kaneda en-aut-mei=Shouichirou kn-aut-name=兼田翔一郎 kn-aut-sei=兼田 kn-aut-mei=翔一郎 aut-affil-num=3 ORCID= en-aut-name=KudouDaizou en-aut-sei=Kudou en-aut-mei=Daizou kn-aut-name=工藤大蔵 kn-aut-sei=工藤 kn-aut-mei=大蔵 aut-affil-num=4 ORCID= en-aut-name=EndouYuuichi en-aut-sei=Endou en-aut-mei=Yuuichi kn-aut-name=遠藤祐一 kn-aut-sei=遠藤 kn-aut-mei=祐一 aut-affil-num=5 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=6 ORCID= en-aut-name=KuramitsuSeiki en-aut-sei=Kuramitsu en-aut-mei=Seiki kn-aut-name=倉光成紀 kn-aut-sei=倉光 kn-aut-mei=成紀 aut-affil-num=7 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=8 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 affil-num=5 en-affil= kn-affil=岡山大学 affil-num=6 en-affil= kn-affil=岡山大学 affil-num=7 en-affil= kn-affil=大阪大学大学院理学研究科 affil-num=8 en-affil= kn-affil=岡山大学 en-keyword=cystathionine γ-synthase kn-keyword=cystathionine γ-synthase en-keyword=pyridoxal 5’-phosphate kn-keyword=pyridoxal 5’-phosphate en-keyword=thermoacidophilic archaea kn-keyword=thermoacidophilic archaea en-keyword=Sulfolobus tokodaii kn-keyword=Sulfolobus tokodaii END start-ver=1.4 cd-journal=joma no-vol=143 cd-vols= no-issue=4 article-no= start-page=467 end-page=473 dt-received= dt-revised= dt-accepted= dt-pub-year=2008 dt-pub=20080626 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Selenite Assimilation into Formate Dehydrogenase H Depends on Thioredoxin Reductase in Escherichia coli en-subtitle= kn-subtitle= en-abstract= kn-abstract=

Escherichia coli growing under anaerobic conditions produce H-2 and CO2 by the enzymatic cleavage of formate that is produced from pyruvate at the end of glycolysis. Selenium is an integral part of formate dehydrogenase H (FDHH), which catalyses the first step in the formate hydrogen lyase (FHL) system. The genes of FHL system are transcribed only under anaerobic conditions, in the presence of a sigma(54)-dependent transcriptional activator Fh1A that binds formate as an effector molecule. Although the formate addition to the nutrient media has been an established procedure for inducing high FDHH activity, we have identified a low-salt nutrient medium containing <0.1% NaCl enabled constitutive, high expression of FDHH even without formate and D-glucose added to the medium. The novel conditions allowed us to study the effects of disrupting genes like trxB (thioredoxin reductase) or gor (glutathione reductase) on the production of FDHH activity and also reductive assimilation of selenite (SeO32-) into the selenoprotein. Despite the widely accepted hypothesis that selenite is reduced by glutathione reductase-dependent system, it was demonstrated that trxB gene was essential for FDHH production and for labelling the FDHH polypeptide with Se-75-selenite. Our present study reports for the first time the physiological involvement of thioredoxin reductase in the reductive assimilation of selenite in E. coli.

en-copyright= kn-copyright= en-aut-name=TakahataMuneaki en-aut-sei=Takahata en-aut-mei=Muneaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=AbeKatsumasa en-aut-sei=Abe en-aut-mei=Katsumasa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MiharaHisaaki en-aut-sei=Mihara en-aut-mei=Hisaaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=KurokawaSuguru en-aut-sei=Kurokawa en-aut-mei=Suguru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=YamamotoYoshihiro en-aut-sei=Yamamoto en-aut-mei=Yoshihiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=NakanoRyuhei en-aut-sei=Nakano en-aut-mei=Ryuhei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=EsakiNobuyoshi en-aut-sei=Esaki en-aut-mei=Nobuyoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil= kn-affil=Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University affil-num=2 en-affil= kn-affil=Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University affil-num=3 en-affil= kn-affil=Laboratory of Molecular Microbial Science, Institute for Chemical Research, Kyoto University affil-num=4 en-affil= kn-affil=Laboratory of Molecular Microbial Science, Institute for Chemical Research, Kyoto University affil-num=5 en-affil= kn-affil=Laboratory of Molecular Microbial Science, Institute for Chemical Research, Kyoto University affil-num=6 en-affil= kn-affil=Department of Genetics, Hyogo College of Medicine affil-num=7 en-affil= kn-affil=Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University affil-num=8 en-affil= kn-affil=Department of Genetics, Hyogo College of Medicine affil-num=9 en-affil= kn-affil=Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University en-keyword=formate dehydrogenase H kn-keyword=formate dehydrogenase H en-keyword=selenite assimilation kn-keyword=selenite assimilation en-keyword=thioredoxin reductase kn-keyword=thioredoxin reductase END start-ver=1.4 cd-journal=joma no-vol=109 cd-vols= no-issue= article-no= start-page=1 end-page=6 dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Recombinant expression and characterization of quinone-containing novel glycine oxidase from Marinomonas mediterranea kn-title=Marinomonas mediterranea由来キノン含有新規グリシンオキシダーゼの大腸菌発現系の確立と性質検討 en-subtitle= kn-subtitle= en-abstract=, , , kn-abstract= 　Novel glycine oxidase (GlyOX) from Marinomonas mediterranea depends on cysteine tryptophilquinone (CTQ) and catalyzes the oxidative deamination of glycine to produce a glyoxylate, ammonia, and hydrogen peroxide. M. mediterranea