Jul 2026· Journal of Agricultural and Food Chemistry· 0 citations· 35 references
Medicine
Abstract
d-Tagatose is a rare hexose sugar with excellent properties, and its synthesis catalyzed by d-tagatose 4-epimerase (T4E) represents a competitive novel pathway. In this study, EbT4E derived from the Eubacteriales bacterium was screened and systematically characterized. By reshaping the microenvironment of the active pocket, mutant M3(S131D/H410W/T279S) was constructed, which showed a 3.89-fold higher conversion rate compared with the wild-type (WT) enzyme. Kinetic parameter analysis and molecular dynamics (MD) simulations revealed that M3 had enhanced substrate affinity, hydrogen bond network, charge properties, and channel accessibility. Finally, the conversion rates of d-fructose to d-tagatose catalyzed by the purified M3 enzyme and M3 whole-cell catalysts reached 29.46% and 26.2%, respectively. Additionally, the dual-enzyme cascade reaction of M3 with glucose isomerase (GI) TEGI-M-L38M-V137L was constructed, achieving a 13.16% yield of d-tagatose from d-glucose. This study demonstrates that EbT4E-M3 is a promising biocatalyst for d-tagatose production, laying the foundation for its subsequent industrial application.
Mogroside VI (Mog VI) is a rare triterpene glycoside from Siraitia grosvenorii with promising bioactivities. However, its biosynthesis is limited by a single rate-limiting glycosylation step converting mogroside V, catalyzed by the inherently low-activity plant glycosyltransferase UGT73-327-2. In this study, we applied a structure-guided engineering strategy to overcome this catalytic bottleneck. By combining substrate-channel expansion with catalytic pocket remodeling, the double mutant W192F/K206E was generated, showing a 22.2-fold increase in catalytic activity. Molecular dynamics simulations and kinetic analyses indicated that the enhanced performance results from an enlarged substrate-access channel, improved substrate-binding stability, and a more favorable active-site geometry that reduces key catalytic distances. Furthermore, coupling the engineered UGT with Arabidopsis thaliana sucrose synthase enabled an in situ UDP-glucose regeneration system, achieving a Mog VI titer of 5.6 g·L-1 with a 76.8% molar conversion. This work establishes an efficient biocatalytic route for Mog VI production and highlights the potential of structure-based glycosyltransferase engineering for the synthesis of rare natural glycosides.
Dong Guo, Yan Zhang, Xupeng Guo et al.· Journal of Agricultural and...· 0 citations
This study establishes a robust chassis for sustainable DPA production and provides a generalizable framework for engineering other valuable biochemicals.
Qing Yang, Tao-Shun Zhou, Bo Zhang et al.· Biotechnology and Bioenginee...· 0 citations
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, Xin-Rui Tang, Wen-Jing Sun et al.· Journal of Agricultural and...· 0 citations
Lignocellulosic biomass is an abundant renewable resource that is rich in aldohexoses and aldopentoses. Efficient biocatalysts for aldopentose oxidation remain limited, and few enzymes exhibit high catalytic efficiency toward both aldohexoses and aldopentoses. Here, a naturally promiscuous pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) from Pseudomonas fragi NL20W was selected as an engineering template and tailored by semirational design to expand aldopentose oxidation while preserving native d-glucose activity. The V706P/L723 M mutant exhibited markedly enhanced catalytic efficiency toward d-xylose and l-arabinose, reaching 8.0- and 2.4-fold those of the wild-type, respectively, while its efficiency toward d-glucose was 1.3-fold that of the wild-type. Molecular simulations suggested that the improved catalysis was associated with an optimized substrate positioning and enhanced conformational flexibility of the active-site region. This study highlights the significance of natural enzyme scaffold selection in substrate scope engineering and expands the application potential of PQQ-GDH in the sustainable valorization of lignocellulosic sugars.
Yue-Hua Chen, Peng Liu, Binbin Sheng et al.· Journal of Agricultural and...· 0 citations
Isobutene is an important platform chemical that is still predominantly produced from petroleum-derived feedstocks. The ferulic acid decarboxylase (Fdc) catalyzed decarboxylation of 3-methylcrotonic acid provides a green and sustainable route to bio-based isobutene. However, the poor activity of native Fdc toward this non-natural substrate severely limits its practical application. Here, we developed an integrated enzyme-engineering strategy that combines sequence-cluster mining with distal-site engineering to improve catalytic performance. The engineered triple-site mutant V132C/Y298F/S484A (3MUT) exhibited 5.3-fold the isobutene production of WT. Coupling this mutant with reaction-process optimization and cofactor engineering increased the isobutene yield to 67.9%, representing the highest reported yield to date. Mechanistic studies revealed that distal-site mutations in 3MUT reshaped the active site by disrupting the M293-mediated hydrogen bond that blocks the substrate to active site, then further promoting favorable hydrogen-bond interactions with the substrate via R183 and E292, respectively. These findings establish an efficient route for sustainable bio-based isobutene production and demonstrate the potential of distal-site engineering for improving UbiD-family decarboxylases.
Ting Feng, Xuanyu Cao, Li-Ran Yang et al.· Bioresource Technology· 0 citations
: Glucose dehydrogenase (GDH) from Bacillus megaterium IWG3 is a NAD(P)⁺ -dependent oxidoreductase widely used in biosensing and biocatalytic NADPH regeneration. However, its intrinsic preference for NAD⁺ over NADP⁺ limits its application in NADPH -driven processes. Here, we report a structure-guided rational design to invert the coenzyme specificity of GDH by targeting a single residue within the conserved GXXXGXG motif of the Rossmann fold. Molecular docking and structural analysis identified Thr17 as the key residue forming a hydrogen bond with the 2′ - hydroxyl of NAD⁺, thereby discriminating against the 2′ -phosphate of NADP⁺. Three -point mutants—T17G, T17K, and T17R—were constructed, expressed, and kinetically characterized. The T17G mutation dramatically inverted cofactor preference, increasing the catalytic efficiency ratio (NADP⁺/NAD⁺) from 0.78 (wild -type) to 7.5, driven by a 2.4-fold decrease for NADP⁺ and a 4.6 - fold increase in for NAD⁺. Remarkably, the T17K mutant not only shifted preference toward NADP⁺ (specificity ratio 0.96) but also enhanced turnover numbers for both coenzymes by up to 5.2-fold, achieving c atalytic efficiencies of 6.39 mM⁻¹·s⁻¹ (NAD⁺) and 6.15 mM⁻¹·s⁻¹ (NADP⁺) —the highest among all variants tested. In contrast, the T17R mutation severely impaired NADP⁺ binding ( k m = 97.18 mM) and abolished activity. Structural modeling revealed that glycine c reates space to accommodate the 2′ -phosphate, while lysine establishes a favorable electrostatic interaction with the phosphate group; arginine’s bulky guanidinium group causes steric clash. This study demonstrates that a single, rationally designed mutation at position 17 can simultaneously broaden cofactor specificity and improve catalytic efficiency, with the T17K mutant emerging as a superior biocatalyst for NADPH regeneration. The strategy provides a generalizable framework for engineering cofactor preference in short-chain dehydrogenase/reductase family enzymes.
Y. Shen, Keju Jing· International Journal of Fro...· 0 citations
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