*3.2. Production of Recombinant PgMA in E. coli*

To produce the recombinant *Pg*MA, an expression plasmid pETDuet-*Pg*MA was constructed (Figure 2a), and the recombinant *Pg*MA was produced in recombinant *E. coli* BL21 (DE3) and purified as a major band shown by SDS-PAGE with a Ni2+ affinity chromatography (Figure 2b). The purified *Pg*MA showed an estimated 68 kD molecular weight in the SDS-PAGE. The production yield was 18.73 mg/L, and the specific activity of the purified enzyme was determined to be 91.46 U/mg by using β-CD as a substrate at pH 7 and 60 ◦C.

**Figure 2.** Expression of *Pg*MA from *Parageobacillus galactosidasius* DSM 18751T in *Escherichia coli* (DE3). (**a**) Recombinant repression plasmid pETDuet-*Pg*MA. (**b**) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of expressed and purified proteins from recombinant *E. coli* harboring pETDuet-*Pg*MA. Lane 1: molecular marker; lane 2: total protein before induction; lane 3: total protein after 20 h of induction; and lane 4: purified protein.
