Optimizing acid stability and catalytic activity of Aspergillus flavus uricase (AfUOX) via surface charge engineering and B-factor guided design.

Dual Engineering of the Hydrophobic Core and Functional Loop Reshapes the Conformational Energy Landscape for Significantly Enhanced Xylanase Activity and Thermostability.

The industrial application of enzyme catalysts is often constrained by the trade-off between thermostability and catalytic activity. Here, a region-focused engineering strategy was applied to a thermophilic GH10 xylanase to simultaneously improve both properties. The strategy integrates qProtein-guided hydrophobic cluster design for scaffold stabilization and dynamic loop analysis for active-site optimization. The resulting triple mutant A206S-N209D-F130L exhibited substantially improved thermostability, with a 5.79 °C increase in melting temperature and an 18.8-fold extension of the half-life at 60 °C. Its optimum temperature increased from 60 to 70 °C, accompanied by a 129.4% enhancement in catalytic activity at 70 °C relatively to the wild type. Molecular dynamics simulations indicated that these mutations reshape the conformational energy landscape by stabilizing hydrophobic packing and modulating loop dynamics. This study provides a generalizable framework for simultaneously improving enzyme stability and catalytic performance.

Thermostability modification and simulated application of acidic catalase

Site-directed mutagenesis of α-L-rhamnosidase boosts its deglycosylation activity for the production of bioactive flavonoid glycosides.

The rational engineering of an α-L-rhamnosidase (DthRha) to address limitations and enhance its performance for flavonoid production highlights the R783A mutant as a robust and thermally stable biocatalyst with great potential for the sustainable production of bioactive flavonoids.

Computer-aided multi-shell electrostatic remodeling of BhS7Xyl for enhanced activity and Thermostability.

Xylanases with high catalytic efficiency and environmental robustness are important for lignocellulosic biomass valorization, but many enzymes are rapidly inactivated under the alkaline and high-temperature conditions used in industrial processes. In this study, a computationally guided rational-design strategy was developed to improve the catalytic performance and stability of the alkaline xylanase BhS7Xyl. Constant-pH molecular dynamics, isothermal compressibility perturbation analysis, and ECNet-assisted fitness prediction were integrated to identify alkaline-sensitive and structurally unstable residues for engineering. The triple mutant H51R/D150N/E287K showed the best overall performance, with a specific activity of 1045.29 U/mg, representing a 3.73-fold increase compared with the wild type. Its melting temperature increased from 55.82 °C to 64.58 °C, while its half-life at pH 10.0 increased from 33.96 to 95.84 min. The thermal half-life at 75 °C was extended from 10.97 to 215.42 min, corresponding to a 19.64-fold improvement. Structural analyses suggested that the improved performance of H51R/D150N/E287K was associated with a more continuous xylohexaose-binding interface, increased hydrogen-bonding contacts, additional electrostatic/polar interactions, strengthened local interaction networks and enhanced dissipation of local thermal perturbation. Under optimized hydrolysis conditions, the triple mutant produced higher levels of xylose and xylooligosaccharides from standard xylan, corn cob xylan, and hardwood pulp xylan than the wild type. These work demonstrates that multi-shell electrostatic remodeling is a useful strategy for improving the activity, alkaline tolerance, and thermal stability of xylanase for xylooligosaccharide production.

Engineering of a Carbonic Anhydrase from Hydrogenimonas thermophila Through Fusion Tags and Surface Mutagenesis Enhances Solubility While Revealing Stability–Function Relationships

Protein solubility can limit enzyme performance in industrial applications. This is the case for some carbonic anhydrases (CAs), key enzymes for CO2 capture and utilization. In this study, we investigated an α-class CA from the thermophilic bacterium Hydrogenimonas thermophila (HtCA), which was predominantly expressed as an insoluble protein in Escherichia coli. Surface analysis using Molecular Operating Environment (MOE) revealed extensive hydrophobic regions, suggesting a basis for its poor solubility. To improve solubility, three C-terminal fusion tags were evaluated (Gb1, ng3-NEXT, and T7B9). All tagged variants showed markedly increased soluble expression as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis. To reduce surface hydrophobicity, selected residues were substituted with charged amino acids. Most variants displayed improved solubility, and V136D showed enhanced thermostability, retaining 76% activity after exposure to 90 °C for an hour. However, the F177D variant completely lost all enzymatic activity, highlighting the importance of evaluating both solubility and catalytic function during protein engineering. Molecular dynamics simulations supported the experimental findings, revealing that thermostable variants exhibited reduced structural fluctuations and favorable free-energy landscapes, while the inactive F177D mutant sampled a broader conformational space and higher-energy conformations, consistent with decreased structural stability and loss of catalytic activity.

Enhancing the Robustness of Nitrilase from Betaproteobacteria bacterium by Multistrategy Synergistic Evolution.

Nicotinic acid is vital in pharmaceuticals, feed, food, and cosmetics. Nitrilase can catalyze the conversion of 3-cyanopyridine to nicotinic acid, but its industrial application is limited by poor thermostability and substrate tolerance. In this study, nitrilase from Betaproteobacteria bacterium was engineered via multistrategy synergistic evolution, including C-terminal loop truncation, consensus mutation, loop engineering and protein surface engineering. Through iterative saturation mutagenesis, we obtained mutant 4 M (L194F/A201Q/S208L/M180Q-Δ47). Its activity reached 12.78 U/mL. After 1 h at 50 °C, 4 M retained 10.46 U/mL residual activity─3.25-fold higher than WT. Its melting and aggregation temperatures increased by 8.3 and 6.6 °C, respectively. Molecular dynamics revealed that enhanced thermostability resulted from reduced loop flexibility, strengthened hydrophobic interactions, and new hydrogen bonds. An engineered Vibrio natriegens strain produced 564.3 g/L nicotinic acid, a 5.8-fold increase over WT. This work provides mechanistic insights into nitrilase thermostability.