Enzymatic characterization of TbrA, the first identified aconitate isomerase from Bacillus thuringiensis
Enzymatic characterization of TbrA, the first identified aconitate isomerase from Bacillus thuringiensis
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TbrA 的酶学表征，TbrA 是第一个从苏云金芽孢杆菌中鉴定出的乌头酸异构酶
DOI：
10.13343/j.cnki.wsxb.20200175
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Cao Zheng
中科院分区：
文献类型：
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作者：
Wanqian Yan;Xinyuan Mao;Yujun Dai;Lihua Wang;Cuiying Du;Cao Zheng
[客观]辅助异构酶（AI）可以介导反式酸（TAA）的生物合成，这是一种具有许多应用电位的多生物活性小分子。 AI的属性尚不清楚。它具有Ni〜（2+）亲和力色谱。 C和25 mmol/L分别暴露于冷耐受性，几乎将其酶活性保留在10°C下。由Mg〜（2+），Ca〜（2+）和DTT，但受到Fe〜（2+），Cu〜（2+），Zn〜（2+）和Mn〜（2+）的强烈抑制，v_（max），kcat和k_（cat）/k_m，用于tbra正向反应[从顺式 - aconitic酸（CAA）到taa]为6.25 mmol/l，1.39μmol/（l·s），4.08 1/s和s and s和s and s and s and s and和0.65 l/（mmol·s），对于TBRA反应（从TAA到CAA）为71.50 mmol/L，4.17 μmol/（l·S），12.25 1/s和0.17 l/（Mmol·S）分别（结论）AI酶TBRA可以在轻度条件下实现最大的活性，并对TAA形成描述了TBRA蛋白的催化特性，该特性为其在TAA工业生物生产中的潜在应用提供了基础。
[Objective] Aconitate isomerase (AI) can mediate the biosynthesis of trans-aconitic acid (TAA), a multi-bioactive small molecule with many application potentials. Although the first AI gene (tbrA) was just identified in the bacterium Bacillus thuringiensis, the property of AI remains unclear. Here we characterized TbrA to understand the AI catalysis. [Methods] We expressed His6-tagged TbrA protein in Escherichia coli Rosetta strain and purified it with Ni~(2+) affinity chromatography. We characterized TbrA in vitro and applied HPLC to determine product formation and enzyme activity. [Results] The optimal pH, temperature and NaCl ionic strength for TbrA activity were determined to be 8.0, 37 °C and 25 mmol/L, respectively. TbrA exhibited a cold tolerance, retaining nearly 60% of its enzymatic activity at 10 °C. Notably, TbrA activity was significantly enhanced by Mg~(2+), Ca~(2+) and DTT, but strongly inhibited by Fe~(2+), Cu~(2+), Zn~(2+) and Mn~(2+). Km, V_(max), kcat and k_(cat)/K_m for TbrA forward reaction [from cis-aconitic acid (CAA) to TAA] were 6.25 mmol/L, 1.39 μmol/(L·s), 4.08 1/s and 0.65 L/(mmol·s), and for TbrA reverse reaction (from TAA to CAA) were 71.50 mmol/L, 4.17 μmol/(L·s), 12.25 1/s and 0.17 L/(mmol·s), respectively. [Conclusion] The AI enzyme TbrA can achieve maximum activity under mild conditions, and favors for TAA formation. This study quantitatively described the catalytic properties of TbrA protein, which provided the basis for its potential application in TAA industrial bioproduction.