Abstract: In this study, coevolutionary residue analysis was used to identify key functional sites affecting the robustness of C57E10 (derived from Nit6803), an enzyme that efficiently catalyzes the production of nicotinic acid from 3-cyanopyridine. Saturation combinatorial mutations were designed directly on the coevolutionary residues and the potential thermostability-enhanced mutations were evaluated by the change of folding free energy (ddG). Two mutants, K44Y and Q120L/A166L, were screened out, which maintained high catalytic activity with significantly enhanced thermostability, and the half-life in C57E10 at 50 ℃ was increased from 15.1 min to 34.5 min and 21.4 min, respectively. However, the further combination of K44Y and Q120L/A166L resulted in a dramatic decrease in thermostability, revealing strong negative epistasis. Structural analysis showed that K44Y enhanced its hydrophobic interaction with I274, whereas Q120L/A166L established denser hydrophobic interaction networks, thereby improving the structural stability. In this study, the nitrilase with high thermostability was obtained, and the strategy based on combination of coevolutionary residue analysis and mutation calculations also provided an applicable reference for the rational design of other enzymes.

Key words: nitrilases, coevolutionary residues, rational design, thermostability, molecular dynamics simulation