Improving thermostability of (R)-selective amine transaminase fromAspergillus terreusthrough introduction of disulfide bonds

To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design (DbD) and Modelling of Disulfide Bonds in Proteins (MODIP) to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilised mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild-type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3.1-fold, and 3.6-fold increase in half-life (t1/2) at 40°C, and a 4.6°C, and 5.1°C increase in T5010. In addition, the combination of mutant R131C-D134C and M150C-M280C displayed the largest shift in thermostability with a 4.6-fold increase in t1/2 at 40°C and a 5.5°C increase in T5010. Molecular dynamics (MD) simulation indicated that mutations of N25C-A28C and M150C-M280C lowered the overall root mean square deviation (RMSD) for the overall residues at elevated temperature, and consequently increased the protein rigidity. The stabilised mutation of R131C-D134C was in the regions of high mobility and on the protein surface, and the disulfide bond constraints the flexibility of loop 121–136. This article is protected by copyright. All rights reserved