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

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.

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.

Structure-informed engineering of a laccase with enhanced catalytic activity and thermostability for facilitating lignocellulosic biomass saccharification.

Lignocellulose is an abundant renewable feedstock for biofuels and value-added bioproducts, yet its efficient bioconversion is hindered by the recalcitrant lignin barrier. While laccases show great potential for lignin modification and delignification, their limited thermostability restricts their application in high-temperature lignocellulose biorefinery processes. To address this constraint, an integrated computational engineering strategy was employed to rationally improve the thermostability and catalytic performance of a laccase from Bacillus aryabhattai TCCC 11368. The optimal variant, S281E/N387D, obtained through the combination of FireProt, PROSS, and supercharge-based engineering, exhibited enhanced thermal stability and catalytic efficiency. Its half-life at 60 °C increased 3.7-fold to 330  min compared with the wild type, accompanied by a 35.4% improvement in catalytic efficiency (kcat/Km). Structural analysis suggested that the improved performance may result from enhanced hydrogen-bond networks, strengthened electrostatic interactions, improved hydrophobic packing, and optimized substrate-binding interactions, which collectively contribute to its enhanced capability in lignocellulosic biomass saccharification. Under optimized conditions, enzymatic treatment of wheat straw using the S281E/N387D variant combined with cellulase yielded 8.63 mg/mL reducing sugars, representing a 10.86% increase over the wild-type laccase treatment. This study provides an effective computational framework for developing robust laccases and demonstrates their potential for improving lignocellulosic biomass conversion in biorefinery applications.

Thermostability modification and simulated application of acidic catalase

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.

A multi-dimensional engineering strategy integrating computational design, self-cyclization and whole cell biocatalysis for efficient epilactose production

Epilactose is a promising functional disaccharide, but its biomanufacturing is limited by insufficient enzyme activity, poor thermostability, and costly catalyst preparation. We first used the REME platform to computationally evaluate candidate enzymes. Among them, cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) showed the highest epilactose synthesis activity. We therefore developed an integrated strategy combining computational design, SpyTag/SpyCatcher-mediated cyclization, and ethanol-permeabilized whole-cell catalysis. By combining enzyme ligand binding energy analysis, protein stability prediction, and catalytic constant prediction, the V52N variant was obtained. Its epilactose synthesis activity was 3.45 times that of the wild type, while lactulose formation was reduced to 14.8% of the wild-type level. Cyclized CCT increased the optimum temperature to 80 °C and extended the half-life at 85 °C by 5.52-fold. The optimized whole-cell process produced 65.81 g/L epilactose from 200 g/L lactose within 20 min, corresponding to 32.90% conversion. This strategy provides a practical route for efficient epilactose biomanufacturing.