*3.3. Determination of Hydrolysis Activity by Recombinant PgMA*

To determine the optimal pH and temperature, β-CD was used as a substrate, and the reaction was performed at different pH levels and temperatures. The results showed that the optimal pH and temperature for the *Pg*MA catalytic reaction were pH 7 (Figure 3a) at 65 ◦C (Figure 3b). Moreover, the addition of Mg2+, K+, or ethylenediaminetetraacetic acid did not significantly affect the activity of *Pg*MA; by contrast, DMSO decreased the activity of *Pg*MA (Figure 3c).

**Figure 3.** Effects of pH (**a**), temperature (**b**), and metal ion or dimethyl sulfoxide (DMSO) (**c**) on the hydrolytic activity of *Pg*MA. The reaction was performed with 1% (*w*/*v*) β-cyclodextrin (CD), 5.6 μg/mL of *Pg*MA, and 50 mM of a different buffer at the tested temperature in the absence or presence of the tested metal ion or DMSO for 30 min. After the reaction was stopped by boiling, the hydrolytic activity of *Pg*MA was determined by measuring the reducing sugars produced from the reaction as described in Section 2.

One of the specific features of MA is that the enzyme prefers CD as its substrate over other polysaccharides, such as starch or pullulan. To characterize the *Pg*MA hydrolysis activity, different polysaccharides were used as a substrate for the *Pg*MA catalytic reaction. The results showed that *Pg*MA exhibited almost equally high specific activities toward α-CD, γ-CD, and β-CD. The specific activities of *Pg*MA toward CD were 65- and 650-fold higher than those toward pullulan and starch, respectively (Figure 4). This CD preference was consistent with other known MAs [20–27]. The 130 residues at the N-terminal of the MA are key for the enzymes to form dimers and largely increase hydrolysis activities toward CD [18,19]. In addition, some recombinant MAs have been purified using Nterminal His-tag fusion [18–27]. The N-terminal His-tag fusion did not seem to affect its dimerization; herein, the recombinant *Pg*MA also remained as the CD preference.

**Figure 4.** Substrate specificity of the hydrolytic activity of *Pg*MA. The reaction was performed with 1% (*w*/*v*) of the tested sugar substrate, 5.6 μg/mL *Pg*MA, and 50 mM of PB at pH 7 and 65 ◦C for 30 min. The hydrolytic activity of *Pg*MA was determined as described in the legend of Figure 3.
