The Small Cycloamylose (CA15) Synthesizing Properties of 4-α-Glucanotransferase from Hyperthermophilic Archaeon Pyrobaculum arsenaticum with Its Distinct Disproportionation Activity

4-α-Glucanotransferase (4-α-GTase, EC 2.4.1.25) facilitates the transfer of α-1,4-linked glucan to another acceptor molecule. This enzyme is widely used during starch modification to produce unique materials, such as thermoreversible gel and cyclic glucan. Because most industrial processing of starch is conducted at elevated temperatures, hyperthermophilic enzymes have received considerable attention. However, only a few of the 4-α-GTases in the glycoside hydrolase family 77 have been isolated from hyperthermophilic archaea. Here, we report for the first time the cycloamylose-forming properties of an archaeal 4-α-GTase (ParGT) isolated from Pyrobaculum arsenaticum. ParGT exhibited optimal activity at pH 6.0 and 95 °C. In particular, ParGT can synthesize small cycloamyloses (CA15-18) with unique disproportionation patterns based on its low transglycosylation activity. Structural modeling with long-chain maltooligosaccharides revealed distinct amino acid residues at the acceptor and second acarbose-binding sites of ParGT. Mutations at Y322 and P231 at the acceptor binding site reduced the disproportionation activity for long-chain maltooligosaccharides, whereas E55 at the second acarbose-binding site influenced the cycloamylose size by affecting the positioning of the 460s loop. These findings provide valuable insights into the structural features and catalytic properties of hyperthermophilic archaeal 4-α-GTase, enabling future modifications of enzymes to improve their capacity to alter starch in diverse biotechnological processes.