Jul 2026· Journal of Agricultural and Food Chemistry· 0 citations· 33 references
Medicine
Abstract
Thermostability is critical for the industrial applications of xylanase, including paper production, animal feed, and lignocellulosic biomass conversion. Here, we report that the rational introduction of surface-exposed glutamic acid (Glu) residues significantly enhances the thermostability of GH10 xylanases. Engineered variants of XT6, BhS7Xyl, and FXYN exhibited prolonged half-lives that were elevated by 2.5-, 1.5-, and 3-fold relative to their respective wild-type enzymes. The stabilization arises from strengthened conformational rigidity due to the formation of numerous new salt bridges. This strategy was further validated in two novel xylanases of Xyn466 and Xyn486 from Cellulomonas bogoriensis 69B4T. In contrast to disulfide bond engineering and ΔΔG-based engineering, surface Glu modification provides superior stabilization (Xyn466-9QE of 5.9-fold and Xyn486-11QE of 9.7-fold increased half-life at 60 °C) with lower mutational load. Our results provide a more efficient strategy with a higher success rate and lower activity trade-off for improving the thermostability of GH10 xylanases.
Deoxynivalenol (DON), a trichothecene mycotoxin commonly found in cereal grains and their derived products, poses significant risks to human and animal health. In previous work, a fusion enzyme composed of the dehydrogenase DADH and the aldo-keto reductase AKR13B3 was engineered to convert DON into the non-toxic 3-epi-DON in a single step. However, the poor thermal stability of this fusion enzyme limited its industrial application. In this study, EVcouplings and the GRAPE-WEB platform were utilized to identify key amino acid residues governing the thermal stability of the fusion enzyme AKR13B3–DADH. Through single-point mutation screening and the combination of beneficial mutation sites, a triple mutant M361L/T508Y/Y603F (M1) was obtained. The half-life of M1 at 50 °C reached about 500 min, representing a 16.4-fold increase compared with the wild type, while its catalytic activity increased by 2.7-fold. The apparent melting temperature increased by approximately 4 °C. Molecular dynamics simulations verified that the improved thermostability results from reduced conformational flexibility in key regions, enhanced structural packing, and a strengthened hydrogen bond network. These results demonstrate the successful development of a thermostable DON-detoxifying fusion enzyme and provide a practical basis for its industrial application.
Yi-Ting Pan, Hao Zhu, Qing-Wei Jiang et al.· International Journal of Mol...· 0 citations
BbAS is established as a thermostable and industrially promising biocatalyst for efficient turanose production through sequence-based analysis and molecular dynamics simulations.
Jeon-Uk Kang, Ye-Jin Kim, Dong-Ho Seo et al.· Journal of Agricultural and...· 0 citations
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.
Lei Zhao, Dianqing Liu, Yi-Min Cai et al.· Bioresource Technology· 0 citations
Xylanases play important roles in lignocellulosic biomass degradation and have broad industrial applications. The discovery of new xylanases expands our understanding of these enzymes and provides new opportunities for industrial applications. This study performed biochemical and structural characterization of the xylanase Xyn11D from Hypocrea virens (HviXyn11D) and evaluated its potential for XOS production from agro-food by-products. HviXyn11D exhibited optimal hydrolytic activity against beechwood xylan at 50 °C and pH 5. Its enzymatic activity was enhanced in the presence of the biomass-derived compounds furfural, 5-hydroxymethylfurfural, and vanillin. The crystal structure of HviXyn11D exhibited a typical β-jelly roll fold and showed a unique substrate-binding cleft compared with homologous xylanases, adopting a closed conformation of the substrate-binding cleft with rigid thumb and finger domains. Molecular dynamics simulations demonstrated that higher temperatures increased the flexibility of the thumb and finger domains, leading to an open conformation of the substrate-binding cleft. Recombinant HviXyn11D enabled the hydrolysis of agro-biomass by-products derived from rice straw, corn cob, and sugarcane bagasse, resulting in successful XOS production. Together, these findings highlight the potential industrial applications of HviXyn11D in biomass degradation and provide new insights into the molecular and structural functions of HviXyn11D and the GH11 xylanase family.
Yena Kim, Bo-Min Jung, Kyeong-Min Kim et al.· International Journal of Bio...· 0 citations
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.· Journal of Agricultural and...· 0 citations
The deacetylase repertoire is expanded and provides a framework for engineering stable industrial enzymes and molecular dynamics simulations revealed that T93P reduces backbone flexibility, C153T enhances β-sheet rigidity via hydrogen bonding, and A244M improves hydrophobic packing by filling a core cavity.
Zechang Sun, Yuxin Xia, Yiran Li et al.· Journal of Agricultural and...· 0 citations
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