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 (ATATA) were constructed by consensus mutagenesis. Using Consensus Finder (http://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, H210N, N245D, G292D, and I295 V) using site-directed mutagenesis. Compared to that of the wildtype, the thermostability of all six single variants was improved. The H210N variant displayed the largest shift in thermostability, with a 3.3-fold increase in half-life (t(1/2)) at 40 degrees C, and a 4.6 degrees C increase in T-50(10) among the single variants. In addition, the double mutant H210N/I77L displayed an even larger shift with 6.1-fold improvement of t(1/2) at 40 degrees C, and a 6.6 degrees C increase in T-50(10). Furtherly, the H210N/I77L mutation was introduced into the previously engineered thermostable AT-ATA by the introduction of disulfide bonds, employing B-factor and folding free energy (Delta Delta G(fold)) calculations. Our results showed that the combined variant H210N/I77L/M150C-M280C had the largest shift in thermostability, with a 16.6-fold improvement of t(1/2) and a 11.8 degrees C higher T-50(10).