GlyOX genes (goxA and goxB) were cloned and recombinant GlyOX was heterologously expressed by E. coli. The purification of recombinant GlyOX was carried out by metal affinity and DEAE-Toyopearl 650M column chromatographies. M. mediterranea GlyOX was homotetramic with a molecular mass of 76kDa and showed optimum activity around 30°C and at pH 5.0, and stability below 50°C and between pH 5.0 to 9.0. M. mediterranea GlyOX shows a strict substrate specificity toward glycine, and the Michaelis constant for glycine was 0.5mM. M. mediterranea GlyOX could determine the quantity of glycine in human serum and human blood plasma with high sensitivity. This study revealed the catalytic and structural properties of M. mediterranea GlyOX with high substrate specificity. en-copyright= kn-copyright= en-aut-name=KajiyamaYuki en-aut-sei=Kajiyama en-aut-mei=Yuki kn-aut-name=梶山雄輝 kn-aut-sei=梶山 kn-aut-mei=雄輝 aut-affil-num=1 ORCID= en-aut-name=MizobataSatsuki en-aut-sei=Mizobata en-aut-mei=Satsuki kn-aut-name=溝端佐津紀 kn-aut-sei=溝端 kn-aut-mei=佐津紀 aut-affil-num=2 ORCID= en-aut-name=AkajiShusaku en-aut-sei=Akaji en-aut-mei=Shusaku kn-aut-name=赤地周作 kn-aut-sei=赤地 kn-aut-mei=周作 aut-affil-num=3 ORCID= en-aut-name=NemotoMichiko en-aut-sei=Nemoto en-aut-mei=Michiko kn-aut-name=根本理子 kn-aut-sei=根本 kn-aut-mei=理子 aut-affil-num=4 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=5 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=6 ORCID= affil-num=1 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil=岡山大学大学院環境生命科学研究科 affil-num=2 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil=岡山大学大学院環境生命科学研究科 affil-num=3 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil=岡山大学大学院環境生命科学研究科 affil-num=4 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil=岡山大学大学院環境生命科学研究科 affil-num=5 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil=岡山大学大学院環境生命科学研究科 affil-num=6 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil=岡山大学大学院環境生命科学研究科 en-keyword=glycine oxidase kn-keyword=glycine oxidase en-keyword=Marinomonas mediterranea kn-keyword=Marinomonas mediterranea en-keyword=cysteine tryptophilquinone kn-keyword=cysteine tryptophilquinone en-keyword=recombinant expression kn-keyword=recombinant expression en-keyword=enzymatic glycine assay kn-keyword=enzymatic glycine assay END start-ver=1.4 cd-journal=joma no-vol= cd-vols= no-issue= article-no= start-page= end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200603 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases en-subtitle= kn-subtitle= en-abstract= kn-abstract=Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms. en-copyright= kn-copyright= en-aut-name=NemotoMichiko en-aut-sei=Nemoto en-aut-mei=Michiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=IwakiSayako en-aut-sei=Iwaki en-aut-mei=Sayako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=MoriyaHisao en-aut-sei=Moriya en-aut-mei=Hisao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MondenYuki en-aut-sei=Monden en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=MayamaShigeki en-aut-sei=Mayama en-aut-mei=Shigeki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=ObuseKiori en-aut-sei=Obuse en-aut-mei=Kiori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=6 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=7 en-affil=Department of Biology, Tokyo Gakugei University kn-affil= affil-num=8 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= en-keyword=Biomineralization kn-keyword=Biomineralization en-keyword=Diatom kn-keyword=Diatom en-keyword=Silica kn-keyword=Silica en-keyword=Transcriptome kn-keyword=Transcriptome en-keyword=Proteome kn-keyword=Proteome END start-ver=1.4 cd-journal=joma no-vol=101 cd-vols= no-issue= article-no= start-page=1 end-page=6 dt-received= dt-revised= dt-accepted= dt-pub-year=2012 dt-pub=20120201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Purification and Characterization of Thermostable Amidase from Thermus sp.O-3-1 kn-title=好熱性細菌Thermus sp.O-3-1由来耐熱性アミダーゼの精製及び性質検討 en-subtitle= kn-subtitle= en-abstract=好熱性細菌Thermus sp.O-3-1 由来の耐熱性アミダーゼ遺伝子を大腸菌中にクローニングし，その塩基配列を決定した．ami 遺伝子は930 bp からなり，310アミノ酸をコードしていた．本酵素の分子量は33，089 Daであると予想された．Thermus sp.O-3-1 由来アミダーゼを大腸菌で生産させ，熱処理とDEAE-トヨパール650M陰イオン交換カラム等により精製した．ゲル濾過クロマトグラフィーとSDS-PAGE の結果から本酵素は分子質量33 kDa のサブユニット2分子からなるダイマー構造を有していることが明らかとなった．精製酵素の熱安定性は80℃まで，pH 安定性は7.0〜10.0であり，安定性の 高い酵素であった．最適温度は90℃，最適 pH は9.0であ った．EDTA により活性が著しく阻害され，Co(2+)やNi(2+)，Mn(2+)によって活性の回復，向上が見られたため，本酵素は金属酵素であることが示唆された．基質特異性の検討 の結果，L-Leu-pNA よりもD-Leu-pNA に対して高い活性を示したため，本酵素がＤ-アミノ酸基質に特異性を持つアミダーゼであることが判明した．本酵素は耐熱性を有するユニークなＤ-アミノ酸アミダーゼであり，今後産業利用が期待される． kn-abstract=The gene encoding a thermostable amidase (EC 3.5.1.4) from thermophilic bacterium Thermus sp.O-3-1, was cloned and expressed in Escherichia coli JM109. The cloned amidase gene (ami) is 930 bp and encodes a protein composed of 310 amino acids. The protein is predicted to have a molecular mass of 33,089 Da. The amidase from Thermus sp.O-3-1 was purified by heat treatment and DEAE Toyopearl 650M column chromatography. The molecular mass of the native enzyme was estimated to be about 70 kDa by gel filtration chromatography, indicating that the enzyme has a homodimeric structure. The purified enzyme was stable up to 80°C and within a pH range from 7.0 to 10.0. The optimum temperature and pH for enzyme activity were 90°C, and 9.0, respectively. The enzyme was strongly