Biochemical characterisation of the 4-α-glucanotransferase from the hyperthermophilic archaeon Pyrobaculum arsenaticum and its formation of high-amylose resistant starch

High-amylose starch (HAS) is gaining attention in biotechnology for its thermal stability, structural resilience and health benefits. Its dense crystalline structure hinders hydrolysis by human gut enzymes, making it a promising source of type 2 resistant starch for hydro-thermal and enzymatic upgrading. 4-α-Glucanotransferases (4αGTs) of glycoside hydrolase family 77 catalyse disproportionation of α-1,4-glucan chains in HAS, enhancing functionality and nutritional properties. Here, a 4αGT, ParGT from the hyperthermophilic archaeon Pyrobaculum arsenaticum, identified in a metagenomic dataset from Pisciarelli hot spring (85 °C, pH 5.5; Naples, Italy), showed highest activity at 100 °C and pH 5.5, and specific activity of maltotriose disproportionation at 75 °C of 1170 U/mg. ParGT effectively modified HAS granules under controlled heating (annealing) at 75 °C, altering crystallinity, surface order and chain length. Comparative analysis of native, heat-treated and ParGT-modified HAS granules from wheat, potato, maize, and barley revealed distinct effects of botanical source, enzymatic modification, and heating. Notably, ParGT increased the resistant starch (RS) contents in wheat and potato HASs subjected to in vitro digestion. Interfacial kinetics correlated the increased resistance to decreased density of glucoamylase attack sites. Overall, ParGT showed strong potential in enzyme- and hydro-thermal modifications developing starch-based ingredients for health and food applications.