p-nitrophenyl esters (pNP-esters) and triacylglycerol were obtained from Sigma–Aldrich (St. Louis, MO, USA). Q-Sepharose was obtained from Pharmacia (Uppsala, Sweden). All other chemicals and materials used were of analytical grade.
2.2. Screening of methanol-tolerant lipase producing bacteria
Soil and water samples were collected from different oil-contaminated locations placed in Kerman region, Iran. Methanol was added to the 20 ml enrichment culture medium (K2HPO4, 3.5 g/L; KH2PO4, 1.0 g/L; MgSO4.7H2O, 0.25 g/L; tween-80 5.0 g/L; (NH4)2SO4, 10.0 g/L; olive oil, 50.0 g/L) at a concentration of 30% (v/v). The mentioned medium were incubated at 30 °C with shaking at 160 rpm. After three days, the culture was inoculated to different medium two times. Lastly, culture samples were spread on tributyrin agar plates (yeast extract 1.0%, tryptone, 0.25%, NaCl 0.5%, tributyrin 0.5%, and agar 1.0%). Colonies displaying higher lipase activity (clear zone) were collected and re-spread on the olive-oil medium (yeast extract 0.1%, MgSO4·7H2O 0.05%, K2HPO4 0.1%, phenol red 0.005%, olive oil 6%, and agar 1.5%). The mentioned plates were placed at 30 °C for two days. The bacteria display great ratios of pink area diameter to colony diameter were chosen as latent lipase source. Strain MG40 produced the lipase with great methanol stability was designated and identified based on 16S rDNA sequence, morphological and biochemical tests according to our earlier reports (Ramezani-Pour et al., 2015; Azadian et al., 2015).
2.3. Production and purification of lipase
The strain MG40 was cultured in medium having KH2PO4, 1.0 g/L; K2HPO4, 3.5 g/L; MgSO4.7H2O, 0.25 g/L; tween-80 5.0 g/L; (NH4)2SO4, 10.0 g/L; olive oil, 50.0 g/L. It was incubated on a shaker incubator at 170 rpm at 30 °C. After two days of incubation, supernatant was centrifuged at 12,000 rpm for 15 min at 4 °C. The obtained supernatant was concentrated by 85 % (W/V) ammonium sulphate and maintained at 4 °C for 5 h. After that, the obtained pellet was collected by centrifugation at 13,000 rpm for 25 min at 4 °C and re-suspended in a minor volume of 45 mM Tris–HCl buffer (pH 7.8). Subsequently, it was dialyzed against 45 mM Tris–HCl buffer (pH 7.8) for 24 h and the buffer was changed after 8h intervals. The obtained dialyzed enzyme was laden onto a Q-Sepharose FF column (1.6 cm × 10 cm) which equilibrated with 45 mM Tris–HCl buffer (pH 7.5). The bounded proteins were eluted with the previous buffer having a linear gradient of NaCl from 0.0 to 0.5 M at a flow rate of 1.0 mL/min. The samples with high enzyme action were collected and dialyzed against the mentioned buffer. Lastly, the purity of enzyme was considered by SDS-PAGE (Laemmli, 1970) and protein value was also measured by Bradford process (Bradford, 1976).
2.4. Lipase hydrolytic activity assay
Lipase activity was investigated by hydrolysis rate of the p-nitrophenyl palmitate (pNPP) as a substrate. This substrate was dissolved in isopropanol to 20 mM concentration. In assay reaction, 20 µL of a pNPP solution was added to 460 µL of Tris–HCl buffer (40 mM, pH 7.8). Then, the reaction was started by addition of 20 µL of the lipase. The assay blend was incubated at 37 °C and after 30 min of incubation, it was stopped by chilling on ice. The absorbance value of the formed para nitrophenol (p-NP) was measured at 410 nm. To consider any spontaneous hydrolysis of substrate, control samples without lipase were also used. All these tests were performed three times. One unit of enzyme activity was considered as the quantity of lipase which liberating one micromole of p-NP per minute below the assay situations.
