Functional Characterization of a Thermostable Amylosucrase from Bifidobacterium boum for Efficient Turanose Production.

Characterization of a Novel GH3 β-Glucosidase with Alkali-Tolerant and Halophilic Properties from Pontixanthobacter luteolus SW-109.

Local enrichment of acidic residues on the PlGH3 surface could generate a negative electrostatic potential, which enables adaptation to high-salt and alkaline environments, thereby sustaining the enzyme's catalytic activity under such extreme conditions.

[Enhancing the thermostability of Pseudoalteromonas sp. κ-carrageenase through computer-aided rational design].

Surface Engineering of Glutamic Acid Residues to Enhance the Thermostability of GH10 Xylanases.

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.

Enzymatic Characterization of a Novel GH3 β-Glucosidase from Lentilactobacillus buchneri: EDTA-Mediated Enhancement of Thermostability

This study presents the first characterization of a GH3 β-glucosidase from L. buchneri and reveals a non-classical stabilizing effect of EDTA, offering valuable insights for enzyme engineering and biocatalytic applications.

Site-directed mutagenesis of α-L-rhamnosidase boosts its deglycosylation activity for the production of bioactive flavonoid glycosides.

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.

Rational Thermostabilization of the Fe(II)/α-Ketoglutarate-Dependent Halogenase BesD While Preserving Halogenation Selectivity.

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.