Magnetic CLEAs of lipase were obtained as follows. The amino coated magnetite nanoparticles (4.

0 mg) were mixed with lipase MG40 in phosphate buffer (3.0 mg/mL, 100 mM and pH 7.5) and stirred for 20 min. After that, saturated ammonium sulphate (5.0 ml) was added with the stirring for 1h at 4 °C. For making cross-linking, glutaraldehyde solution (40 mM) was supplemented and stirred for 4 h at 30 °C.

The magnetic CLEAs lipase were gathered using magnet, and washed four times by phosphate buffer (100 mM, pH 7.5) and stored in the mentioned buffer at 4 °C. Lipase was finally immobilized on the functionalized nanomaterials rendering to the method designated in scheme 1. The magnetic CLEAs of lipase (3 mg) were mixed with functionalized mGO in phosphate buffer solution (3 mg/mL, 100 mM and pH 7.5) and stirred for 20 min at 30 °C. Subsequently, glutaraldehyde solution (40 mM) was supplemented into the resulting blend and stirred for 180 min at 30 °C (Talekar et al., 2012; Xie and Huang, 2018).

Functionalized mGO-CLEAs lipase were gathered using a magnet, washed four times by phosphate buffer (100 mM, pH 7.5) and stored in the mentioned buffer at 4 °C. Temporarily, in the immobilization procedure, the washing products were gathered to consider the remaining lipase. The protein quantities in the nanocomposite and washing results was measured by using Bradford technique (Bradford, 1976). 2.7.

Characterization of immobilized enzyme Scanning electron micrographs (SEMs) of magnetic graphene oxide (mGO), coated magnetic graphene oxide (mGO) nanocomposites and coated magnetic graphene oxide (mGO) CLEA lipase (cMGO-CLEA-lipase) were acquired on JEOL JSM6360 (Germany) scanning electron microscope (SEM) run at 5 kV. EDX investigation of cMGO-CLEA-lipase was also done from select inside SEM image. FT-IR spectra were identified on a Shimadzu IR-Prestige-21 spectrometer in the spectrum of 400–4000 cm?1 subsequent the KBr pellet methods. 2.8.

Biochemical characterization of free and immobilized enzyme2.8.1.

Effect of temperature and pH on the lipase activity and stability The temperature activity profile of the lipase MG10 and cMGO-CLEA-lipase was investigated by considering the lipase activity at diverse temperatures from 10 to 70 °C at Tris-HCl buffer (100 mM, pH 7.5). For thermal stability investigation, the free and immobilized lipase was pre-incubated in 100 mM Tris/HCl buffer (pH 7.5) at the different temperatures from 10 to 70 °C for 3h.

At diverse time interval, a sample of lipase solution was picked up and residual enzyme activity was measured. Lipase activity was also investigated at different pH values. Different buffers (50 mM) such as sodium acetate (pH 4.5–6.5), sodium phosphate (pH 6.0–8.0), Tris/HCl (8.0–9.

0), Glycine/NaOH buffer (9.0–11.0) were used. 2.

8.2. Determination of the thermal stability of immobilized lipase To examine the irreversible thermo-inactivation of free and immobilized lipase, the enzyme solution was incubated at mentioned temperature for 3 h.

At different time interval, samples were picked up and analyzed for residual activity. Storage stabilities of the free and coated-MGO-magnetic CLEAs lipase were also investigated by incubating enzyme solutions in phosphate buffer (100 mM, pH 7.5) without substrate at 4 °C. Every 2 days, cMGO-CLEAs lipase was picked up by a magnetic and washed by distilled water. After that, the lipase activity in free and immobilized enzyme was measured as described previously. The remaining lipase activities were measured by counting the initial lipase activity as 100%.2.

8.3. Determination of Kinetic parametersKinetic factors of both free and coated-MGO-magnetic CLEAs lipase were examined using diverse concentrations of pNPP in phosphate buffer (100 mM, pH 7.

0) at 45 °C. In both forms, 2 mg of lipase was used in each assay reaction. The amounts of Vmax, Km factors for free and coated-MGO-magnetic CLEAs lipase were considered from line Waver-Burk plot of the initial reaction rates equivalent to different substrate concentrations.2.9.

Biodiesel production Enzymatic transesterification reactions were carried out by free and cMGO-CLEAs lipase and maintained for 48 h with a stirring speed of 160 rpm. The reaction consists of 0.4 g oil (oil from Ricinus communis), methanol (1:3 molar ratio between R. communis oil and methanol) and 0.

2% enzyme (free or correspond lipase on support) (w/w, based on the oil weight, g). At diverse time intervals (6, 12, 24 and 28 h), 100 µl of reaction blend was picked up and diluted with the same volume of n-hexane solvent. Afterward, the sample was gathered and the upper layer (10 µL) was performed to gas chromatography (GC) investigation for biodiesel measurement (Ji et al., 2010; Wang et al., 2017; Malekabadi et al., 2018).