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

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.