inhibited by the metal-chelating compound EDTA. The activity of the EDTA-treated enzyme was reactivated by the addition of Co(2+), Ni(2+) and Mn(2+) ions. Therefore the enzyme was predicted to be metalloenzyme. Finally, as a result of investigation into substrate specificity, the purified enzyme was suggested to be D-amino acid specific amidase, as it showed higher activity toward D-Leu-pNA than L-Leu-pNA. en-copyright= kn-copyright= en-aut-name=KobayashiFumiaki en-aut-sei=Kobayashi en-aut-mei=Fumiaki kn-aut-name=小林史明 kn-aut-sei=小林 kn-aut-mei=史明 aut-affil-num=1 ORCID= en-aut-name=AomineHiroki en-aut-sei=Aomine en-aut-mei=Hiroki kn-aut-name=青峰弘起 kn-aut-sei=青峰 kn-aut-mei=弘起 aut-affil-num=2 ORCID= en-aut-name=MizunashiWataru en-aut-sei=Mizunashi en-aut-mei=Wataru kn-aut-name=水無渉 kn-aut-sei=水無 kn-aut-mei=渉 aut-affil-num=3 ORCID= en-aut-name=YuFujio en-aut-sei=Yu en-aut-mei=Fujio kn-aut-name=湯不二夫 kn-aut-sei=湯 kn-aut-mei=不二夫 aut-affil-num=4 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=5 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil= affil-num=2 en-affil= kn-affil= affil-num=3 en-affil= kn-affil=(株)三菱レイヨン affil-num=4 en-affil= kn-affil= affil-num=5 en-affil= kn-affil=岡山大学 affil-num=6 en-affil= kn-affil=岡山大学 en-keyword=amidase kn-keyword=amidase en-keyword=thermostable enzyme kn-keyword=thermostable enzyme en-keyword=Thermus kn-keyword=Thermus en-keyword=D-amino acid specific amidase kn-keyword=D-amino acid specific amidase END start-ver=1.4 cd-journal=joma no-vol=97 cd-vols= no-issue=1 article-no= start-page=1 end-page=7 dt-received= dt-revised= dt-accepted= dt-pub-year=2008 dt-pub=200802 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Purification, Characterization and Crystal Structure of Isoamylase from Thermophilic Bacteria Rhodothermus marinus kn-title=海産性好熱性細菌 Rhodothermus marinus 由来イソアミラーゼ の精製，性質検討及びＸ線結晶構造解析 en-subtitle= kn-subtitle= en-abstract=Rhodothermus marinus 由来イソアミラーゼ遺伝子を組み込んだプラスミド pBX２を使用し，大腸菌 Top10株を形質転換し，16時間の前培養，24時間の本培養後，菌体破砕し，得られた無細胞抽出液を熱処理（80℃，10 min），50ｵ硫安分画，陰イオン交換カラムクロマトグラフィー（DEAEﾝトヨパール），ハイドロキシアパタイトカラムクロマトグラフィーに供して本酵素の精製を行った．本精製酵素の性質検討を行った結果，本酵素の最適反応温度は70℃，pH４であり，また本酵素は60℃で１時間処理しても活性が低下することが無く，Pseudomonas amyloderamosa 由来イソアミラーゼよりも高い耐熱性を有することが判明した．本酵素の結晶化・X線結晶構造解析を行った結果，本酵素は P. amyloderamosa 由来イソアミラーゼと同様Ｎドメイン・ＡドメインＣドメインの３つのドメインから構成されており，活性残基（Ｄ359，Ｅ395，Ｄ467）など活性中心付近のアミノ酸残基も P. amyloderamosa 由来イソアミラーゼと同様，高度に保存されていた．本酵素の熱安定性が P. amyloderamosa 由来イソアミラーゼよりも高 い要因として，P. amyloderamosa 由来イソアミラーゼよりもループの長さが全体的に短いことと，カルシウムイオン結合サイトの欠如が挙げられた．今後さらに構造解析を進めることにより，本酵素の熱安定性機構，反応 機構など更なる知見が得られることが期待される． kn-abstract=The isoamylase gene from Rhodothermus marinus was cloned into and expressed in Escherichia coli Top 10. As a result of characterization of purified R. marinus isoamylase. the enzyme had an optimum pH of 4.0 and optimum temperature of 70℃. Thermal inactivation studies of the purified R. marinus isoamylase revealed the enzymatic activity to be uninfluenced after one hour incubation at 60℃. These results suggest that R. marinus isoamylase has high thermostability. The crystallization and crystal structure analysis of R. marinus isoamylase was performed. The three-dimensional structure at 1.9? resolution was determined in complex with the panose. R. marinus isoamylase is composed of three domains N, A and C, and, has a (β/α)8-barrel in domain A. The secondary structural alignments of the R. marinus isoamylase and P. amyloderamosa isoamylase was carried out. They have the four active-site consensus regions characteristic of the α-amylase family. And the essential residue of the α-amylase family (D359, E395, and D467) was conserved in these enzymes. R. marinus isoamylase has shorter loops than P. amyloderamosa isoamylase. And R. marinus isoamylase had no Ca2+ binding site. These results are thought to be factors of thermostability of R. marinus isoamylase. en-copyright= kn-copyright= en-aut-name=TachibanaAkiko en-aut-sei=Tachibana en-aut-mei=Akiko kn-aut-name=立花亜紀子 kn-aut-sei=立花 kn-aut-mei=亜紀子 aut-affil-num=1 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=2 ORCID= en-aut-name=ImadaKatsumi en-aut-sei=Imada en-aut-mei=Katsumi kn-aut-name=今田勝巳 kn-aut-sei=今田 kn-aut-mei=勝巳 aut-affil-num=3 ORCID= en-aut-name=KinoshitaMiki en-aut-sei=Kinoshita en-aut-mei=Miki kn-aut-name=木下実紀 kn-aut-sei=木下 kn-aut-mei=実紀 aut-affil-num=4 ORCID= en-aut-name=NambaKeiichi en-aut-sei=Namba en-aut-mei=Keiichi kn-aut-name=難波啓一 kn-aut-sei=難波 kn-aut-mei=啓一 aut-affil-num=5 ORCID= en-aut-name=TsutsumiNoriko en-aut-sei=Tsutsumi en-aut-mei=Noriko kn-aut-name=堤紀子 kn-aut-sei=堤 kn-aut-mei=紀子 aut-affil-num=6 ORCID= en-aut-name=HashidaMiyoko en-aut-sei=Hashida en-aut-mei=Miyoko kn-aut-name=橋田みよ子 kn-aut-sei=橋田 kn-aut-mei=みよ子 aut-affil-num=7 ORCID= en-aut-name=SakaguchiHiromichi en-aut-sei=Sakaguchi en-aut-mei=Hiromichi kn-aut-name=坂口博脩 kn-aut-sei=坂口 kn-aut-mei=博脩 aut-affil-num=8 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=9 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=大阪大学 affil-num=4 en-affil= kn-affil=大阪大学 affil-num=5 en-affil= kn-affil=大阪大学 affil-num=6 en-affil= kn-affil=ノボザイムズ ジャパン affil-num=7 en-affil= kn-affil=ノボザイムズ ジャパン affil-num=8 en-affil= kn-affil=ノボザイムズ ジャパン affil-num=9 en-affil= kn-affil=岡山大学 en-keyword=isoamylase kn-keyword=isoamylase en-keyword=Rhodothermus marinus kn-keyword=Rhodothermus marinus en-keyword=crystal structure kn-keyword=crystal structure