*3.8. Kinetic Constant*

The Michaelis constant (Km) and the maximum velocity (Vmax) of Mal-mPEG5000-SPA and SPA was defined by Lineweaver-Burk plot. Different concentrations of substrates (1–20 mg/mL) in 20 mM disodium-hydrogen phosphate-citric acid buffer pH 5.8 were used and specific activity was assessed by the DNS method. The values of Km and Vmax were calculated respectively based on the double reciprocal plot [25].

#### *3.9. Activation Energy (Ea)*

*Ea* of the enzyme was measured by Arrhenius equation (lnkcat = lnk0 − *Ea*/RT), and the temperature was set from 25 ◦C to 65 ◦C. Representing 1/T in K on the axis of x and natural log of specific activity on the axis of y, Arrhenius plot was generated. *Ea* was defined based on the value of the slope.
