N-Ethylmaleimide-resistant acyl-coenzyme A oxidase from Arthrobacter ureafaciens NBRC 12140:: Molecular cloning, gene expression and characterization of the recombinant enzyme
N-Ethylmaleimide-resistant acyl-coenzyme A oxidase from Arthrobacter ureafaciens NBRC 12140:: Molecular cloning, gene expression and characterization of the recombinant enzyme
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DOI：
10.1016/j.bbapap.2006.10.008
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发表时间：
2007-01-01
影响因子：
3.2
通讯作者：
Kajiyama, Naoki
中科院分区：
文献类型：
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作者：
Bakke, Mikio;Setoyama, Chiaki;Kajiyama, Naoki
N-乙基马来酰亚胺 (NEM) 抗性酰基辅酶 A 氧化酶 (ACO) 一直是游离脂肪酸 (FFA) 测定的理想选择。为了满足这一需求，我们从Arthrobacter ureafaciens NBRC 12140中制备了重组ACO。该基因的编码区为2109，编码703个氨基酸的蛋白质，预测分子量为76.5 kDa。大肠杆菌中的异源表达水平比天然菌株高520倍。纯化的酶在 10 mM NEM 存在下于 37°C 孵育 4 小时后保留了超过 60% 的活性，而其他市售 ACO 在相同的 NEM 处理后仅显示出不到 10% 的活性。我们推测这是由于 A. ureafaciens 的 ACO 中仅存在三个半胱氨酸。定点诱变研究和对其他相关 ACO 三维结构的仔细审查表明，这些半胱氨酸被埋藏在蛋白质中，并且与 NEM 不发生反应。重组酶用于比色测定游离脂肪酸，得到线性校准。 (c) 2006 Elsevier B.V. 保留所有权利。
N-Ethylmaleimide (NEM)-resistant acyl-coenzyme A oxidase (ACO) has been desired for the determination of free fatty acids (FFAs). In order to meet this demand, we prepared recombinant ACO from Arthrobacter ureafaciens NBRC 12140. The coding region of the gene was 2109, encoding a protein of 703 amino acids with a predicted molecular mass of 76.5 kDa. The heterologous expression level in Escherichia coli was 520-fold higher than that in the native strain. The purified enzyme retained more than 60% activity after incubation in the presence of 10 mM NEM at 37 degrees C for 4 h, while other commercially available ACOs showed only less than 10% activities after the same NEM treatment. We presume that this is due to the presence of only three cysteines in ACO from A. ureafaciens. Site-directed mutagenesis studies and close scrutiny of the three-dimensional structures of other related ACOs Suggested that these cysteines were buried in the protein and unreactive to NEM. The recombinant enzyme was used for the colorimetric determination of free fatty acid, which gave a linear calibration. (c) 2006 Elsevier B.V. All rights reserved.