Improving thermostability of (R)-selective amine transaminase from Aspergillus terreus by evolutionary coupling saturation mutagenesis

As a class of efficient and industrially-desired biocatalysts, amine transaminases can catalyze asymmetric amination of ketones for production of chiral amines. However, one of the main factors hampering the application of amine transaminase is the low storage and operational stability. To further enhance the thermostability of mutant L118T of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), three strongly interacting residues (F115, L181, W184) with L118 site in the co-evolving subnetwork were identified as the evolutionary mutational hotspots. After screening 600 colonies by saturation mutagenesis, a double mutant F115L-L118T was found to exhibit increased values of the temperature for 50 % enzymatic activity after 10-min heating (T-50(10)), half-life (t(1/2)) at 40 degrees C, the melting temperature (T-m), and urea concentration (C-1/2(Urea)) for 50 % unfolding, showing higher thermostability. All-atom molecular dynamics simulations reveal that the mutations reduce the overall flexibility of the AT-ATA, which may have the stabilizing effects on the double mutant F115L-L118T. For asymmetric amination of aromatic ketones with halogenated substitutions, the evolutionary coupling mutation introducing F115L-L118T had almost no effect on R-enantioselectivity of AT-ATA with excellent e. e. values (>99 %).

Automated summary

The study identified three strongly interacting residues as evolutionary mutational hotspots to enhance the thermostability of (R)-selective amine transaminase, resulting in a double mutant with increased stability. Molecular dynamics simulations showed that the mutations reduced the overall flexibility of the enzyme. The evolutionary coupling mutation had minimal effect on the enantioselectivity of the enzyme in asymmetric amination reactions with halogenated substitutions.