2.5. Preparation of magnetic graphene oxide composites
Graphene oxide (GO) was produced by oxidizing graphite substrate according to the Hummers process with slight modification (Sun et al. 2012). Usually, graphite powder (2.0 g), NaNO3 (1.0 g), KMnO4 (6.0 g), and concentrated H2SO4 (98%, 50 mL) were blended and strongly stirred in an ice-bath for 120 min. After that, the resultant blend was incubated in a 37 °C and preserved for 120 min, tracked by the mild addition of ultra-pure water (150 mL). Subsequently, the temperature was increased to 98 °C and the reaction was permitted to continue at the mentioned temperature for 40 min. When the temperature diminished to 60 °C, H2O2 (30%, 10 mL) was subsequently added and auxiliary stirred for 120 min. The resulting blend was filtered and then washed with hydrochloric acid (5.0 %) and distilled water for numerous times pending the pH of the washing solution was neutral, and then dehydrated in a vacuum oven at 60 °C. Exfoliation of graphene oxide was approved out by sonication for 120 min in an aqueous solvent.
Magnetite nanoparticles were synthesized and activated with amino functional groups (Reza et al. 2010). These particles were prepared by addition of ammonium hydroxide into a mixed solution of FeSO4 (0.7 g) and FeCl2 (1.4 g) in 30 ml deionized water pending gaining a brown precipitate. To eliminate the remaining ions, the gained product was gathered and washed with deionized water up to a pH value of 7.0 was attained; the resulting product was dried at 100 °C for 120 min. Then, these nanoparticles were functionalized with 3-aminopropyl triethoxysilane to acquire amino activated magnetic nanoparticles. 0.02 g of Magnetite nanoparticles, 100 µL of 3-aminopropyl trimetoxysilane, were dissolved in 30 µL of deionized water and 2.5 mL of methanol. So, the resulting blend was sonicated for 25 min. Afterward, 1.5 mL of glycerol was added to the mixture, and the obtained solution was heated at 85 °C for 6 h. The acquired precipitate was washed with deionized water and methanol for three times (Talekar et al., 2012; Xie and Huang, 2018).
The coated magnetic GO were equipped by co-precipitation manner with adding of ammonia solution (Deng et al., 2013). GO (0.5 g) was distributed in 220 mL distilled water and subsequently imperiled to ultrasound for 3 h. Then, the GO solution was gradually added into the coated magnetite nanoparticles solution. Subsequently, the obtained blend was ultrasonicated for 60 min. Finally, the obtained nanocomposite were gathered by using an external magnetic field, washed four times with distilled water, and lastly dried in a vacuum oven at 60 °C.
2.6. Lipase immobilization
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 stirring for 1h at 4 °C. For making cross-linking, glutaraldehyde solution (40 mM) was added 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 according to the method designated in scheme 1. The magnetic CLEAs of lipase (3 mg) were mixed with coated-mGO in phosphate buffer solution (3 mg/mL, 100 mM and pH 7.5) and stirred for 20 min at 30 °C. After that, glutaraldehyde solution (40 mM) was added into the resulting blend and stirred for 3 h at 30 °C (Talekar et al., 2012; Xie and Huang, 2018). Coated-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. Meanwhile, in the immobilization procedure, the washing solutions were gathered to consider the remaining lipase. The protein quantities in the lipase and washing solution were 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 enzyme
2.8.1. Effect of temperature and pH on the lipase activity and stability
The temperature activity profile of the lipase MG40 and cMGO-CLEA-lipase was investigated by considering the lipase activity at different 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, the lipase samples were 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 Fe-MCM-41 lipase
To investigate the irreversible thermo-inactivation of free and immobilized lipase, the enzyme solution were 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 samples 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. Then, the lipase activity in free and immobilized enzyme was measured as described previously. The residual lipase activities were measured by taking the initial lipase activity as 100%.
2.8.3. Determination of Kinetic parameters
Kinetic parameters of free and coated-MGO-magnetic CLEAs lipase were examined using diverse pNPP substrate concentrations in the range of 0.2–3.0 mg/mL in phosphate buffer (100 mM, pH 7.0) at 45 °C. In each form, 2 mg of lipase was used. Vmax, Km values of 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
Transesterification reactions were carried out by free lipase MG and cMGO-CLEAs lipase in 15 mL tubes and maintained for 24 h at room temperature with a stirring rate of 160 rpm. The reaction consist of 0.4 g oil (oil from Ricinus communis), methanol (1:3 molar ratio between 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 (4, 6, 8, 12, 16, 20, 24 and 28 h), 100 µl of reaction blend were picked up and diluted with the same volume of n-hexane. 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).