en-keyword=thermostability kn-keyword=thermostability END start-ver=1.4 cd-journal=joma no-vol=5 cd-vols= no-issue= article-no= start-page=100044 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20211231 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Structural basis of enzyme activity regulation by the propeptide of l-lysine α-oxidase precursor from Trichoderma viride en-subtitle= kn-subtitle= en-abstract= kn-abstract=Harmuful proteins are usually synthesized as inactive precursors and are activated by proteolytic processing. l-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid to produce a 2-oxo acid with ammonia and highly toxic hydrogen peroxide and, therefore, is expressed as a precursor. The LAAO precursor shows significant variation in size and the cleavage pattern for activation. However, the molecular mechanism of how the propeptide suppresses the enzyme activity remains unclear except for deaminating/decarboxylating Pseudomonasl-phenylalanine oxidase (PAO), which has a short N-terminal propeptide composed of 14 residues. Here we show the inactivation mechanism of the l-lysine oxidase (LysOX) precursor (prLysOX), which has a long N-terminal propeptide composed of 77 residues, based on the crystal structure at 1.97?? resolution. The propeptide of prLysOX indirectly changes the active site structure to inhibit the enzyme activity. prLysOX retains weak enzymatic activity with strict specificity for l-lysine and shows raised activity in acidic conditions. The structures of prLysOX crystals that soaked in a solution with various concentrations of l-lysine have revealed that prLysOX can adopt two conformations; one is the inhibitory form, and the other is very similar to mature LysOX. The propeptide region of the latter form is disordered, and l-lysine is bound to the latter form. These results indicate that prLysOX uses a different strategy from PAO to suppress the enzyme activity and suggest that prLysOX can be activated quickly in response to the environmental change without proteolytic processing. en-copyright= kn-copyright= en-aut-name=KitagawaMasaki en-aut-sei=Kitagawa en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=ItoNanako en-aut-sei=Ito en-aut-mei=Nanako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=MatsumotoYuya en-aut-sei=Matsumoto en-aut-mei=Yuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=SaitoMasaya en-aut-sei=Saito en-aut-mei=Masaya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=KusakabeHitoshi en-aut-sei=Kusakabe en-aut-mei=Hitoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=ImadaKatsumi en-aut-sei=Imada en-aut-mei=Katsumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Department of Macromolecular Science, Graduate School of Science, Osaka University kn-affil= affil-num=2 en-affil=Department of Macromolecular Science, Graduate School of Science, Osaka University kn-affil= affil-num=3 en-affil=Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=4 en-affil=Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=5 en-affil=Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=6 en-affil=Enzyme Sensor Co., Ltd. kn-affil= affil-num=7 en-affil=Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=8 en-affil=Department of Macromolecular Science, Graduate School of Science, Osaka University kn-affil= en-keyword=L-Lysine α-oxidase kn-keyword=L-Lysine α-oxidase en-keyword=Crystal structure kn-keyword=Crystal structure en-keyword=Precursor kn-keyword=Precursor en-keyword=Substrate recognition kn-keyword=Substrate recognition END start-ver=1.4 cd-journal=joma no-vol=103 cd-vols= no-issue= article-no= start-page=5 end-page=9 dt-received= dt-revised= dt-accepted= dt-pub-year=2014 dt-pub=20140201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Characterization of L-Arginine Oxidase Made from L-Glutamate Oxidase kn-title=高基質特異性L-グルタミン酸オキシダーゼより作成した基質特異性改変酵素L-アルギニンオキシダーゼの性質検討 en-subtitle= kn-subtitle= en-abstract= kn-abstract=　L?Glutamate oxidase (LGOX) from Streptomyces sp. X?119?6 has strict substrate specificity toward L?glutamate. Recently, we solved the X?ray crystal structure of LGOX and this revealed that Arg305 in the active site is the key residue involved in substrate recognition. Therefore, we created 19 mutant enzymes of R305X?LGOX by saturation mutagenesis. One of them R305D?LGOX, Arg305 substituted with Asp exhibited oxidase activity for L?Arg. Optimum pH of R305D?LGOX mutant enzyme was pH 8.5. Interestingly, the activity of R305D?LGOX toward L?Arg was inhibited by phosphate. And furthermore, the substrate specificity of R305D?LGOX was affected by using buffer. The results of inhibition analysis suggest, that phosphate is a competitive inhibitor of R305D?LGOX when L?Arg is used as substrate. Kinetic analysis of R305D?LGOX showed that Km value and kcat value of R305D?LGOX toward l-Arg were 0.68 mM and 6.7 s-1 respectively. In this study, we showed that R305D?LGOX mutant enzyme is a novel l-arginine oxidase and useful for l-arginine biosensor. en-copyright= kn-copyright= en-aut-name=NakaiRyuichiro en-aut-sei=Nakai en-aut-mei=Ryuichiro kn-aut-name=中井隆一郎 kn-aut-sei=中井 kn-aut-mei=隆一郎 aut-affil-num=1 ORCID= en-aut-name=FujinoShihoko en-aut-sei=Fujino en-aut-mei=Shihoko kn-aut-name=藤野志保子 kn-aut-sei=藤野 kn-aut-mei=志保子 aut-affil-num=2 ORCID= en-aut-name=UtsumiTomohiro en-aut-sei=Utsumi en-aut-mei=Tomohiro kn-aut-name=内海友宏 