Computer-aided design for enhancing the tolerance of ω-amine transaminase against methanol as a cosolvent

A significant challenge hindering the application of enzymes in industrial biotechnology is their inadequate stability during processing. Employing organic solvents rather than aqueous systems for enzymatic catalysis presents several key benefits. However, excessive organic solvents as cosolvent that added to reaction system often inactivate enzymes. Exploiting highly active and methanol-resistant ω-amine transaminase from Aspergillus terreus (AtATA) is of great significance for (R)-chiral amines production. Herein, a computer-aided directed evolution method was employed for selecting the potential mutation sites, and the "best" combinatorial variant M4 (AtATA-T23I/E117V/T200G/Q294K) was acquired with higher catalytic activity and methanol tolerance than wild type of AtATA (WT). The catalytic activity and half-life (t_1/2) in 30 % (v/v) methanol of M4 was 19.0- and 3.5-fold higher than that of WT. Molecular dynamics simulations demonstrated that enhanced methanol resistance primarily stemmed from reduced methanol occupancy within the substrate-binding pocket. The decrease of total solvent accessibility surface area and the increase of substrate-binding pocket hydrophobicity in M4 were also the main reason for improving the methanol resistance and activity compared to WT. Moreover, in the 50-mL scale experiment, the conversion of 30 mM substrate was increased from 29.1 % to 76.7 % with a high (R)-(+)-1(1-naphthyl)-ethylamine e.e._p of >99 %.