Jul 2026· International Journal of Biological Macromolecules· pp.
153482
· 0 citations· 52 references
Medicine
TL;DR
The rational engineering of an α-L-rhamnosidase (DthRha) to address limitations and enhance its performance for flavonoid production highlights the R783A mutant as a robust and thermally stable biocatalyst with great potential for the sustainable production of bioactive flavonoids.
Abstract
α-L-Rhamnosidases are valuable biocatalysts for the synthesis of bioactive flavonoids, however, their industrial application is often restricted by limited catalytic efficiency and suboptimal thermal stability. This study reports the rational engineering of an α-L-rhamnosidase (DthRha) to address these limitations and enhance its performance for flavonoid production. A library of mutants was constructed via substrate-binding pocket modeling and site-directed mutagenesis. Among them, the R783A variant exhibited a 3.56-fold increase in enzymatic activity relative to the wild-type enzyme (WT), along with remarkable thermal stability, retaining over 92% of its initial activity after 2 h incubation at 60-90 °C， whereas the WT rapidly lost activity above 60 °C. Kinetic assays revealed a 1.46-fold increase in kcat/Km over the WT, coupled with enhanced substrate specificity. Molecular docking and MD simulations suggested that the enhanced catalytic performance of R783A may arise from favorable steric conformation and a more accessible catalytic tunnel. The practical applicability of the R783A was demonstrated at a 50 mL laboratory scale, affording prunin and isoquercitrin in >98% yield with space-time yields of 3.58 and 1.92 g/L/h, respectively. These findings highlight the R783A mutant as a robust and thermally stable biocatalyst with great potential for the sustainable production of bioactive flavonoids.
Fe(II)/α-ketoglutarate (αKG)-dependent halogenases that catalyze site-selective C-H halogenation of free substrates without carrier proteins are attractive biocatalysts for diversifying pharmaceuticals and agrochemicals. However, their application remains limited by the narrow diversity of natural halogenases, poor stability, and restricted substrate scope. Protein stabilization is a common strategy to enhance mutational tolerance during enzyme engineering; however, Fe(II)/αKG-dependent halogenases are structurally closely related to hydroxylases, and consensus-based stabilizing mutations risk shifting activity toward competing hydroxylation. To address this challenge, a workflow was designed to improve structural stability while preserving substrate and product specificity by combining computational identification of substrate-recognition residues with Rosetta-based stabilization. This approach was applied to the l-lysine 4-chlorinase BesD from Streptomyces cattleya as a model enzyme. The resulting variants exhibited a T50 increase of more than 45 °C with no loss of substrate specificity or regioselective chlorination activity and served as stable seed enzymes for subsequent substrate scope expansion. This strategy, which systematically excludes substrate recognition- and/or reaction-selectively related residues from the mutation space to preserve native enzyme function, may provide a versatile platform for stabilizing enzymes without substantially compromising catalytic activity.
Teppei Niide, Keita Miyawaki, Hyuga Miyamoto et al.· ACS Chemical Biology· 0 citations
Rhamnosyltransferases are remarkable biocatalysts for the synthesis of rhamnosylated natural products with valuable physicochemical properties and bioactivities. However, their application is hindered by poor stability and low catalytic efficiency. Here, we achieved simultaneous enhancement of catalytic efficiency and stability of a 1,2-rhamnosyltransferase by a distal mutational engineering strategy. The variant M9 exhibited a 589.43-fold extension in half-life, a 2.5 °C increase in Tm, a 13.6 °C increase in T50, and an 8- to 763-fold increase in activity toward diverse flavonoids compared with the wild type. Molecular dynamics simulations provided insights into enhanced thermostability and catalytic efficiency. To demonstrate its synthetic utility, a whole-cell biocatalytic system was constructed in E. coli by coexpressing M9 and UDP-rhamnose synthase, enabling a neohesperidin titer of 1.14 g L–1 without exogenous sugar donor supplementation. This study presents a practical enzyme engineering strategy for simultaneous activity–stability enhancement in glycosyltransferases and provides a promising biocatalyst for rhamnosylated natural product biosynthesis.
