Biochemical characterisation of aconitase from Corynebacterium glutamicum
Biochemical characterisation of aconitase from Corynebacterium glutamicum
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DOI：
10.1016/j.jbiotec.2010.07.002
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发表时间：
2011-07-10
影响因子：
4.1
通讯作者：
Bott, Michael
中科院分区：
文献类型：
--
作者：
Baumgart, Meike;Bott, Michael
在这项工作中，乌头酸酶的生物技术相关的微生物谷氨酸棒杆菌的特点。葡萄糖生长的细胞提取物中乌头酸酶的比活性通过四种不同的测定法测定。在其中三个顺乌头酸的形成或消失进行了测量，而在第四次测定的乌头酸酶反应与异柠檬酸脱氢酶。以顺乌头酸盐为底物测定的活性最高（0.433 +/- 0.054 Umg（-1）），以柠檬酸盐为底物测定的活性最低（0.134 +/- 0.026 Umg（-1））。只有后一种测定法涵盖了完整的乌头酸酶反应，因此给出了体内活性的最相关信息。为测定动力学常数，乌头酸酶heterogously过度生产，纯化，再活化和生化特性。大小排阻色谱表明蛋白质是单体。该酶显示Michaelis-Menten动力学，柠檬酸的Km值为480 +/- 200 μ M，异柠檬酸的Km值为552 +/- 302 μ M，顺乌头酸的Km值为18.5 +/- 3.4 μ M。顺乌头酸水合的Vmax最高，为40.6Umg（-1）。酶在很宽的pH和温度范围内有活性，在pH 7.5和7.75之间以及在约50 ℃下具有最大活性。(C)2010 Elsevier BV保留所有权利。
In this work, aconitase of the biotechnologically relevant microorganism Corynebacterium glutamicum was characterised. The specific activity of aconitase in extracts of glucose-grown cells was determined by four different assays. In three of them the formation or disappearance of cis-aconitate was measured, whereas in the fourth assay the aconitase reaction was coupled with isocitrate dehydrogenase. The highest activity was determined with cis-aconitate as substrate (0.433 +/- 0.054 Umg(-1)) and the lowest one with the coupled assay and citrate as substrate (0.134 +/- 0.026 Umg(-1)). Only the latter assay covers the complete aconitase reaction and thus gives the most relevant information on in vivo activity. For the determination of kinetic constants, aconitase was heterologously overproduced, purified, reactivated and biochemically characterised. Size exclusion chromatography indicated that the protein is monomeric. The enzyme showed Michaelis-Menten kinetics with K-m values of 480 +/- 200 mu M for citrate, 552 +/- 302 mu M for isocitrate and 18.5 +/- 3.4 mu M for cis-aconitate. The highest V-max was observed for the hydration of cis-aconitate with 40.6Umg(-1). Aconitase was active over a wide pH and temperature range with maximal activity between pH 7.5 and 7.75 and at about 50 degrees C. (C) 2010 Elsevier B.V. All rights reserved.