kn-aut-sei=内海 kn-aut-mei=友宏 aut-affil-num=3 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=4 ORCID= en-aut-name=KusakabeaHitoshi en-aut-sei=Kusakabea en-aut-mei=Hitoshi kn-aut-name=日下部均 kn-aut-sei=日下部 kn-aut-mei=均 aut-affil-num=5 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 affil-num=5 en-affil= kn-affil=潟Gンザイムセンサ affil-num=6 en-affil= kn-affil=岡山大学 en-keyword=L-glutamate oxidase kn-keyword=L-glutamate oxidase en-keyword=L-arginine oxidase kn-keyword=L-arginine oxidase en-keyword=biosensor kn-keyword=biosensor en-keyword=modified substrate specificity kn-keyword=modified substrate specificity en-keyword=L-amino acid oxidase kn-keyword=L-amino acid oxidase END start-ver=1.4 cd-journal=joma no-vol=14 cd-vols= no-issue=1 article-no= start-page=1730 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230403 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota en-subtitle= kn-subtitle= en-abstract= kn-abstract=An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis. en-copyright= kn-copyright= en-aut-name=Jaunet-LaharyTitouan en-aut-sei=Jaunet-Lahary en-aut-mei=Titouan kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=ShimamuraTatsuro en-aut-sei=Shimamura en-aut-mei=Tatsuro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=HayashiMasahiro en-aut-sei=Hayashi en-aut-mei=Masahiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=NomuraNorimichi en-aut-sei=Nomura en-aut-mei=Norimichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=HirasawaKouta en-aut-sei=Hirasawa en-aut-mei=Kouta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=ShimizuTetsuya en-aut-sei=Shimizu en-aut-mei=Tetsuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=YamashitaMasao en-aut-sei=Yamashita en-aut-mei=Masao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=TsutsumiNaotaka en-aut-sei=Tsutsumi en-aut-mei=Naotaka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=SuehiroYuta en-aut-sei=Suehiro en-aut-mei=Yuta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=IwanariHiroko en-aut-sei=Iwanari en-aut-mei=Hiroko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= en-aut-name=HamakuboTakao en-aut-sei=Hamakubo en-aut-mei=Takao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=14 ORCID= en-aut-name=IwataSo en-aut-sei=Iwata en-aut-mei=So kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=15 ORCID= en-aut-name=OkazakiKei-Ichi en-aut-sei=Okazaki en-aut-mei=Kei-Ichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=16 ORCID= en-aut-name=HiraiTeruhisa en-aut-sei=Hirai en-aut-mei=Teruhisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=17 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=18 ORCID= affil-num=1 en-affil=Research Center for Computational Science, Institute for Molecular Science, National Institutes of Natural Sciences kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Kyoto University kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Kyoto University kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Kyoto University kn-affil= affil-num=6 en-affil=RIKEN SPring-8 Center kn-affil= affil-num=7 en-affil=RIKEN SPring-8 Center kn-affil= affil-num=8 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=9 en-affil=School of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=10 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=11 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=12 en-affil=Graduate School of Environmental and Life Sciences, Okayama University kn-affil= affil-num=13 en-affil=Research Center for Advanced Science and Technology, The University of Tokyo kn-affil= affil-num=14 en-affil=Research Center for Advanced Science and Technology, The University of Tokyo kn-affil= affil-num=15 en-affil=Graduate School of Medicine, Kyoto University kn-affil= affil-num=16 en-affil=Research Center for Computational Science, Institute for Molecular Science, National Institutes of Natural Sciences kn-affil= affil-num=17 en-affil=RIKEN SPring-8 Center kn-affil= affil-num=18 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=94 cd-vols= no-issue=1 article-no= start-page=1 end-page=7 dt-received= dt-revised= dt-accepted= dt-pub-year=2005 dt-pub=20050201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Genetic Analysis, Expression in Eschericia coli of Aconitase from Chemolithotrophic Acidithiobacillus thiooxidans kn-title=化学合成独立栄養細菌 Acidithiobacillus thiooxidans 由来アコニターゼの遺伝子解析と大腸菌での発現 en-subtitle= kn-subtitle= en-abstract= kn-abstract=An aconitase from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The cloned aconitase gene (acn) was expressed in Escherichia coli JM 109; aconitase activity was found in the cell extarct. The acn gene encodes a 646-amino acid polypeptide and is located upstream of the isocitrate dehydrogenase gene (icd). A. thiooxidans aconitase showes high sequence similar to pig heart aconitase and E.coli aconitase B. Twenty-five of twenty-seven active site residues assigned in pig heart aconitase are conserved in A. thiooxidans aconitase. The enzyme was purified by DEAE-Toyopearl 650M column chromatogrophy. The purified enzyme had an optimum pH of 7.5 and an optimum temperature of 60 C. Thermal inactivation studies of the purified enzyme revealed the enzyme activity to be uninfluenced after one hour incubation at 40 c. Enzyme activity was retained 100% after incubation of the enzyme at pH 6.0-9.0 for 