Enhanced thermostability and structural integrity of dextransucrase via oxidized dextran-mediated modification.

Abstract

Dextransucrase DsrD5 synthesizes low-molecular-weight dextran (~5 kDa) but has limited industrial utility because of poor thermal and pH stability. Here, DsrD5 was modified with oxidized dextran (ODX) to improve enzyme robustness while retaining the principal low-molecular-weight product profile. Ammonium sulfate enrichment was assessed by specific activity and SDS-PAGE, and ODX modification conditions were optimized by varying dextran molecular weight, oxidation degree, reaction pH, ODX-solution-to-enzyme-solution ratio, temperature, reaction time, and post-coupling reagent. The optimized condition used 150 kDa ODX prepared at 50% theoretical oxidation, followed by 0.5 mol·L-1 glycine post-treatment, pH 7.5, 30 °C, 12 h, and an ODX-solution-to-enzyme-solution volume ratio of 8:1. FTIR, UV-Vis spectroscopy, and dynamic light scattering provided physicochemical evidence consistent with ODX-mediated modification, whereas molecular dynamics (MD) simulations explored possible pose-dependent structural effects of polymer tethering. Compared with native DsrD5, the modified enzyme showed improved thermal and pH stability, a 10 °C increase in optimum temperature, and a 304-fold longer half-life at 40 °C. Although apparent Km increased and apparent Vmax decreased, GPC analysis of the recovered polysaccharide fraction showed that the principal product remained centered near 5 kDa. Thermal-inactivation analysis showed a higher activation energy and higher ΔH and ΔG values after modification. MD simulations further suggested pose-dependent stabilization, with the Lys1089-linked model showing reduced RMSD, stronger internal hydrogen bonding, lower local flexibility, and extensive polymer-protein contacts. These results support an enzyme-specific soluble polymer-mediated strategy for improving DsrD5 stability while retaining its principal low-molecular-weight dextran product profile.