Dual-enzyme-induced polymerization for the synthesis of hyperbranched, high-molecular-weight α-glucans

Branching enzymes increase the branching density of starch by cleaving internal α-1,4 linkages and forming new α-1,6 bonds. This effect can be further enhanced by the addition of 4-α-glucanotransferase (4αGT). For the incubations consisting of both Rhodothermus obamensis branching enzyme and Thermus thermophilus 4αGT, optimization of α-1,6 glucosidic bond content is achieved through sequential application, whereas simultaneous application appears ineffective. We investigated the distinction between these two modes of enzyme application in promoting branching to elucidate underlying mechanistic differences. Furthermore, we demonstrate that simultaneous use of both enzymes enhances branching and increases molecular weight. Their concurrent activity promotes continuous, alternating chain elongation and branching, efficiently utilizing the low-molecular-weight oligosaccharides generated during the reaction. Accordingly, consistent with a chain-extension polymerization mechanism, there is a substantial rise in molecular weight. Time-dependent supplementation with exogenous α-glucan oligosaccharides further enhances this effect, yielding up to 80 % more product by weight than when only using the branching enzyme. This dual-enzyme polymerization strategy results in the synthesis of highly branched starches with tunable molecular weights, most effectively achieved when using both enzymes simultaneously.