60min. The A. thiooxidans aconitase was composed of a single polypeptide chain with a molecular mass of 66 kDa. en-copyright= kn-copyright= en-aut-name=KanaharaYohei en-aut-sei=Kanahara en-aut-mei=Yohei kn-aut-name=金原陽平 kn-aut-sei=金原 kn-aut-mei=陽平 aut-affil-num=1 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=2 ORCID= en-aut-name=TokudaChizuka en-aut-sei=Tokuda en-aut-mei=Chizuka kn-aut-name=徳田千束 kn-aut-sei=徳田 kn-aut-mei=千束 aut-affil-num=3 ORCID= en-aut-name=NakamuraAtsuo en-aut-sei=Nakamura en-aut-mei=Atsuo kn-aut-name=中村淳雄 kn-aut-sei=中村 kn-aut-mei=淳雄 aut-affil-num=4 ORCID= en-aut-name=MatsukawaHirokazu en-aut-sei=Matsukawa en-aut-mei=Hirokazu kn-aut-name=松川寛和 kn-aut-sei=松川 kn-aut-mei=寛和 aut-affil-num=5 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=オリエンタル酵母工業(株) affil-num=4 en-affil= kn-affil=オリエンタル酵母工業(株) affil-num=5 en-affil= kn-affil=オリエンタル酵母工業(株) affil-num=6 en-affil= kn-affil=岡山大学 en-keyword=Aconitase kn-keyword=Aconitase en-keyword=Acidithiobacillus kn-keyword=Acidithiobacillus en-keyword=Isocitrate dehydrogenase kn-keyword=Isocitrate dehydrogenase END start-ver=1.4 cd-journal=joma no-vol=3 cd-vols= no-issue=4 article-no= start-page=e00715-15 end-page=e00715-15 dt-received= dt-revised= dt-accepted= dt-pub-year=2015 dt-pub=20150709 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Draft Genome Sequence of Streptomyces incarnatus NRRL8089, which Produces the Nucleoside Antibiotic Sinefungin en-subtitle= kn-subtitle= en-abstract= kn-abstract=A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD. en-copyright= kn-copyright= en-aut-name=OshimaKenshiro en-aut-sei=Oshima en-aut-mei=Kenshiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=HattoriMasahira en-aut-sei=Hattori en-aut-mei=Masahira kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=ShimizuHitomi en-aut-sei=Shimizu en-aut-mei=Hitomi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=FukudaKoji en-aut-sei=Fukuda en-aut-mei=Koji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=NemotoMichiko en-aut-sei=Nemoto en-aut-mei=Michiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil= kn-affil=The University of Tokyo affil-num=2 en-affil= kn-affil=The University of Tokyo affil-num=3 en-affil= kn-affil=Graduate School of Life and Environmental Sciences, Okayama University affil-num=4 en-affil= kn-affil=Graduate School of Life and Environmental Sciences, Okayama University affil-num=5 en-affil= kn-affil=Graduate School of Life and Environmental Sciences, Okayama University affil-num=6 en-affil= kn-affil=Graduate School of Life and Environmental Sciences, Okayama University affil-num=7 en-affil= kn-affil=Graduate School of Life and Environmental Sciences, Okayama University END start-ver=1.4 cd-journal=joma no-vol=98 cd-vols= no-issue=1 article-no= start-page=1 end-page=7 dt-received= dt-revised= dt-accepted= dt-pub-year=2009 dt-pub=200902 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Expression, Purification and Properties of Alanine Racemase from Thermus thermophillus HB8 kn-title=高度好熱性細菌 Thermus thermophilus HB8 由来アラニンラセマーゼの大腸菌での発現，精製及び諸性質の検討 en-subtitle= kn-subtitle= en-abstract=高度好熱性細菌 Thermus thermophilus HB8 由来アラニンラセマーゼ遺伝子を大腸菌中にクローニングし、発現させた後に、精製及び性質検討を行った。alr遺伝子は1080bpからなり360アミノ酸残基 HB8をコードしていたので、本酵素は38,596Daの分子量であると予想された。alr遺伝子の [ G + C ] 含量は、72%であり、Tm値は98.8℃であった。T.thermophilus HB8由来アラニンセマーゼを中等度好熱性細菌 Geobacillus stearothermophilus 及び赤痢菌 Shigella sonnei 由来アラニンラセマーゼと一次配列の比較をしたところ、G.stearothermophilus 由来のものと33%、赤痢菌由来の酵素を28%の相同性を示した。T.thermophilus HB8 由来アラニンラセマーゼを、70℃で10分間の熱処理後、DEAE-トーヨーパール陰イオン交換カラム等により精製した。精製酵素の最適温度は、d-アラニンからl-アラニンへの反応で55℃、l-アラニンからd-アラニンへの反応では60℃であり、最適pHは、9.0?10.0であった。また、70℃で30分インキュベーションを行った後にも、活性の低下は見受けられず耐熱性を示した。更に、本酵素は分子量38,000モノマー酵素であると推定され、その反応機構に興味が持たれる。 kn-abstract=An alanine racemase (EC 5.1.1.1) from an extreme thermophilic bacterium Thermus thermophilus HB8, was purified and characterized, and its gene was cloned. The cloned alanine racemase gene (alr) was expressed in Escherichia coli JM 109. The alr gene is composed of a 1080 bp and encoded a 360 amino acid, and was predicted to have a molecular weight of 38,596. The enzyme was purified by heat shock at 70°C for 10min and DEAE Toyopearl 650M column chromatography. The purified enzyme had an optimum pH9.0?10.0 and an optimum temperature of 55°C?60°C. Enzyme activity was retained 100% after incubation of the enzyme at 70°C for 10min. Alanine racemase from Thermus thermophilus HB8 is a monomeric enzyme with a molecular mass of 39 kDa. en-copyright= kn-copyright= en-aut-name=YanagitaniMasahiko en-aut-sei=Yanagitani en-aut-mei=Masahiko kn-aut-name=柳谷昌彦 kn-aut-sei=柳谷 kn-aut-mei=昌彦 aut-affil-num=1 ORCID= en-aut-name=UemaeSatoshi en-aut-sei=Uemae en-aut-mei=Satoshi kn-aut-name=上前智 kn-aut-sei=上前 kn-aut-mei=智 aut-affil-num=2 ORCID= en-aut-name=ShiragaTomoyuki en-aut-sei=Shiraga en-aut-mei=Tomoyuki kn-aut-name=白神智行 kn-aut-sei=白神 kn-aut-mei=智行 aut-affil-num=3 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=4 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=5 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 affil-num=2 en-affil= kn-affil=岡山大学 affil-num=3 en-affil= kn-affil=岡山大学 affil-num=4 en-affil= kn-affil=岡山大学 affil-num=5 en-affil= kn-affil=岡山大学 en-keyword=alanine racemase kn-keyword=alanine racemase en-keyword=pyridoxal 5’-phosphate kn-keyword=pyridoxal 5’-phosphate en-keyword=thermostable enzyme kn-keyword=thermostable enzyme en-keyword=Thermus thermophilus HB8 kn-keyword=Thermus thermophilus HB8 END start-ver=1.4 cd-journal=joma no-vol=6 cd-vols= no-issue= article-no= start-page=19742 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2016 dt-pub=20160128 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico en-subtitle= kn-subtitle= en-abstract= kn-abstract=Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni?Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions. en-copyright= kn-copyright= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=TsunekawaNaoki en-aut-sei=Tsunekawa en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NemotoMichiko en-aut-sei=Nemoto en-aut-mei=Michiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=HiranoToshiyuki en-aut-sei=Hirano en-aut-mei=Toshiyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=SatoFumitoshi en-aut-sei=Sato en-aut-mei=Fumitoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil= kn-affil=Graduate School of Environmental and Life Science, Okayama University affil-num=2 en-affil= kn-affil=Institute of Industrial Science, the University of Tokyo affil-num=3 en-affil= kn-affil=Graduate School of Environmental and Life Science, Okayama University affil-num=4 en-affil= kn-affil=Graduate School of Environmental and Life Science, Okayama University affil-num=5 en-affil= kn-affil=Institute of Industrial Science, the University of Tokyo affil-num=6 en-affil= kn-affil=Institute of Industrial Science, the University of Tokyo END start-ver=1.4 cd-journal=joma no-vol=105 cd-vols= no-issue= article-no= start-page=1 end-page=5 dt-received= dt-revised= dt-accepted= dt-pub-year=2016 dt-pub=20160201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Survey on the undernourished university students who tend to lack breakfast ; a proposal for a novel viewpoint for the improvement kn-title=大学生の朝食欠食習慣の統計解析と改善への新指針 en-subtitle= kn-subtitle= en-abstract= kn-abstract=This study investigated the current status and causes underneath the life of university students who tend to lack breakfast at a relatively high frequency, and statistical analysis on consequences leading to such lack of well-nourished eating habitat in their university life. In October 2014, self-assessed questionnaires were administered to over 150 faculty students. It contained questions about breakfast habits, time allowance for the morning class, and lunchtime setting in their high school timetable. Breakfast states were clearly separated in three groups : 68% of students regularly have breakfast throughout the weekdays, 21% students skipping the breakfast occasionally, and 11% student no habit for breakfast at all. The survey on the high school lives revealed that 70% students used to have lunch 30 min later than the lunchtime set in the university timetable, 7% of them had the lunch time even more than 1 h later. Lunchtime varies among high schools, and statistical significance was revealed (p<0.01) that schools with higher deviation scores tend have late lunch beyond 12: 30. Accordingly, university students were given directions to prepare for the timetable reform on postulation of having lunch time over one o’clock. After continuous survey on the breakfast habits during the second semester, more than 90% of students established the habit of breakfast regularly in their university lives with the improved consciousness toward well-balanced healthy breakfast contents for their higher level of education quality. en-copyright= kn-copyright= en-aut-name=TamuraT. en-aut-sei=Tamura en-aut-mei=T. kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=1 ORCID= en-aut-name=IbiT. en-aut-sei=Ibi en-aut-mei=T. kn-aut-name=揖斐隆之 kn-aut-sei=揖斐 kn-aut-mei=隆之 aut-affil-num=2 ORCID= en-aut-name=InagakiK. en-aut-sei=Inagaki en-aut-mei=K. kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=3 ORCID= en-aut-name=KuboY. en-aut-sei=Kubo en-aut-mei=Y. kn-aut-name=久保康隆 kn-aut-sei=久保 kn-aut-mei=康隆 aut-affil-num=4 ORCID= en-aut-name=OkudaK. en-aut-sei=Okuda en-aut-mei=K. kn-aut-name=奥田潔 kn-aut-sei=奥田 kn-aut-mei=潔 aut-affil-num=5 ORCID= affil-num=1 en-affil= kn-affil=岡山大学教務FD 委員会 affil-num=2 en-affil= kn-affil=岡山大学教務FD 委員会 affil-num=3 en-affil= kn-affil=岡山大学教務FD 委員会 affil-num=4 en-affil= kn-affil=岡山大学教務FD 委員会 affil-num=5 en-affil= kn-affil=岡山大学教務FD 委員会 en-keyword=Undernourished students kn-keyword=Undernourished students en-keyword=breakfast kn-keyword=breakfast en-keyword=lunchtime kn-keyword=lunchtime en-keyword=statistical significance kn-keyword=statistical significance END start-ver=1.4 cd-journal=joma no-vol=100 cd-vols= no-issue= article-no= start-page=3 end-page=7 dt-received= dt-revised= dt-accepted= dt-pub-year=2011 dt-pub=20110201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Purification and Characterization of l-Methionine Decarboxylase from Streptomyces sp. 590 kn-title=放線菌Streptomyces sp.590由来l-メチオニン脱炭酸酵素の精製および性質検討 en-subtitle= kn-subtitle= en-abstract= kn-abstract=L-Methionine decarboxylase [EC 4.1.1.57] catalyzes the decarboxylation of L-methionine and is a pyridoxal 5’-phosohate(PLP)-dependent enzyme. L-Methionine decarboxylase has been purified 630-fold by DEAE-Toyopearl 650M, Phenyl-Toyopearl 650M and Sephacryl S-300 column chromatographies from Streptomyces sp.590. The enzyme has a dimeric structure with identical subunits of Mr 60,000. This enzyme shows