Computational thermostability engineering of a nitrile hydratase using synergetic energy and correlated configuration for redesigning enzymes (SECURE) strategy

Nitrile hydratase (NHase), an excellent biocatalyst, has been widely used for the production of amides, but the exothermic hydration reaction leads to its rapid inactivation, hindering its industrial applications, which requires the thermostability of NHase to be enhanced. In this study, employing NHase from Bordetella petrii DSM 12804 (NHAB) as the object, a computational strategy using synergetic energy and correlated configuration for redesigning enzymes (SECURE) was proposed to prune the reasonable mutant library and assemble effective single mutations, thus maximizing the thermostability of NHAB. Among the mutants, the best variant, A6M/B4M, combined six mutations in its & alpha;-subunit (S30T, A71D, A74D, A78R, S81T, and A133P) and four mutations in its & beta;-subunit (L25F, G27Y, N59P, and A173N), showing an increase in Tm by 13.2 & DEG;C, an 866.0-fold prolonged half-life at 50 & DEG;C, and an 11.2% increase in activity. Then, the catalytic efficiency dramatically increased to 249.5 g L-1 acrylamide in 5 batches compared with that of 166.5 g L-1 by the wild type in 3 batches. Finally, the synergistic effect on the mutant represented by an overall change in structure and newly formed intermolecular interactions through dynamic simulations accounted for the enhanced thermostability. Thus, SECURE was demonstrated to be practical for the redesign of multimeric NHase, which also provided guidance for computational thermostability engineering of other industrial biocatalysts. A computational strategy using synergetic energy and correlated configuration for redesigning enzymes (SECURE) is proposed for the thermostability engineering of multimeric proteins.