High hydrostatic pressure-induced modulation of regioselectivity in amylosucrase from Deinococcus geothermalis

High hydrostatic pressure (HHP) profoundly modulates enzymatic reaction coordinates. This study investigates the pressure-induced functional redirection of Deinococcus geothermalis amylosucrase (DgAS), a biocatalyst capable of producing functional carbohydrates such as sucrose isomers and α-glucans from sucrose. Reactions were performed under hydrostatic pressures ranging from 0.1 to 200 MPa. While DgAS synthesizes long-chain α-glucans at atmospheric pressure, elevating pressure to 200 MPa drastically shifted catalysis toward isomerization, favoring trehalulose. Analysis of pressure-dependence revealed a positive apparent activation volume for polymerization (ΔV^‡_poly > 0), contrasting with a negative value for isomerization (ΔV^‡_iso < 0). Consequently, the biosynthesis of long-chain glucans was suppressed, while the formation of short-chain oligosaccharides was enhanced. Molecular dynamics simulations revealed that HHP induces a "physical capping" effect by increasing gating loop flexibility and compressing the active site volume. This steric restriction hinders the accommodation of bulky growing chains, thereby redirecting catalytic flux toward smaller isomers. These findings highlight HHP as a non-genetic tool to tailor enzyme selectivity for functional food applications.