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 (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 wild-type， the thermostability of all six single va... More
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 (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 wild-type， 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) at 40 °C， and a 4.6 °C increase in T among the single variants. In addition， the double mutant H210N/I77L displayed an even larger shift with 6.1-fold improvement of t at 40 °C， and a 6.6 °C increase in T. 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 (ΔΔG) 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 and a 11.8 °C higher T.
