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Purification and Molcular Cloning of Sinefungin synthetase fom Streptomyces incarnatus NRRL8089.

Research Project

Project/Area Number 11680636
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionOkayama University

Principal Investigator

INAGAKI Kenji  Okayama University, Faculty of Agriculture, Professor, 農学部, 教授 (80184711)

Co-Investigator(Kenkyū-buntansha) TAMURA Takashi  Okayama University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (40253009)
TANAKA Hidehiko  Okayama University, Faculty of Agriculture, Professor, 農学部, 教授 (90065912)
Project Period (FY) 1999 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
KeywordsSinefungin / Rifampicin resistance / Expression vector / DNA methylase Inhibitor / Drug resistance marker / PCR / 放線菌の二次代謝酵素 / メチル化酵素 / 二次代謝 / 放線菌宿主ベクター系 / DNAメチラーゼ阻害試験 / プロトプラスト / 突然変異
Research Abstract

Sinefungin is a nucleoside antibiotic, in which a molecule of L-omithine is linked to the 5'-end of adenosine through a CC bond. The antibiotic, isolated from the culture broth of Streptomyces incarnatus NRRL 8089, has a strong inhibitory effect on various fungi and trypanosome, and is expected to be useful as an anti-malaria drug as well. It has been reported that the cell-free extract prepared from a variant of incarnatus produced sinefungin from L-arginine and ATP in the presence of pyridoxal-5'-phosphate, but the enzymes involved in sinefungin-production have not been characterized due to the instability and low expression in the producer strain. The present study was undertaken to characterize the enzymes responsible for sinefungin production.
In the present research, we have first established DNA methylse inhibition assay for detecting sinefungin. Then, the sinefungin producer strain underwent mutagenesis by ultraviolet light irradiation on the protoplast, and a high-producer stra … More in was selected among the mutants that acquired rifampicin-resistance. Because the enzyme activity of sinefungin synthetase was still not detected in the crude cell extract, we attempted to clone the genes involved in sinefungin production. The genome DNA was partially digested, ligated to a vector plasmid pKU110, and transformed in Streptomyces lividans TK24. Three transformants (#19, #20, and #21) were screened from over 200 of transformants as the candidate strain that produces sinefungin. The transformants were cultivated in 2 L of the medium, and sinefungin was isolated from the culture broth by means of charcoal column chromatography, cation-exchange column chromatography, and anion-exchange column chromatography. The strain #19 produced sinefungin more than the strains #20 and #21. We attempted to isolate the recombinant plasmids from these sinefungin-producing transformants, but the conventional isolation protocol did not yield plasmid. We examined the presence of the thiostrepton-resistance marker sequence, which is located in the vector plasmid, by polymerase chain reaction, but the corresponding DNA fragment was not amplified. It was concluded that the genes of sinefungin production from S. incarnatus were integrated to the genome DNA of S. lividans. Two references were published in the present research. Less

Report

(4 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • Research Products

    (27 results)

All Other

All Publications (27 results)

