Modulation of starch structure at molecular and supramolecular levels via interlinked hydrolytic and branching activities of Rhodothermus obamensis branching enzyme.

Branching enzymes (BE; EC 2.4.1.18) increase starch branching by forming α-1,6 glucosidic bonds. In many BEs, branching is accompanied by a reduction in weight-average molecular weight (Mw). This decrease arises from intramolecular transglycosylation or hydrolytic side activity, depending on the enzyme origin. However, this aspect is often overlooked and its impact on the overall restructuring mechanism and final α-glucan architecture is often underestimated. For the Rhodothermus obamensis BE (RoBE), hydrolysis occurs at the onset of the reaction. We found that hydrolysis and branching are interconnected: branching begins only after the Mw decreases below ~220 kDa and subsequently stabilizes as the branching proceeds. Hydrolysis is thus not merely a secondary activity, but an essential prerequisite for RoBE Mw-dependent branching. The coupling of RoBE activities enables controlled modulation of glucan architecture by tuning branching density with a limited impact on Mw. This governs polymer behavior via supramolecular organization, affecting chain conformation in solution (hydrodynamic radius and intrinsic viscosity) and potentially, solvent-polymer interactions. These changes translate into altered macroscopic properties, including resistance to amylolytic degradation and structural integrity, highlighting the potential of enzymatic α-glucan tailoring for functional applications.