*2.6. Assay of Hydrolysis Activity*

The standard reaction was performed with 1% (*w*/*v*) β-CD, 5.6 μg/mL *Pg*MA, and 50 mM of PB at pH 6 and 60 ◦C for 30 min. After the reaction was stopped by boiling, the amount of reducing sugars produced from each reaction was estimated using the dinitrosalicylic acid method [40]. One unit of MA activity was defined as the amount of the enzyme that released 1 μmol of reducing sugar as maltose per min under the assay condition described earlier. For optimal conditions, the reaction was further performed at different temperatures and pH values, including pH 5 (acetate buffer), pH 6–7 (PB), and pH 8–10 (glycine buffer). Accordingly, the substrate specificity was measured with 1% (*w*/*v*) of the studied sugars, including α-CD, β-CD, γ-CD, soluble starch, and pullulan, performed at pH 7 and 65 ◦C. To realize the effects of metal ions and DMSO on the hydrolysis activity of *Pg*MA, 10 mM of tested metal ion or 5–20% (*v*/*v*) of DMSO was added into the reaction mixture.