optimum activity at pH7.0 and 45°C, and is stable between pH5.7 and pH9.0. L-Methionine decarboxylase has antitumor activity against RERF-LC-AI and HeLa cells. Ten N-terminal amino acid sequence of L-methionine decarboxylase was determined, and the sequence showed no homology with other reported proteins. en-copyright= kn-copyright= en-aut-name=MaemuraTomomi en-aut-sei=Maemura en-aut-mei=Tomomi kn-aut-name=前村知美 kn-aut-sei=前村 kn-aut-mei=知美 aut-affil-num=1 ORCID= en-aut-name=UchitomiKumiko en-aut-sei=Uchitomi en-aut-mei=Kumiko kn-aut-name=内富久美子 kn-aut-sei=内富 kn-aut-mei=久美子 aut-affil-num=2 ORCID= en-aut-name=KusakaChika en-aut-sei=Kusaka en-aut-mei=Chika kn-aut-name=日下知香 kn-aut-sei=日下 kn-aut-mei=知香 aut-affil-num=3 ORCID= en-aut-name=InagakiJunko en-aut-sei=Inagaki en-aut-mei=Junko kn-aut-name=稲垣純子 kn-aut-sei=稲垣 kn-aut-mei=純子 aut-affil-num=4 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name=田村隆 kn-aut-sei=田村 kn-aut-mei=隆 aut-affil-num=5 ORCID= en-aut-name=SodaKenji en-aut-sei=Soda en-aut-mei=Kenji kn-aut-name=左右田健次 kn-aut-sei=左右田 kn-aut-mei=健次 aut-affil-num=6 ORCID= en-aut-name=InagakiKenji en-aut-sei=Inagaki en-aut-mei=Kenji kn-aut-name=稲垣賢二 kn-aut-sei=稲垣 kn-aut-mei=賢二 aut-affil-num=7 ORCID= affil-num=1 en-affil= kn-affil=農芸化学コース affil-num=2 en-affil= kn-affil=農芸化学コース affil-num=3 en-affil= kn-affil=農芸化学コース affil-num=4 en-affil= kn-affil=岡山大学大学院医歯薬学総合研究科 affil-num=5 en-affil= kn-affil=農芸化学コース affil-num=6 en-affil= kn-affil=京都大学 affil-num=7 en-affil= kn-affil=農芸化学コース en-keyword=L-methionine decarboxylase kn-keyword=L-methionine decarboxylase en-keyword=pyridoxal 5’-phosohate kn-keyword=pyridoxal 5’-phosohate en-keyword=Streptomyces kn-keyword=Streptomyces en-keyword=decarboxylation of L-methionine kn-keyword=decarboxylation of L-methionine END start-ver=1.4 cd-journal=joma no-vol=25 cd-vols= no-issue=6 article-no= start-page=1208 end-page=1219 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20231210 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation en-subtitle= kn-subtitle= en-abstract= kn-abstract=Nitzschia is one of the largest genera of diatoms found in a range of aquatic environments, from freshwater to seawater. This genus contains evolutionarily and ecologically unique species, such as those that have lost photosynthetic capacity or those that live symbiotically in dinoflagellates. Several Nitzschia species have been used as indicators of water pollution. Recently, Nitzschia species have attracted considerable attention in the field of biotechnology. In this study, a transformation method for the marine pennate diatom Nitzschia sp. strain NIES-4635, isolated from the coastal Seto Inland Sea, was established. Plasmids containing the promoter/terminator of the fucoxanthin chlorophyll a/c binding protein gene (fcp, or Lhcf) derived from Nitzschia palea were constructed and introduced into cells by multi-pulse electroporation, resulting in 500 μg/mL nourseothricin-resistant transformants with transformation frequencies of up to 365 colonies per 108 cells. In addition, when transformation was performed using a new plasmid containing a promoter derived from a diatom-infecting virus upstream of the green fluorescent protein gene (gfp), 44% of the nourseothricin-resistant clones exhibited GFP fluorescence. The integration of the genes introduced into the genomes of the transformants was confirmed by Southern blotting. The Nitzschia transformation method established in this study will enable the transformation this species, thus allowing the functional analysis of genes from the genus Nitzschia, which are important species for environmental and biotechnological development. en-copyright= kn-copyright= en-aut-name=OkadaKoki en-aut-sei=Okada en-aut-mei=Koki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=MorimotoYu en-aut-sei=Morimoto en-aut-mei=Yu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=ShiraishiYukine en-aut-sei=Shiraishi en-aut-mei=Yukine kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MayamaShigeki en-aut-sei=Mayama en-aut-mei=Shigeki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=KadonoTakashi en-aut-sei=Kadono en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=AdachiMasao en-aut-sei=Adachi en-aut-mei=Masao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=IfukuKentaro en-aut-sei=Ifuku en-aut-mei=Kentaro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=NemotoMichiko en-aut-sei=Nemoto en-aut-mei=Michiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Environmental and Life Science, Okayama University kn-affil= affil-num=4 en-affil=Faculty of Environmental, Life, Natural Science and Technology, Okayama University kn-affil= affil-num=5 en-affil=The Advanced Support Center for Science Teachers, Tokyo Gakugei University kn-affil= affil-num=6 en-affil=Faculty of Agriculture and Marine Science, Kochi University kn-affil= affil-num=7 en-affil=Faculty of Agriculture and Marine Science, Kochi University kn-affil= affil-num=8 en-affil=Graduate School of Agriculture, Kyoto University kn-affil= affil-num=9 en-affil=Faculty of Environmental, Life, Natural Science and Technology, Okayama University kn-affil= en-keyword=Diatom kn-keyword=Diatom en-keyword=Genetic transformation kn-keyword=Genetic transformation en-keyword=Nitzschia kn-keyword=Nitzschia en-keyword=Multi-pulse electroporation kn-keyword=Multi-pulse electroporation END