Construction of super-stable enzymes : distinct reversibility of structure and its mechanism

超稳定酶的构建：独特的结构可逆性及其机制

• 批准号: 16580280
• 负责人: TOKUNAGA Masao
• 金额: $ 2.53万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16580280/
• 关键词: halophilic enzyme; halophilic bacteria; nucleoside dinhosnhate kinase; chimeric protein; solubility; 好塩性; 酸性アミノ酸; 変異; サブユニット; 蛋白質; 高次構造形成; フォールディング; キメラ蛋白質

(1) We have succeeded in the expression of haloarchaeal nucleoside diphosphate in Escherichia coil and X-ray structure analysis. The structure of main chain of polypeptide is quite similar to non-halophilic counterpart, indicating elegant distribution of side chains of acidic amino acid residues.(2) We have cloned nucleoside diphosphate kinase gene from moderately halophilic bacteria and expressed it in E. coil. We found for the first time it forms dimeric structure.(3) We have succeeded in the construction, expression, purification and characterization of chimeric nucleoside diphosphate kinase molecules from the enzymes isolated from Halomonas and Pseudomonas, which is halophilic and non-halophilic bacteria, respectively.Consequently, the landmark of halophilic protein is found to be(1) higher optimum salt concentration for enzymatic activity(2) higher stability at higher salt environment(3) highly reversible characteristics due to its high solubility(4) slow mobility on SDS-polyacrylamide gel electrophoresis.

(1)成功地在大肠杆菌中表达了嗜盐古菌核苷二磷酸，并对其进行了X射线结构分析。多肽的主链结构与非嗜盐性多肽的主链结构非常相似，表明酸性氨基酸残基的侧链分布优雅。(2)我们从中度嗜盐菌中克隆了核苷二磷酸激酶基因，并在大肠杆菌中进行了表达。(3)成功构建、表达、纯化和鉴定了嗜盐单胞菌和非嗜盐假单胞菌的嵌合核苷二磷酸激酶分子，发现嗜盐蛋白具有以下特点：(1)较高的酶活性最适盐浓度；(2)较高盐环境下的稳定性；(3)高度可逆性；(4)SDS-PAGE迁移率低。

项目成果

融合タンパク質発現ベクター

融合蛋白表达载体

• 发表时间: 2005

Dimeric structure of nucleoside diphosphate kinase from moderately halophhilic bacterium:contrast to the tetrameric Pseudomonas counterpart

中度嗜盐细菌核苷二磷酸激酶的二聚结构：与四聚假单胞菌对应物的对比

• 发表时间: 2007
• 期刊: FEMS Microbiol.Lett. 268
• 作者: Y.Yonezawa;K.Izutsu;H.Tokunaga;K.Maeda;T.Arakawa;and M.Tokunaga
• 通讯作者: and M.Tokunaga

Electrostatic and hydrophobic interactions play a major role in the stability and refolding of halophilic proteins

• DOI: 10.2174/0929866043478220
• 发表时间: 2004-04-01
• 期刊: PROTEIN AND PEPTIDE LETTERS
• 影响因子: 1.6
• 作者: Arakawa, T;Tokunaga, M
• 通讯作者: Tokunaga, M

Residue 134 detrmines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

残基 134 决定中度嗜盐细菌核苷二磷酸激酶的二聚体-四聚体组装

• 发表时间: 2008
• 期刊: FEBS Lett 582
• 作者: Tokunaga H.;Ishibashi;M.;Arisaka;F.;Arai;S.;Kuroki;R.;Arakawa;T.;Tokunaga;M.
• 通讯作者: M.

Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium : contrast to the tetrameric Pseudomonas counterpart

中度嗜盐细菌核苷二磷酸激酶的二聚结构：与四聚假单胞菌对应物的对比

• 发表时间: 2007
• 期刊: FEMS Microbiol. Lett 268
• 作者: Y. Yonezawa;K. Izutsu;H. Tokunaga;H. Maeda;T. Arakawa;M. Tokunaga
• 通讯作者: M. Tokunaga