Enhancing the solubility and thermostability of nitrilase through surface hydrophobic residue engineering

Nitrilase is an important industrial enzyme that catalyzes the one-step conversion of nitriles to carboxylic acids. Due to its diverse origins, heterologous expression of nitrilase in model microbial chassis often leads to the formation of inactive inclusion bodies, which severely hampers its screening, characterization, and industrial application. However, unlike improvements in activity and stability, engineering for enhanced solubility lacks well-defined quantitative metrics, making rational design challenging. Fusion expression with fluorescent proteins provides an effective means to monitor the spatiotemporal characteristics and yield of target proteins. In this study, we established a screening platform for quantitatively assessing the distribution of nitrilase between soluble and insoluble fractions by fusing it with the green fluorescent protein (sfGFP). Based on this platform, we constructed saturation mutation libraries targeting six solvent-exposed hydrophobic residues on the surface of a previously obtained high-activity mutant PD1, aiming to isolate variants with improved solubility. Four positive single-point mutations (I5S, F25N, V69C, and I201T) were identified, which not only increased nitrilase solubility but also enhanced whole-cell activity and thermostability. Furthermore, by exhaustively exploring all combinatorial patterns of double, triple, and quadruple mutations, we ultimately obtained the optimal triple mutant PD1Mut3 (I5S/V69C/I201T). Compared to PD1, PD1Mut3 demonstrated significantly improved soluble yield, a 5°C higher optimal temperature (45°C), a 1.8-fold longer half-life at 50°C, and superior whole-cell catalytic performance. Molecular dynamics simulations revealed that the introduced mutations reduce structural flexibility, promote a more compact oligomeric assembly, and decrease local solvent exposure, collectively explaining the enhanced thermostability. This work establishes a general integrated strategy combining surface hydrophobic residue engineering and GFP-fusion-based screening to simultaneously improve enzyme solubility and catalytic performance, thereby facilitating further industrial applications.