Enhancing thermostability of a psychrophilic alpha-amylase by the structural energy optimization in the trajectories of molecular dynamics simulations.

The cold-adapted alpha-amylase (PHA) from Pseudoalteromonas haloplanktis is a psychrophilic enzyme which demonstrates high activity at low temperatures, but poor thermostability. Most of the method only employed the crystal structure to design the target protein. However, the trajectory of protein molecular dynamics (MD) simulation contained clues about the protein stability. In this study, we combined MD simulation and energy optimization methods to design mutations located at non-conserved residues. Two single point mutants (S255K, S340P) and one integrated mutant (S255K/S340P) enhanced thermostability without affecting the optimal catalytic activity. After incubation at 40 °C for 80 min, the residual activities of mutants S255K, S340P and S255K/S340P were 1.6-, 2.4-, and 2.6-fold greater than that of the wild type (WT). Additionally, the catalytic efficiency values (kcat/Km) of S255K, S340P, and S255K/S340P also increased 1.9-, 2.0-, and 2.7-fold when compared to WT.