*3.6. Characterization of the Purified rPsLeuDH*

The optimal temperature of the purified rPsLeuDH was determined with the standard assay at temperatures from 0 ◦C to 60 ◦C. To evaluate the thermostability, the purified enzyme was incubated at three different temperatures (30, 40, and 50 ◦C) for 120 min, and the residual activity was measured by the standard enzyme assays. The optimal pH of the purified enzyme was determined at 30 ◦C using Citric acid/Na2HPO4 buffer (0.2 M) and NH4Cl-NH4OH buffer (0.2 M) for pH ranges 4.0–8.0 and 8.0–10.0, respectively. To assess pH stability, the rPsLeuDH was pretreated at pH 4.0–11.0 in the absence of substrate at 30 ◦C for 30 min, and the residual activity was measured by the standard enzyme assays. The purified rPsLeuDH was incubated at 0–3.0 M NaCl at 30 ◦C for 30 min, and remaining activity was assayed with the standard enzyme assays. The effects of different reagents on the rPsLeuDH activity were assayed with the standard enzyme assay after pre-incubating enzyme in different metal ions at 30 ◦C for 30 min. Enzyme activity assayed without any reagent was defined as control (100%).

#### *3.7. Kinetic Parameter of the rPsLeuDH*

To assess the kinetics parameters, the Lineweaver-Burk plot method was used to calculate the *K*<sup>m</sup> and *V*<sup>m</sup> of rPsLeuDH [31]. The kinetic constants of NADH (0.025 mM–0.4 mM), L-leucine (0.05 mM–2 mM), L-tyrosine (0.05 mM–2 mM), L-proline (0.05 mM–2 mM), DL-methionine (0.05 mM–2 mM), L-arginine (0.05 mM–2 mM), TMP (0.05 mM–2 mM), and NAD<sup>+</sup> (0.025 mM–0.4 mM) were determined by the above method in rPsLeuDH.
