*2.1. Preparation of Nanoparticles*

Fe3O4 nanoparticles were prepared/synthesized by solvothermal method by Guoet et al. [22] with some modifications. One gram of FeCl3·6H2O was added in 20 mL of ethylene glycol and stirred to ge<sup>t</sup> a clear solution, then 3 g of sodium acetate and 10 mL of ethylene diamine were added in the solution, which was stirred for 30 min at room temperature. The mixture was then enclosed in a 50 mL Teflon lined autoclave and it was heated in oven for 8 h at 180 ◦C. The black colour product obtained was washed with successive distilled water/ethanol rinses followed by product drying using a desiccator. Black magnetic powder was obtained after drying.

#### *2.2. Grafting of Dopamine on Fe3O4 Nanoparticles*

Dopamine was coated on nanoparticles using "graft from" technique [23]. 0.1 g of Fe3O4 nanoparticles were suspended in 20 mL of distilled water. Afterwards, 20 mL of 20 milli molar tris-HCl bu ffer having pH 8.5 was introduced in the suspension. Then, 0.1 g of dopamine hydrochloride was slowly added in the suspension (2 mg/15 s) and the suspension was stirred for 1 h. Nanoparticles coated with polydopamine were formed by the self-polymerization of dopamine in alkaline conditions.

#### *2.3. Characterization of Nanoparticles and PDA-Nanoparticles Complex*


#### *2.4. Lipase Immobilization on Modified Nanoparticles*

Lipase was produced from *Aspergillus terreus* AH-F2 and purified using the method described in previous work of our research group [24]. Into 40 mL of phosphate bu ffer with pH 7, 0.40 g of lipase was added. The suspension of polydopamine coated magnetic nanoparticles was added slowly to the mixture of lipase with vigorous stirring for 3 h at 4 ◦C. The nucleophilic groups present in lipase such as thiol and amines reacted with the residual catechole and quinone groups present at the surface of Fe3O4-PDA nanoparticles by Schi ff base and Michael addition mechanisms and were immobilized without the need of any coupling and activating agents (Figure 1). The protein content before and after the immobilization process was determined to check the immobilization yield using Bradford's method. The formed nano-biocatalyst was washed several times with phosphate bu ffer to remove any unreacted lipase and then freeze dried [23,25].

**Figure 1.** Schematic representation of lipase immobilization on dopamine coated.

#### *2.5. Lipase Activity Assay (Free and Immobilized Form)*

Lipase assay for free and immobilized lipase was performed by using the titrimetric method [24]. The activity of lipase was measured by the titration of fatty acids, which were produced from oil while reacting with enzyme. The assay reaction mixture (consisted of 1 mL of free lipase (produced from *Aspergillus terreus* AH-F2) while 0.1 g of nano-biocatalyst in the case of immobilized lipase and 10 mL of olive oil (10%)) was homogenized in 10 g of gum acacia and phosphate buffer (5 mL) with pH 7 followed by the addition of 0.6% calcium chloride (2 mL). This mixture was then incubated for 1 h in a water bath shaker at 37 ◦C. After incubation, the enzymatic reaction was ceased by adding 20 mL of ethanol/acetone mixture (1:1) following the addition of few phenolphthalein drops. The quantity of fatty acids released during this period by the action of lipase was titrated by using 0.1 N NaOH solution.

One unit of lipase activity (U) was defined as the amount of enzyme which released one micro mole (μmol) of fatty acid per minute under specified assay conditions.

Lipase units were determinedLipase units were determined as follows:

$$\text{Lipsse Activity} \left( \text{U}/\text{mg}/\text{min} \right) = \frac{\Delta \text{V} \times \text{N}}{\text{m} (\text{sample})} \times \frac{1000}{60} \text{s}$$

where Δ V = V2 − V1; V1 = volume of NaOH consumed against control flask; V2 = volume of NaOH consumed against experimental flask; N = normality of NaOH; m(sample) = mass of enzyme extract; 60 = time of incubation (min) for bacterial lipase.

The e ffects of pH and temperature on the activity of free and immobilized lipase were studied using the above-mentioned assay method. The impact of pH on catalytic activity was investigated by incubating the sample at 37 ◦C with the phosphate bu ffer within pH range 5 to 10, whereas the impact of temperature in the range 25–50 ◦C was also studied at optimum pH levels.

#### *2.6. Collection and Characterization of Feedstock*

WCO (waste cooking oil) was collected from the local restaurant for the biodiesel production. The feedstock was purified by filtration to remove any food chunks and inorganic material in the waste oil. The physico-chemical properties of WCO biodiesel were analysed to reveal the quality of the used oil. Iodine number, specific gravity, density, peroxide value, acid value, saponification value and refractive index of the oil were calculated using standard analytical AOCS (American oil chemist's society) methods.
