Abstract: L-threonine dehydrogenase (L-TDH) is one of the key enzymes in L-threonine conversion value-added pathway, which can catalyze the dehydrogenation of L-threonine to synthesize ethyl L-2-aminoacetate, and is the only key enzyme in the synthesis of food flavor 2,5-dimethylpyrazine (2,5-dimethylpyrazine, 2, 5-DMP) with L-threonine as the substrate. In order to improve the catalytic efficiency of this reaction, the highly active L-threonine dehydrogenase from E. coli was first mined by gene mining, and then soluble expression and identification in Escherichia coli BL21 (DE3) by pACYCDuet-1 expression system. The results showed that L-TDH achieved a high level of soluble expression in E. coli BL21 (DE3), and the enzyme activity in the lysate after determination and conversion was 19.13 IU/mL, which was about 79 times the background expression level of E. coli. The purified specific enzyme activity was 12.77 IU/mg, the optimal reaction temperature of the enzyme was 45 °C, the optimal reaction pH was 9.0, and the residual enzyme activity could still reach more than 90% after 120 min at 35-40 °C. In addition, the kinetic parameters of EcTDH are also superior to other reported threonine dehydrogenases, which has a greater advantage in the enzymatic synthesis of L-threonine synthesis of 2,5-DMP. This study laid a solid foundation for the conversion and value-added of L-threonine and the efficient synthesis of 2,5-DMP.

Key words: L-Threonine, L-Threonine dehydrogenase, Escherichia coli, expression, biotransformation