3

3.1. Screening and identification of bacterial producing the methanol-tolerant lipase

Lipase producing bacteria were screened in enrichment culture medium supplemented with olive oil as a sole source of carbon. Furthermore, methanol (30%, v/v) was also used to acquire the methanol tolerant lipase. The clear area around the colonies on the tributyrin agar plate was evaluated as lipase production. The greatest lipolytic strains were also examined on the olive oil plate complemented with phenol red, as a pH indicator. Results showed that MG isolate was a strain which displayed the maximum pink halo around the colony. The 16S rDNA gene of MG isolate was amplified and sequenced (Genbank Accession No. MF927590.1) and compared by BLAST investigation to other bacteria in the NCBI database. The results proposed a near relationship between MG40 isolate and the other members of the Enterobacter genus with a maximum sequence homology (99%) to Enterobacter cloacae. The phylogenetic tree (Fig. 1) designated that the strain MG was associated with Enterobacter species and used for following study.

3.2. Purification and immobilization of the lipase
Cell free supernatant of MG stain was exposed to ammonium sulfate precipitation (85% saturation) and Q-sepharose chromatography. Lipase MG was eluted from the Q-Sepharose column with a 19.5-fold purification and a 38.1 % yield, and it displayed a specific activity of 442.6 U/mg. This yield of MG lipase was analogous to that of lipase from S. maltophilia CGMCC 4254 (33.90%) (Li et al., 2013) and lower than lipase from P. aeruginosa PseA (51.6%) (Gaur et al., 2008), but higher than lipase from B. licheniformis strain SCD11501 (8.4 %) (Sharma and Kanwar, 2017). SDS–PAGE analysis of the purified MG40 lipase shown that it has a single band about 33 kDa, which it is different from the other Enterobacter cloacae.
Results of protein measurement with Bradford technique displayed that protein loading on these coated magnetite nanomaterials was succeeded. Moreover, the results of quantitative determination of protein loading on these nanomaterials shown that, immobilization efficiency was achieved about 73%. mGO-CLEAs lipase were dispersed in phosphate buffer. After a magnet was positioned sidewise, mGO-CLEAs Lipase showed fast response (60 seconds) to the peripheral magnetic field. It means that the magnetic CLEAs-Lip particles were shown suitable magnetic concern even though layers of CLEAs-Lipase were covered on their surfaces, in which it is significant in term of lipase immobilization.

3.3. Analytical characterization
Lipase MG40 was immobilized on the surface of magnetic functionalized graphene oxide, in which aldehyde groups of glutaraldehyde making linkage between amine of lipase and amino coated magnetite nanomaterials (Xie and Huang, 2018). Fig. 2a and b display SEM images of magnetic functionalized graphene oxide and mCLEAs-Lipase on magnetic graphene oxide, respectively. The SEM analysis of graphene oxide on Fig. 2a shown an irregular circular structure which was similar to the previous reports (Wang et al. 2015; Dwivedee et al. 2017), providing a bulky specific surface zone of the nanomaterials. Results of SEM image in Fig. 2b shown that lipase immobilization seem to reduce the construction of stacked GO structures. These results designated that the glutaraldehyde linkage successfully have been occurred between the amine surface of magnetic functionalized graphene oxide and amino groups of lipase.
Elemental EDX investigation from particular part of SEM image of magnetic CLEAs-Lipase for elemental plotting obviously specifies the existence of associated atoms of support including C, N, O, Si, P, S and Fe which displays the effective functionalization of APTES, particularly by noticing Si atom (Heidarizadeh et al., 2017). Furthermore, the remarkable attendance of phosphorous atom can intensely endorse the effective lipase immobilization (Fig. 3).
Presence of functional groups on surface of graphene and immobilization of lipase MG10 onto these nanoparticles were investigated by FTIR spectroscopy. FTIR spectra of graphene oxide (A), magnetic functionalized graphene oxide (B) and magnetic functionalized graphene oxide-CLEA lipase (C) have been shown in Fig. 4. The peak around 532-614 cm?1 could be evaluated to the stretching vibration of Fe–O in Fe3O4 nanoparticles (Fig. 5B, C), indicating the presence of Fe3O4 in the graphene oxide which directed that the preparation of Fe3O4-graphene oxide nanoparticles was effective (Thangaraj et al., 2016; Xie and Huang, 2018).
Moreover, peaks at 1635 and 1636 cm?1 resemble C=O vibrations of the present carbonyl and carboxyl groups on the mGO and presence of amide link between glutaraldehyde with Fe3O4 nanoparticles and CLEAs (Cui et al., 2015; Xie and Huang, 2018). Additionally, a characteristic adsorption band achieved at 3447 cm?1 equivalent to the adsorbed H2O and OH group on the mGO surface (Paludo N, 2015), which shown excessive absorbance in all of these nanoparticles and the magnetic functionalized graphene oxide-CLEA (Mehrasbi et al., 2017). FTIR spectrum of magnetic functionalized graphene oxide shows the presence of a peak in 2922 cm?1 spreads to aliphatic chain of functionalized APTES (Heidarizadeh, et al., 2017).
After lipase immobilization on the mGO (Fig. 5c), the 614 cm?1 band owing to the stretching vibration of Fe–O in Fe3O4 nanoparticles was practically vanished, which signifying the covering of Fe3O4 by lipase. Moreover, FTIR spectrum of magnetic functionalized graphene oxide-CLEA lipase also shown two absorption peaks at 2840 and 2922 cm?1 mentioning C-H stretching in -CH3 and -CH2-, which demonstrate the immobilization of enzyme on the support. In addition, the appearance two new FTIR absorption bands at 1404 and 1514 cm?1 because of the lipase were detected as associated with the mGO support, so indicating that the enzyme was covalently bound to the mGO composites via amide links.

