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

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.

Parallel Multidimensional Engineering of Acetylxylan Esterase Enables Concurrent Enhancement of Thermostability and Catalytic Efficiency.

Simultaneously enhancing the thermostability and catalytic activity of acetylxylan esterases (AxEs) remains a significant challenge due to the inherent stability-activity trade-off. Here, a previously uncharacterized CE7 family acetylxylan esterase (TsAxE) from Thermoclostridium stercorarium was identified and engineered through a parallel multidimensional engineering strategy integrating consensus motif engineering, molecular docking, and interface engineering. The best-performing mutant BCF (D218L/D245P/G183Y) exhibited a 44.10 ± 0.44% increase in activity toward p-nitrophenyl acetate (pNPA). Notably, the thermostability of BCF was substantially improved, with the half-life (t1/2) at 60 °C extended from 0.63 ± 0.04 h to 43.82 ± 3.90 h. In addition, BCF showed improved catalytic efficiency toward p-nitrophenyl butyrate (pNPB). Molecular docking and molecular dynamics analyses suggested that these mutations may reshape substrate-binding pocket and improve structural stability. This study demonstrates the effectiveness of integrating complementary engineering strategies for the rational improvement of AxEs activity and thermostability.

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.

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.

Functional Characterization of a Thermostable Amylosucrase from Bifidobacterium boum for Efficient Turanose Production.

Amylosucrase efficiently synthesizes sucrose isomers, including turanose, a low-glycemic functional sweetener. In this study, an amylosucrase from Bifidobacterium boum (BbAS) was identified through sequence-based analysis, which predicted favorable thermostability. Molecular dynamics simulations revealed that the B'-domain residues at the active-site entrance exhibited reduced flexibility in the turanose-bound state compared to the apo form, suggesting that turanose-induced stabilization of the active-site entrance contributes to the enhanced isomerization efficiency of BbAS. Experimental characterization confirmed these computational predictions by demonstrating optimal activity at 50 °C, while maintaining high residual activity during prolonged incubation at 45 °C. Kinetic analysis of BbAS with sucrose further revealed a biphasic non-Michaelian pattern, with a 3-fold increase in kcat,app above 49.1 mM. Notably, fructose supplementation shifted catalysis toward isomerization, achieving a turanose conversion rate of 61.3% while suppressing α-glucan formation to below 3.6%. These findings establish BbAS as a thermostable and industrially promising biocatalyst for efficient turanose production.

[Enhancing the thermostability of Pseudoalteromonas sp. κ-carrageenase through computer-aided rational design].