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.
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
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
Lignocellulosic biomass deconstruction requires robust biocatalysts capable of operating under demanding industrial conditions. The thermophilic fungus Myceliophthorathermophila is a promising biotechnological platform for cellulase production and lignocellulose valorization because of its naturally thermostable CAZyme repertoire. Extensive knowledge has accumulated across diverse research fields, alongside its taxonomic reclassification as Thermothelomyces thermophila, emphasizing the need to integrate findings across research contexts. This review synthesizes knowledge generated using publicly available M. thermophila strains, retaining the historically prevalent name M. thermophila to facilitate cross-disciplinary comparison. Emphasis is placed on the C1 strain, whose longstanding use as a production platform provides a foundation for current advances in strain engineering and protein production. We provide a genetics-focused perspective on this biotechnological platform, integrating advances in genetic engineering, transcriptional regulation, and protein production. We discuss CRISPR systems applied to M. thermophila, strategies to improve protein production through extracellular protease elimination, optimization of secretion and unfolded protein response pathways, carrier proteins, and synthetic expression systems. We further summarize the transcriptional regulation of cellulase expression, the repertoire of characterized thermostable cellulases, and opportunities for protein engineering. Collectively, these advances position M. thermophila as a versatile biotechnological platform for producing industrial enzymes and other value-added bioproducts for biorefinery applications.
J. Ortega, M. Vélez-Mercado, Gabriela Martínez-Machado et al.· Processes· 0 citations
Efficient lignocellulosic biomass conversion under industrially relevant high temperatures is limited by the thermolability of commercial cellulases and a lack of synergistic, system-level thermostable cocktails. To address this gap, a thermostable cellulase cocktail comprising the endoglucanase TpEG, the cellobiohydrolase HmCel6A, and the β-glucosidase TnBglB was established, capable of synergistically hydrolyzing cellulose under high-temperature conditions. To further improve system performance, precise component optimization was performed using an advanced artificial intelligence framework. First, using the protein language model-guided enzyme mining pipeline VenusMine, EG5, an ultra-thermostable endoglucanase, was identified from a large sequence space and exhibited excellent thermostability at 90 °C. Concurrently, protein engineering of HmCel6A guided by the protein language model PRIME generated the variant S347P, yielding a 1.8-fold increase in catalytic activity. Both the wild-type (WT) and engineered cocktails showed excellent high-temperature hydrolytic activity. At 90 °C, DNS assay showed that the filter paper hydrolytic activity of the WT cocktail was approximately sevenfold higher than that of Cellic® CTec3 (Novonesis), while the engineered cocktail exhibited approximately 1.15-fold higher activity than the WT cocktail. HPLC analysis showed that the engineered cocktail released significantly more glucose from corn stover than both CTec3 and the WT cocktail. Furthermore, supplementation of CTec3 with these engineered enzymes led to increased reducing sugar release under the tested supplementation conditions. This study demonstrates the power of integrating artificial intelligence-driven enzyme discovery with protein engineering, expanding the design space for thermostable enzymes while delivering a high-performing cellulase cocktail for industrial-scale, high-temperature biomass saccharification.
Runye Huang, Jin Huang, P. Tan et al.· Bioresource Technology· 0 citations
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.
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.
Jing Tian, Xueting Qu, Wen Huang et al.· Journal of Agricultural and...· 0 citations
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