Engineering thermostable (R)-selective amine transaminase from Aspergillus terreus through in silico design employing B-factor and folding free energy calculations.

Amine transaminases have recently gained a lot of attention for the synthesis of chiral amines. Using (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) as a transaminase model, in silico design was applied employing B-factor and folding free energy (ΔΔGfold) calculations. Mutation sites were selected by targeting flexible regions with the greatest B-factors, and were substituted with amino acids that were determined by folding free energy calculations (ΔΔGfold < 0) to be more rigid than the original ones. By site-directed mutagenesis, we obtained four stabilized mutants (T130M, T130F, E133F and D134L) with improved stability from 19 candidates. Compared to the wild type, the best single mutant (T130M) showed an increase in thermal stability with a nearly 2.2-fold improvement of half-life (t1/2) at 40 °C and a 3.5 °C higher T1/210 min. The optimum catalytic temperature of T130F was increased by 10 °C. In addition, the T130M/E133F double mutant displayed the largest shift in thermostability with 3.3-fold improvement of t1/2 at 40 °C and a 5.0 °C higher T1/210 min. Modeling analysis showed that new hydrophobic interactions and hydrogen bonds might contribute to the observed thermostability improvement.