3.1.4. Lipase Activity Assay

The activity titer of free lipase was found to be 18.32 U/mg/min, while the activity titer of Fe3O4\_PDA\_Lipase was found to be 17.83 U/mg/min. Lipase loading of 0.336 g (84.2% of used enzyme) was achieved by using polydopamine functionalized Fe3O4. The results showed that polydopamine gave high lipase loading and lipase activity because the complex formed by the combination of polydopamine and Fe3O4 nanoparticles was found to be efficient regarding the immobilization of lipase providing wider surface during the reactions, hence gave higher conversion rate in a short period of time [31].

#### 3.1.5. Effect of pH on Nano-Biocatalyst Activity

The impact of pH on the activities of free and immobilized lipase was studied to find the optimum pH of enzyme activity. The effect of pH in the range of 5 to 10 was studied (Figure 5), and it was found that maximum lipase activity was exhibited at pH 7.0 for free lipase (i.e., 18.32 U/mg/min taken as 100% for free lipase) while lipase immobilized on Fe3O4 nanoparticles gave maximum activity at pH 8 (i.e., 17.83 U/mg/min taken as 100% for immobilized lipase). Above and below this pH, lipase activity was reduced. The results showed that, the relationship curve between pH and activity of lipase immobilized on Fe3O4 nanoparticles was shifted towards the right, which demonstrate that due to lipase immobilization on functionalized magnetic nanoparticles, the ability of lipase tolerance increased even if the pH of the reaction mixture varied. This means that by the immobilization of lipase, its adaptability to a wide pH range increased as compared to free lipase. Our studies were in accordance with the studies of Baharfar and Mahajer, in which magnetic nanoparticles were used for the immobilization of lipase, and immobilized lipase gave maximum activity at increased pH as compared to free lipase [31].

**Figure 5.** Effect of pH on the activities of free and immobilized lipase (date points are the mean of triplicate reaction values).

#### 3.1.6. Effect of Temperature on Activity of Nano-Biocatalyst

The impact of temperature on the activity of free and immobilized lipase was studied in the range of 25 to 50 ◦C (Figure 6) and it was depicted that maximum activity of free lipase was obtained at 37 ◦C, while the lipase immobilized on Fe3O4 nanoparticles showed maximum activity at the temperature of 40 ◦C, which means that lipase immobilized on nanoparticles was tolerant to high temperature fluctuations and was stable at a wider temperature range. The covalent bonds formed during immobilization may have increased the stability and tolerability of the lipase. Similar studies were reported by other researchers [23,31].

**Figure 6.** Effect of temperature on the activities of free and immobilized lipase (date points are the mean of triplicate reaction values).
