Nitrilases serve as vital tools in green chemistry, catalyzing the one-step hydrolysis of nitriles to carboxylic acids without the need for harsh chemical reagents. Despite their potential, the industrial deployment of these enzymes is frequently obstructed by low soluble expression in microbial hosts like Escherichia coli. When overexpressed, nitrilases often misfold and aggregate into inactive inclusion bodies, a problem driven largely by surface hydrophobicity. Traditional methods to improve solubility, such as fusion tags or chaperone co-expression, often introduce steric hindrance or complicate the manufacturing process. Addressing this challenge requires a precise engineering approach that enhances solubility without compromising the enzyme’s structural integrity or catalytic power.
In a recent study, researchers from Jiangnan University established a high-throughput screening platform to tackle this issue. The team utilized a fusion strategy with superfolder green fluorescent protein (sfGFP) to visually and quantitatively monitor the distribution of the target nitrilase between soluble and insoluble fractions. This innovative screening method allowed for the rapid identification of beneficial mutations on the enzyme’s surface. By focusing on solvent-exposed hydrophobic residues—key drivers of aggregation—the team constructed saturation mutation libraries to isolate variants with superior solubility profiles.
The screening process yielded four positive single-point mutations, which were further combined to create the optimal triple mutant, PD1Mut3 (I5S/V69C/I201T). This engineered variant demonstrated a marked improvement in performance compared to the parental strain. PD1Mut3 not only achieved a significantly higher soluble yield but also exhibited enhanced thermostability, with an optimal temperature increased by 5 °C and a 1.8-fold longer half-life at 50 °C. Molecular dynamics simulations provided mechanistic insights, revealing that the introduced mutations reduce structural flexibility and promote a more compact oligomeric assembly, thereby minimizing local solvent exposure.
This research highlights the efficacy of surface hydrophobic residue engineering as a dual-purpose strategy to simultaneously improve enzyme solubility and stability. By validating the sfGFP-fusion screening platform, the study provides a scalable and efficient workflow for protein engineers. These findings pave the way for the broader industrial application of nitrilases and offer a valuable reference for the biomanufacturing of other complex enzymes that suffer from aggregation issues.
The work entitled “Enhancing the solubility and thermostability of nitrilase through surface hydrophobic residue engineering” was published in Systems Microbiology and Biomanufacturing (published on April 15, 2026).
DOI:

10.1007/s43393-026-00473-5