Jul 2026· Journal of Agricultural and Food Chemistry· 0 citations· 45 references
Medicine
Abstract
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.
d-Allulose is a promising low-calorie rare sweetener; however, its industrial production is limited by the insufficient catalytic activity and thermostability of d-allulose 3-epimerases (DAEases). In this study, a combinatorial engineering strategy integrating lid-domain modulation and computational design was developed to improve enzyme performance. Lid engineering identified a key mutation (N120D) associated with enhanced catalytic turnover, while computational screening identified beneficial mutations (T69S, D71E, and S128A). The best mutant, T69S/D71E/N120D/S128A, exhibited a 1.9-fold increase in specific activity, a 2.2-fold improvement in catalytic efficiency, and a 34.1% longer half-life at 60 °C. It produced 152 g/L d-allulose from 500 g/L d-fructose within 3 h, achieving a 30.4% conversion yield under mildly acidic and high-temperature conditions. Mechanistic analyses suggested that improved performance resulted from the combinatorial effects of enhanced structural rigidity and altered lid-domain dynamics, consistent with improved catalytic turnover while maintaining thermostability. This work provides an efficient biocatalyst and a generalizable protein engineering strategy.
Qi-Chen Liu, Xinrui Tang, Wenjing Sun et al.· Journal of Agricultural and...· 0 citations
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.
Zhaoran Li, Zhixin Dou, Sha Zhao et al.· Journal of Agricultural and...· 0 citations
L-asparaginase (ASNase) is used both for treating acute lymphoblastic leukemia and as a processing aid to hydrolyze L-asparagine prior to heating, thereby suppressing acrylamide formation in foods and certain traditional Chinese medicinal materials. Industrial pretreatment, however, typically involves high temperature, alkaline conditions, and complex ionic environments, where many enzyme preparations rapidly lose activity, and gains in thermostability often come at the expense of catalytic efficiency. Here, we developed Evolutionary Dynamic Guided Engineering (EDGE), an evolution-and-dynamics-informed strategy that integrates phylogenetic reconstruction with residue-level conformational flexibility profiling to define a target rigidity-flexibility architecture and to select an ancestral starting scaffold. Using EDGE, we performed ancestral sequence reconstruction along the hyperthermophilic branch and identified an inferred ancestral enzyme, designated Anc-ASNase, as the baseline scaffold for engineering. Furthermore, we implemented a two-stage computational pipeline screening, selecting ten single-point candidates for experimental validation. The triple mutant (S282P/A117P/D250M) exhibited a specific activity of 4892.71 U/mg (3.29-fold higher than Anc-ASNase) and a prolonged half-life of 141.6 min at 85 °C. Molecular dynamics simulations indicated that these mutations enhance hydrophobic packing and stabilize interaction networks, suppressing nonproductive fluctuations while retaining functional conformations required for catalysis. Under harsh processing conditions (100 °C, 30 min), the engineered enzyme reduced acrylamide by 70.2% in French fries and by 57.0% in herbal material samples, and it remained tolerant under alkaline conditions and in the presence of several metal ions. Together, these findings support dynamics-guided engineering as a practical route to develop hyperthermophilic ASNase for high-temperature processing and acrylamide mitigation.
Linyu Luo, Shengyu Zhang, Jingbo Zhou et al.· International Journal of Bio...· 0 citations
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.
Yuan Li, Yuwei Ma, Tongli Li et al.· Journal of Agricultural and...· 0 citations
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.· International Journal of Bio...· 0 citations
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.· Journal of Agricultural and...· 0 citations
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