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

Rational Design of Flexible Loops Adjacent to the Active Site Enhances the Catalytic Efficiency of Bacillus β-Mannanase.

This work rationally engineered flexible loops near the active site via molecular dynamics, NMsim, NMsim, sequence conservation analysis, and MAESTRO prediction, and obtained the optimal double mutant M1, which showed 1.54-fold higher specific activity and nearly 2-fold improved pH stability at pH 8, with a nearly doubled kcat while retaining substrate affinity.

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.

Interface-Targeted Rational Design Strategies for Enhancing the Thermostability of d-Allulose 3-Epimerase.

d-Allulose 3-epimerase (DAEase) catalyzes d-fructose conversion to d-allulose, but the poor thermostability of Clostridium cellulolyticum H10 DAEase limits its industrial application. Here, we enhanced DAEase thermostability by targeting the subunit interface using PROSS-guided combinatorial engineering and spatial clustering. Candidate mutations were classified into interface core, interface-adjacent, and distal regions, followed by stepwise iterative combination. Two mutants, M5 and M6, retained WT-like activity but showed markedly improved thermostability. The Tm values of M5 and M6 increased by 11.4 and 12.4 °C, respectively, while their half-lives at 65 °C increased 3-fold and 12-fold. Structural analysis indicated that interface mutations promoted salt-bridge reconstruction, distal mutations stabilized monomers, and interface-adjacent mutations optimized the assembly microenvironment. This spatially coordinated strategy provides an effective approach for engineering thermostable multimeric enzymes.

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

Uricase with improved acid stability is desirable for biomedical and biotechnological applications, yet enhancing the intrinsic acid tolerance of the enzyme while maintaining high catalytic activity remains a challenge. Here, we employed an integrated rational design strategy combining surface charge optimization and B-factor-guided engineering, followed by iterative combinatorial mutagenesis, to engineer urate oxidase from Aspergillus flavus. The final combinatorial variants retained 67% and 64% of their initial activity after 60 min of incubation at pH 4.5, respectively-substantially higher than the 25% retained by the wild-type enzyme-while also exhibiting enhanced specific activities. Mechanistic analyses combining biophysical characterization, molecular dynamics simulations, residue interaction network analysis, and electrostatic calculations suggested that the enhanced acid tolerance may be associated with surface charge redistribution and strengthened van der Waals interaction networks, which may help alleviate electrostatic-repulsion-driven conformational changes and contribute to conformational stabilization. The enhanced catalytic performance may be linked to improved substrate binding and restructuring of the substrate channel. These variants and the underlying design logic illustrate a practical approach to engineering acid-resistant uricase and other pH-sensitive oligomeric enzymes.

Multidimensional Computational Engineering of Pectate Lyase for Enhanced Thermostability and Moderately Improved Catalytic Efficiency.

This study provides a practical strategy for engineering thermostable pectate lyases with improved catalytic performance by developing a multidimensional consensus computational framework integrating sequence conservation, structural dynamics, and thermodynamic prediction to identify functional mutation hotspots in PcPel1834.

Thermostability modification and simulated application of acidic catalase