Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis.

Amine transaminases are a class of efficient and industrially-desired biocatalysts for the production of chiral amines. In this study, stabilized variants of the (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) were constructed by consensus mutagenesis. Using Consensus Finder (https://cbs-kazlab.oit.umn.edu/), six positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these six residues were individually mutated to match the consensus sequence (I77 L, Q97E, H210 N, N245D, G292D, and I295 V) using site-directed mutagenesis. Compared to that of the wild-type, the thermostability of all six single variants was improved. The H210 N variant displayed the largest shift in thermostability, with a 3.3-fold increase in half-life (t1/2 ) at 40 °C, and a 4.6 °C increase in T50 10 among the single variants. In addition, the double mutant H210 N/I77 L displayed an even larger shift with 6.1-fold improvement of t1/2 at 40 °C, and a 6.6 °C increase in T50 10 . Furtherly, the H210 N/I77 L mutation was introduced into the previously engineered thermostable AT-ATA by the introduction of disulfide bonds, employing B-factor and folding free energy (ΔΔGfold ) calculations. Our results showed that the combined variant H210 N/I77 L/M150C-M280C had the largest shift in thermostability, with a 16.6-fold improvement of t1/2 and a 11.8 °C higher T50 10 .