Expression in Escherichia coli of an Unnamed Protein Gene from Aspergillus oryzae RIB40 and Cofactor Analyses of the Gene Product as Formate Oxidase
Expression in Escherichia coli of an Unnamed Protein Gene from Aspergillus oryzae RIB40 and Cofactor Analyses of the Gene Product as Formate Oxidase
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DOI：
10.1271/bbb.90497
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发表时间：
2009-12
期刊：
影响因子：
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通讯作者：
Y. Maeda;D. Doubayashi;M. Oki;Hiroaki Nose;A. Sakurai;K. Isa;Y. Fujii;H. Uchida
中科院分区：
文献类型：
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作者：
Y. Maeda;D. Doubayashi;M. Oki;Hiroaki Nose;A. Sakurai;K. Isa;Y. Fujii;H. Uchida
在大肠杆菌中以C-His 6-标记形式产生了曲霉RIB 40的未命名蛋白质（登录号XP_001727378），其氨基酸序列显示出与由德巴利酵母Vanjiriae MH 201产生的甲酸氧化酶同种型的氨基酸序列高度相似。该基因产物，通过亲和柱层析纯化，催化甲酸氧化产生过氧化氢，但没有表现出对其他底物的活性的证据。在30 °C和pH 4.5下的Km和Vmax值分别为7.9 mM和26.3 μmole/min mg。纯化的酶显示出普通黄素蛋白的非典型紫外可见光谱。该酶的紫外-可见光谱和通过煮沸纯化的酶获得的提取物的紫外-可见光谱、荧光光谱和质谱表明，该酶具有非共价结合的FAD类似物，其预期为8-甲酰基-FAD。
An unnamed protein of Aspergillus oryzae RIB40 (accession no. XP_001727378), the amino acid sequence of which shows high similarity to those of formate oxidase isoforms produced by Debaryomyces vanjiriae MH201, was produced in Escherichia coli in C-His6-tagged form. The gene product, purified by affinity column chromatography, catalyzed the oxidation of formate to yield hydrogen peroxide but showed no evidence of activity on the other substrates tested. The K m and V max values at 30 °C at pH 4.5 were 7.9 mM and 26.3 μmole/min mg respectively. The purified enzyme showed UV-visible spectra atypical of ordinary flavoproteins. The UV-visible spectra of the enzyme and the UV-visible spectra, fluorescence spectra, and mass spectrometry of the extract obtained by boiling the purified enzyme suggested that the enzyme has a non-covalently bound FAD analog, which is expected to be 8-formyl-FAD.