Wenjuan Dai, Chaorong Guo, Hongyan Yang et al.· Journal of Agricultural and...· 0 citations
Background Prunin is scarce in nature and suffers from low extraction efficiency, and industrial-scale production of prunin and L-rhamnose remains technically inadequate. Therefore, developing an α-L-rhamnosidase capable of efficient and specific naringin hydrolysis is of great practical significance. Methods An α-L-rhamnosidase gene from Aspergillus nidulans was expressed recombinantly in E. coli Rosetta (DE3). Expression conditions were optimized, and random mutagenesis coupled with high-throughput screening was performed to obtain a beneficial mutant (R11). Enzyme properties were characterized, and fed-batch fermentation in a 2-L bioreactor was carried out for scale-up. Results The purified recombinant enzyme (120.7 kDa) showed optimal activity at 75 °C and pH 5.0, with a Km of 5.23 mM toward pNPR. A key mutant, R11, exhibited 22.2% higher relative activity than the wild type. After tank fermentation, enzyme activities of both wild-type and R11 were increased approximately two-fold, and the conversion rate of naringin to prunin by R11 was 2.77-fold higher than that of the unoptimized wild-type control. Conclusion The recombinant α-L-rhamnosidase possesses broad temperature adaptability and excellent catalytic properties. This study provides a novel integration of random mutagenesis and bioreactor scale-up for efficient naringin-to-prunin bioconversion, laying a theoretical foundation for industrial production.
Chuncheng Wang, Li Liang, Bei Ni et al.· Frontiers in Microbiology· 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
The 1,4-α-glucan branching enzyme found in Bifidobacterium longum can modify the physicochemical properties of starch by forming denser and shorter branches. To enhance its activity and modification function with regard to pea starch, ten mutants were engineered using site-directed mutagenesis based on multiple sequence alignment, molecular docking, and dynamic simulation. The mutants E395D, S321D and M509L exhibited specific activities 1.32-, 1.23- and 0.81-fold higher than the wild type (WT). The catalytic efficiencies of S321D and E395D were 18.36 and 14.00 mg-1·min-1·mL-1, respectively, which were significantly higher than the WT's efficiency of 11.19 mg-1·min-1·mL-1. Docking analysis revealed that the mutations reorganised the hydrogen-bonding and electrostatic microenvironment at the active site, producing mutation-specific effects on substrate binding and transition-state stabilisation. These locally altered polar and nucleophilic properties ultimately dictated the catalytic efficiency of each mutant. Compared with WT, E395D and S321D increased DP 2 and 3 short branches, reduced FT-IR R₁₀₄₇/₁₀₂₂ and XRD crystallinity at 200-800 U/g starch, which expanded the amorphous region of starch. After seven days' storage at 4 °C, the retrogradation enthalpy of S321D-modified starch was 0.63 J/g lower than that of WT-modified starch of 0.71 J/g. Glucose release from S321D-modified starch was significantly lower than from WT-modified starch at the level of small intestinal α-glucosidase. The slowly digestible fraction of S321D-modified starch was 38.51%, significantly higher than the 29.19% observed in WT-modified starch. Fresh and stored pea cake modified by S321D had significantly lower hardness and higher slowly digestible properties than the WT-modified equivalent.
Dan Li, Yue Su, Yuhang Meng et al.· International Journal of Bio...· 0 citations
Gallic acid is a bioactive polyphenol with poor solubility and stability. Enzymatic α-glucosylation can address these limitations, but regioselective synthesis among multiple phenolic hydroxyls remains challenging. Here, we engineer a sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) for the switchable synthesis of gallic acid 4-O- and 3-O-α-d-glucopyranosides. Structure-guided rational design identifies four key regions controlling substrate orientation. The double mutant I231A/D400E achieves >90% selectivity for the 4-O isomer with 82% yield in 2 h. Strikingly, the mutant L341 V/T157G completely reverses regioselectivity, producing the 3-O isomer with >98% yield and >99% selectivity. Molecular docking reveals that these mutations remodel the active pocket and substrate channel, positioning the target hydroxyl group adjacent to the catalytic center. The resulting α-glucosides show up to 12-fold improved water solubility and superior cytoprotective, anti-inflammatory (IL-1β), and antioxidant (HO-1/NQO1) activities compared to gallic acid. This work provides two high-performance, regiocomplementary biocatalysts for green synthesis of pharmaceutically relevant gallic acid glycosides and offers mechanistic insights into regioselectivity control in sucrose phosphorylases.