  • [Publications] T.Tamura, A.Kataoka, L-Y.Shu, A.Ashida, H.Tanaka, K.Inagaki: "An in vitro screening method for DNA cytosine-C5 methyl's inhibitor"Natural Product Letter. 16(1). 25-27 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Tamura, L-Y.Shu, A.Ashida H.Tanaka, K.Inagaki: "Improvement of sinefungin-producing strain of Streptomyces incarnatus by conferring rifampicin-resistance through ultraviolet light irradiation and protoplast regeneration"Sci.Rep.Fac.Agric.Okayama Univ. 91. 1-5 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] K.Inagaki, N.Ueno, T.Tamura, H.Tanaka: "Purification and characterization of acid trehalase from Acidobacterium capsulatum"J.Biosci.Bioeng.. 91. 141-146 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 田村 隆, 李 銀淑, 片岡 睦, 稲垣賢二, 田中英彦: "DNAシトシンC5メチラーゼ阻害剤のスクリーニング法の確立"生化学. 72. 968 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] J.Yanagitani, T.Tamura, K.Inagaki, K.Soda, H.Tanaka: "Purification and characterization of NAD : penicillamine ADP transferase from Bacillus sphaericus ; a novel NAD-dependent enzyme catalyzing phosphoramide bond formation"J.Biol.Chem.. 274. 795-800 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 片岡 睦, 田村 隆, 芦田顕彦, 稲垣賢二, 田中英彦: "DNAメチラーゼ阻害物質のアッセイ法の確立"日本農芸化学会誌. 73. 352 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Tamura, A. Kataoka, L-Y. Shu, A., Ashida, H. Tanaka and K. Inagaki: "An in vitro screening method for DNA cytosine-C5 methylase inhibitor"Natural Product Letters. 16. 25-27 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Tamura, L-Y. Shu, A. Ashida, H. Tanaka and K. Inagaki: Sci. Rep. Fac. Agric. Okayama Univ.. 91. 1-5 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] K. Inagaki, N. Ueno, T. Tamura and H. Tanaka: J. Biosci. Bioeng.. 91. 141-146 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] J. Yanagitani, T. Tamura, K. Inagaki, K. Soda and H. Tanaka: J. Biol. Chem.. 274. 795-800 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Tamura, A.Kataoka, L-Y.Syu, A.Ashida, H.Tanaka, K.Inagaki: "An in vitro screening method for DNA cytosine-C5 methylase inhibitor"Natural Product Letter. (印刷中). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Tamura, L-Y.Shu, A.Ashida, H.Tanaka, K.Inagaki: "Improvement of sinefungin-producing strain of Streptomyces incarnatus by conferring rifampicin-resistance through ultraviolet light irradiation and protoplast regeneration"Sci. Rep. Fac. Agric. Okayama Uni. 91. 1-5 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] K.Inagaki, N.Ueno, T.Tamura, H.Tanaka: "Purification and characterization of acid trehalase from Acidobacterium capsulatum"J. Biosci. Bioeng. 91(2). 141-146 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Tamura, M.Hasegawa, M.Sugimoto, K.Inagaki, H.Tanaka: "Molecular cloning of thioredoxin reductase isozymes, TrxR1 and TrxR2, from human lung adenocarcinoma cell line, NCI-H441"Biryo-eiyouso-kenkyu. 18. 149-153 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] K.Goto, Y.Tanimoto, T.Tamura, K.Mochida, D.Arai, M.Asahara, M.Suzuki, H.Tanaka, K.Inagaki: "Identifiation of thermoacidophilic bacteria and a new Alicyclobacillus genomic species isolated from acidic enviroments"Extremphiles. 6(印刷中). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] 稲垣賢二: "抗がん酵素メチオニンγ-リアーゼとNAD依存イソプロピルリンゴ酸脱水素酵素の立体構造に基づく機能解析"ビタミン. 76(印刷中). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kawaguchi,H.,K.Inagaki,H.Matsunami,Y.Nakayama,T.Tano,H.Tanaka: "Purification and Characterization of 3-Isopuropylmalate Dehydrpgenase from Thiobacillus thiooxidans"J.Biosci.Bioeng.. 90(4). 459-461 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Inoue,H.,K.Inagaki,N.Adachi,T.Tamura,N.Esaki,K.Soda and H.Tanaka: "Role of Tyrosine 114 of L-Methionine γ-Lyase from Pseudomonas putida"Biosci.Biotech.Biochem. 64・11. 2332-2339 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Ohhira,I.,T.Tamura,H.Kurokawa,H.Nakaue,K.Inagaki,and H.Tanaka: "Purification of anti-Escherichia coli O-157 components produced by Enterococcus faecalis TH10, an isolate from Malaysian fermentation food, Tempeh"Milk Science. 49・2. 81-88 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Seow,T.-K.,K.Imagaki,T.Nakamura,R.Maeda,T.Tamura,and H.Tanaka: "Purification and Some Characteristics of a Monomeric Alanine Racemase from an Extreme thermophile, Thermus thermophilus"J.Biosci.Bioeng. 90・3. 344-346 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tamura,T.,Y.Oki,A.Yoshida,T.Kuriyama,H.Kawakami,H.Inoue,K.Inagaki and H.Tanaka: "Noncompetitive, reversible inhibition of aminoacylase-1 by a series of 1-α-hydroxyl-and 1-α-fluoro-fatty acids ; the ligand specificity of Aspergillus oryzae and porcine kidney enzymes"Archives Biochem.Biophys.. 379・2. 261-266 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Lim,L.-C.,T.Tamura,R.Rahim,M.Ali,H.Ghazaki,K.Inagaki and H.Tanaka: "A colourimetric screening method for microorganisms that have methionine γ-lyase activity"Asia Pacific J.Mol.Biol.Biotech.. 8・1. 95-97 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Motoshima,M.,K.Inagaki,T.Kumasaka,M.Furuichi,H.Inoue,T.Tamura,N.Esaki,K.Soda,N.Tanaka,M.Yamamoto,and H.Tanaka: "Crystal Structure of the Pyridoxal-5'-phosphate Dependent L-methionine γ-lyase from Pseudomonas putida"J.Biochem.,. 128・3. 349-354 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] 片岡 睦、田村 隆、芦田顕彦、稲垣賢二、田中英彦: "DNAメチラーゼ阻害物質のアッセイ法の確立"日本農芸化学会誌. 73(臨時増刊号). 352-352 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 柳谷 純、田村 隆、稲垣賢二、左右田健次、田中英彦: "Purification and Characterization of NAD : Penicillamine ADP Transferase from Bacillus sphaericus ; a Novel NAD-dependent Enzyme Catalyzing Phosphoramide Bond Formation"Journal of Biological Chemistry. 274(2). 795-800 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 田村 隆、大場耕造、稲垣賢二、田中英彦: "Streptomyces cattleyaのモノフルオロ酢酸生合成の研究"生化学. 71(8). 939-939 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 稲垣賢二、岸本憲明、白石竹彦、杉尾 剛、田中英彦、田野達男: "Purification and Properties of an Acid α-Glucosidase from Acidobacterium capsulatum"Journal of Applied Glycoscience. 46. 121-127 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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