Engineering dual α-1,3/α-1,6 glycosidic bonds in starch via a novel maltotriosyl transferase for enhanced slow digestion and stability

Designing starch with tailored digestibility and stability remains a challenge, as achieving multi-faceted structural modifications typically requires complex multi-enzyme processes. To address this, we report on a recombinant maltotriosyl transferase (MTase) from Aeribacillus pallidus , efficiently expressed in a food-grade host and exhibited robust catalytic activity. This enzyme serves as a dual-specificity biocatalyst, uniquely enabling the concurrent introduction of both α-1,3 (17%) and α-1,6 (33%) glycosidic linkages into waxy corn starch in a single, streamlined step. This catalytic action triggered a profound multi-scale structural transformation, leading to a short-chain, hyper-branched polymer (DP ≤13 increased to 76.9%), a marked reduction in molecular weight, and the reassembly of fragments into a dense lamellar network as visualized by cryo-SEM. These changes synergistically enhanced functional properties: the contents of slowly digestible starch (SDS) and resistant starch (RS) increased by approximately 129% and 54%, respectively, while the modified starch also achieved high cold-water solubility (90.01%) and exceptional freeze-thaw stability (water release <20% after 25 cycles). This study establishes a direct correlation between the engineered hybrid-branched (α-1,3/α-1,6), short-chain architecture and the concurrent improvement in nutritional and physicochemical properties of starch.