Key aromatic residues at subsites + 2 and + 3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates

► Phe236 in the N-loop of SBG is involved in longer-maltooligosaccharide specificity. ► Mutation of Phe236 lowered subsite affinities at + 2 and + 3. ► Giving the N-loop of ANG a Phe residue somewhat altered substrate specificity. ► The Phe residue in the N-loop of ANG raised the subsite affinity at + 3. ► Phe residue in the N-loop implicates the chain-length specificity in GH31AGs.