Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.
Keywords: amine transaminase; differential scanning fluorimetry (DSF); mutual information (MI); saturation mutagenesis; thermostability.