分离自苏云金芽胞杆菌的首例细菌乌头酸异构酶TbrA的酶学性质研究

Enzymatic characterization of TbrA, the first identified aconitate isomerase from Bacillus thuringiensis

摘要:

[目的] 乌头酸异构酶（aconitate isomerase，AI）可介导具有多重生物学活性及应用潜力的小分子物质反式乌头酸（trans-aconitic acid，TAA）的合成。本文通过表征来自苏云金芽胞杆菌的生物体首条AI基因（tbrA）的产物——TbrA蛋白的催化性质，填补人们对于AI酶学特性的认识。[方法] 我们利用大肠杆菌Rosetta菌株和Ni²⁺柱亲和纯化获得了His₆-TbrA蛋白，并在体外通过HPLC检测了产物生成及对应酶活。[结果] TbrA蛋白的最适pH、温度与离子强度分别为8.0，37℃和25 mmol/L。TbrA在10℃时仍保留约60%的活性，展现了较好的耐低温特性。金属离子Mg²⁺、Ca²⁺与还原剂DTT可显著增强TbrA活性，而Fe²⁺、Cu²⁺、Zn²⁺、Mn²⁺则强烈抑制TbrA活性。TbrA将顺式乌头酸（cis-aconitic acid，CAA）异构为TAA的正反应K_m、V_max、k_cat、k_cat/K_m值分别为6.25 mmol/L、1.39 μmol/（L·s）、4.08 1/s、0.65 L/（mmol·s），将TAA异构为CAA的逆反应的上述数值分别为71.50 mmol/L、4.17 μmol/（L·s）、12.25 1/s、0.17 L/（mmol·s）。[结论] AI酶蛋白TbrA可以在温和的理化条件下表现出最高活性，且更倾向于合成TAA。本研究定量描述了TbrA催化特性，为其今后应用于TAA工业生物合成奠定基础。

Abstract:

[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 Ni²⁺ 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℃ and 25 mmol/L, respectively. TbrA exhibited a cold tolerance, retaining nearly 60% of its enzymatic activity at 10℃. Notably, TbrA activity was significantly enhanced by Mg²⁺, Ca²⁺ and DTT, but strongly inhibited by Fe²⁺, Cu²⁺, Zn²⁺ and Mn²⁺. K_m, V_max, k_catand 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.