Engineering Nitrilase for Enhanced Industrial Biocatalysis through Oligomer State Modulation and Active-Site Redesign

Oligomerization is a notable post-translational self-assembly process in proteins. While enzyme oligomerization often enhances stability, its mechanisms remain unclear. Nitrilases are particularly prone to oligomerization, making precise regulation of this process critical for advancing the biochemo catalytic route to gabapentin and for broader applications in pharmaceutical synthesis and green chemistry. Here, we applied computational protein engineering to optimize a nitrilase from Acidovorax facilis (AcN). Using AlphaFold 3 for structural analysis and ESM-DLkcat for activity estimation, we first explored C-terminal engineering as a means to modulate AcN oligomerization, identifying proline content and net charge as important contributors to changes in filament length and stability. While C-terminal modifications primarily improved thermal robustness, they were accompanied by reduced or modestly altered catalytic activity. To overcome this limitation, we subsequently applied FoldX-guided mutagenesis to the β-sheet and active-site regions of the thermally stable AcNΔC30 variant, yielding the optimized mutant M1₅ (F307Y/F168V/T201M/V305L/I320A). M1₅ exhibited a 36. 9% increase in activity and a 6. 8-fold longer half-life (40. 77 h). Molecular dynamics simulations suggest that improved substrate channel accessibility and enhanced structural rigidity contribute to the observed performance gains. Application of similar active-site redesign principles to an alternative variant (M2) resulted in M2₄, which showed substantially increased activity, highlighting the broader utility of this stepwise engineering framework. Notably, M1₅ enabled complete conversion of 1 M substrate 1-cyanocyclohexane acetonitrile under industrial conditions (an increase from 67. 99% to 100%), underscoring its potential for sustainable biomanufacturing.