Preparation and characterization of sinefungin synthetase

辛芬净合成酶的制备及表征

• 批准号: 14560069
• 负责人: TANAKA Hidehiko
• 金额: $ 2.3万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560069/
• 关键词: sinefungin; Streptomyces incarnates; anti-malarial activity; PLP enzymes; Capillary electrophoresis; シネフギン; メチラーゼ阻害剤; 異種発現ベクター; ネオマイシン耐性; シャトルベクター

Sinefungin is a nucleoside antibiotic, in which a molecule of L-methionine is linked to the 5'-end of adenosine through a C-C bond. The antibiotic, isolated from the culture broth of Streptomyces incarnates NRRL8089, 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 S. incarnates produced sinefungin from L-arginine and ATP in the presence of pyridoxal-5'-phosphate, but the enzyme(s) involved in sinefungin-production have not been characterized clue to the instability and low expression of the responsible enzymes in the producer strain. In the present study we have constructed a new expression-vector of pTRA502, which derives from pKU110 shuttle vector. The present study was undertaken to identify genes involved in the sinefungin biosynthesis. We have constructed an original cloning vector of pTRA502 by replacing thiostrepton resistant gene with that of neomycin resistant gene of pKU110. Limited digestion of genome DNA was carried out with Sau3AI restrictions enzyme to obtain genetic fragments of about 5-6 kb. Optimal conditions were examined by restriction enzyme concentrations, enzyme concentration and incubation time. There was a problem that the collection of DNA was low in the process to extract DNA. The amount of collection and purity was improved by using the kit of glass beads. Shot-gun cloning is now being carried out with pTRA502 vector. The transformants were examined for the sinefungin producing ability by a capillary electrophoresis.

Sinefungin是一种核苷抗生素，其中L-甲硫氨酸分子通过C-C键连接到腺苷的5 '端。该抗生素是从Streptomyces incarnates NRRL 8089的培养液中分离出来的，对各种真菌和锥虫具有很强的抑制作用，预计也可用作抗疟疾药物。据报道，从S.在存在吡哆醛-5 '-磷酸的情况下，incarnates从L-精氨酸和ATP生产sinefungin，但是涉及sinefungin生产的酶还没有被表征，这是由于生产菌株中负责酶的不稳定性和低表达。本研究从pKU 110穿梭载体出发，构建了一个新的表达载体pTRA 502。本研究旨在鉴定参与西奈芬净生物合成的基因。我们用pKU 110的新霉素抗性基因替换硫链丝菌素抗性基因，构建了原始克隆载体pTRA 502。用Sau 3AI限制性内切酶对基因组DNA进行有限消化，以获得约5-6 kb的遗传片段。通过限制性内切酶浓度、酶浓度和孵育时间考察了最佳条件。在提取DNA的过程中，存在DNA的收集率低的问题。通过使用玻璃珠试剂盒，提高了收集量和纯度。目前正在用pTRA 502载体进行鸟枪克隆。通过毛细管电泳检测转化子的西奈芬净产生能力。

项目成果

Inoue, H., Nishito, A., Eriguchi, S., Tamara, T., Inagaki, K., Tanaka, H.: "Purification and substrate characterization of α-Ketobutyrate decarboxylase from Pseudomonas putida"J.Mol.Catal.B. 23. 265-271 (2003)

Inoue, H.、Nishito, A.、Eriguchi, S.、Tamara, T.、Inagaki, K.、Tanaka, H.：“恶臭假单胞菌 α-酮丁酸脱羧酶的纯化和底物表征”J. Mol。 B.23.265-271 (2003)

Takakura, T., Akita, K., Takimoto, A., Inagaki, K., Esaki, N., Soda, K., Mitsushima, K: "Quantitative measurement of L-methionine γ-lyase in activity in the presence of L-methionine was approached from progress curve analysis."Anal.Biochem.. (in press). (

Takakura, T.、Akita, K.、Takimoto, A.、Inagaki, K.、Esaki, N.、Soda, K.、Mitsushima, K：“在存在以下物质的情况下定量测量 L-蛋氨酸 γ-裂合酶的活性L-蛋氨酸是从进度曲线分析中得出的。“Anal.Biochem..（正在出版）。

Tamura, T., A.Kataoka, Y.S.Li, A.Ashida, H.Tanaka, K.Inagaki: "An in vivo screening method for DNA cytosine 5'-methylase inhibitor"Natural Product Lett. 16. 25-27 (2002)

Tamura, T., A.Kataoka, Y.S.Li, A.Ashida, H.Tanaka, K.Inagaki：“DNA胞嘧啶5-甲基化酶抑制剂的体内筛选方法”天然产物快报。

Tamura, T., L.Y.Shu, H.Tanaka, K.Inagaki: "Improvement of sinefungin producing strain of Streptomyces incarnatus by conferring ritampicin resistance through ultraviolet light irradiation and protoplast regeneration"Sci.Rep.Fac.Agric.Okayama Univ. 91. 1-5

Tamura, T., L.Y.Shu, H.Tanaka, K.Inagaki：“通过紫外线照射和原生质体再生赋予利坦平抗性，改进链霉菌产辛芬净菌株”Sci.Rep.Fac.Agric.冈山大学。

高橋竜也, 田村 隆, 篠原寛明, 日下部均, 田中英彦, 稲垣賢二: "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センシングの応用"岡山大学学術報告. 91. 15-22 (2002)

Tatsuya Takahashi、Takashi Tamura、Hiroaki Shinohara、Hitoshi Kusakabe、Hidehiko Tanaka、Kenji Inagaki：“使用 L-谷氨酸氧化酶的谷氨酸传感器的开发和 GOT/GPT 传感的应用”冈山大学学术报告 91. 15-22 (2002)。