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Jul 2026

A Novel Cold-Adapted and Heat-Resistant Alginate Lyase: Unveiling Its Characteristics and Mechanism of Thermostability.

Alginate lyases are important for producing bioactive oligosaccharides and combating Pseudomonas aeruginosa biofilms, demonstrating significant application potential in medicine, food, industry, and other sectors. However, few cold-adapted enzymes with high thermostability exist. Here, we identified a novel alginate lyase, PpAly7A (26.75% sequence identity), which adapts to various pH, temperatures, salt concentrations, metal ions, and surfactants. PpAly7A effectively disrupts P. aeruginosa PAO1 biofilms (36.51% removal) and exhibits exceptional thermostability (half-life 4.39 days at 50 °C). Site-directed mutagenesis and molecular dynamics simulations revealed that synergistic interactions among disulfide bonds, proline residues, salt bridges, and hydrogen bond networks maintain thermostability by enhancing global stability and compactness. Notably, the E180Q mutation further extends the half-life to 7.25 days while retaining most catalytic activity, demonstrating a favorable activity-stability trade-off. This study deepens the understanding of the structure-function relationships of enzymes and provides a viable molecular template for the rational design of thermostable industrial enzymes.

Luyao Tang, Shuhong Lin, Congyu Li et al. · 0 citations
Aug 2026

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.

Chun-Lin Tan, Xin Yu, Lanxi Sun et al. · 0 citations
Aug 2026

Insights into thermostability enhancement of a PL-5 alginate lyase through rational design.

Efficient utilization of natural alginate is restricted by its high viscosity. Alginate lyase degrades alginate via β-elimination to reduce viscosity, supporting its development and application. However, natural alginate lyases exhibit poor thermostability, limiting their application in high-temperature environments. Enhancing the thermostability of natural alginate lyase would better meet the demands of industrial production. We cloned and characterized RwAly5A, a PL-5 family alginate lyase from Ralstonia wenshanensis 56D2. Recombinant RwAly5A exhibited maximal catalytic activity at pH 8.0, with robust tolerance over the pH range of 4.6-10.6. Its maximum activity is observed at 50 °C, and although the protein remains fairly stable at 0-40 °C, its thermostability deteriorates rapidly at temperatures above 50 °C. Additionally, RwAly5A exhibited optimal activity at 100 mM NaCl. Its activity was promoted by a broad spectrum of cations, including monovalent (K+, NH4+, and Li+) and divalent (Ca2+ and Mg2+) ions. In contrast, Mn2+, Ba2+, Fe3+, and EDTA suppressed the enzyme, and SDS almost completely abolished its function. Based on molecular dynamics (MD) simulations, residues predicted to be located in the flexible-loop region were chosen as targets for proline substitution, and the resulting variants were subjected to thermostability mechanism studies. Proline substitutions were introduced in flexible loops, generating D20P, D227P, and A229P mutants, which exhibited half-lives at 50 °C that increased to 1.14‑, 4.69‑, and 2.28‑fold, respectively, while retaining most of the catalytic activity. These findings provide new strategies for improving alginate lyases for commercial applications.

Shuhong Lin, Xin Li, Yiming Tang et al. · 0 citations
Jul 2026

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.

Qi Zhu, Lanxin Xiao, Yijie Sun et al. · 0 citations
Aug 2026

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.

Yu-Yue Li, Dan-Yao Zhou, Qi Wen et al. · 0 citations
Aug 2026

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.

Ziqi Hou, Gen Lu, Tong Shu et al. · 0 citations