Function and structure of GH 13_31 α‐glucosidase with high α‐(1→4)‐glucosidic linkage specificity and transglucosylation activity

α‐Glucosidase hydrolyzes α‐glucosides and transfers α‐glucosyl residues to an acceptor through transglucosylation. In this study, GH 13_31 α‐glucosidase Bsp AG 13_31A with high transglucosylation activity is reported in Bacillus sp. AHU 2216 and biochemically and structurally characterized. This enzyme is specific to α‐(1→4)‐glucosidic linkage as substrates and transglucosylation products. Maltose is the most preferred substrate. Crystal structures of Bsp AG 13_31A wild‐type for the substrate‐free form and inactive acid/base mutant E256Q in complexes with maltooligosaccharides were solved at 1.6–2.5 Å resolution. Bsp AG 13_31A has a catalytic domain folded by an (β/α) 8 ‐barrel. In subsite +1, Ala200 and His203 on β→α loop 4 and Asn258 on β→α loop 5 are involved in the recognition of maltooligosaccharides. Structural basis for specificity of GH 13_31 enzymes to α‐(1→4)‐glucosidic linkage is first described.