3.4. Biochemical characterization of free and immobilized enzyme
3.4.1. Effect of temperature and pH on the lipase activity
As shown in Fig. 5A, the maximum activity of free and immobilized lipase was obtained at pH 8.0 and 9.0, respectively. Moreover, relative lipase activity of immobilized lipase was faintly lower than free enzyme in acidic pH, but slightly higher than in basic pH. Therefore, the immobilization process seems to expand the tolerance of the lipase in harsh basic conditions. Lipase activity in different temperatures was shown in Fig. 5B. The immobilized lipase showed a broad range of maximum temperature activity about 40-60 °C, compare to free enzyme. These results indicating the development of covalent links between protein and support, which may diminish conformational flexibility and result in preserve lid opening (Perez et al., 2011; Lu et al., 2009).
3.4.2. Thermal stability of free and immobilized lipase
Immobilization method is one of the most promising strategies to improve catalytic activity for the applied application. Consequently, to explore the thermal stability, free and immobilized enzyme were maintained in phosphate buffer (100 mM, pH 7.5) for 3h at 60 °C, and then the remaining activities were measured in the phosphate buffer (100 mM, pH 7.5) with pNPP as substrate. The lipase activity of both free and immobilized lipases was highest up to 45 min of incubation at 60 °C. The remaining activity of the free lipase is 50 % while the immobilized lipase reserved 85 % of its initial activity after 3h of incubation at 60 °C (Fig. 6a). These results evidently designate that the immobilization of lipases into mGO can avoid their conformation transition at high temperature, and improving their thermal stability.
3.4.3. Determination of Km and Vmax
Kinetic parameters of free and mGO-lipase were investigated by calculating initial reaction rates with different substrate concentrations. As shown in Fig. 6B and Table 1, Vmax values of mGO-CLEA-lipase was slightly higher than free enzyme about 0.1 µmol/min, which directed the rate of pNPP hydrolysis was not significantly changed after mGO-CLEAs-lipase preparation. The same results were also observed for magnetic CLEAs of the other enzyme. In the case of mGO-CLEAs-lipase, the detected lower Km value state a greater lipase affinity for the pNPP substrate, about 2.25 folds. It approves that conformational changes by reason of enzyme immobilization assistance the protein to appropriately turn its active site concerning the substrate (Aytar and Bakir, 2008; Sangeetha and Abraham, 2008; Talekar et al., 2012).

3.4.4. Reusability assay
Reusability of immobilized lipase preparation is a dominant factor for its commercial use in biotransformation reaction. The reusability of mGO-CLEAs lipase was measured up to 8 cycles. Enzyme activity of mGO-CLEAs lipase was the highest up to 5 cycles, but it continuously decrease over 5 cycles (Fig. 7a). Protein leaking was also investigated during reusability test of mGO-CLEAs lipase. Results exhibited no lipase activity was detected in reaction mixture up to 4 cycles of lipase reusability test. These results recommend that suitable cross-linking of enzyme and mGO nanomaterials produced stable MGO-CLEAs lipase (Talekar et al., 2012).
Storage stability of free and mGO-CLEAs lipase were also investigated by storing these enzyme in phosphate buffer at 4 °C and considering the lipase activity. Results displayed mGO-CLEAs-lipase retained about 75 % of its original activity after 30 days of incubation, in which free enzyme missed its initial activity at the same time (Fig. 7b). These results verified that mGO-CLEAs lipase had chief protection on the storage stability of lipase. These results designated that an active mGO-CLEAs lipase prevent protein leaking from mGO-CLEAs nanomaterials (Yong et al., 2008).

3.5. Biodiesel production from non-edible
Nowadays, non-edible oil resources as a favorable source for biodiesel synthesis have been admired for researchers. Ricinus communis is a small and fast-growing tree which is a highly productive and precocious maker of toxic seeds. In addition, it is very adjustable to diverse situations and has been broadly distributed. The highest biodiesel synthesis (26 %) from R. communis oil was gained at room temperature after 24 h of incubation by Entrobacter Lipase MG40 (10 mg) (Fig. 8). Mehrasbi and co-workers described using of free C. antarctica lipase B (100 mg) constructing 34% of biodiesel from waste cooking oil at 50 °C after 72 h of incubation (Mehrasbi et al., 2017). Some excellent properties of MG40 lipase such as methanol-tolerant, and short time transesterification make it capable as a latent enzyme for biodiesel synthesis from non-edible oils.
Remarkably, mGO-CLEAs lipase formed the highest biodiesel construction (78 %) from R. communis oil after 24 h (Fig. 5). Furthermore, the immobilized MG40 lipase improved biodiesel construction from R. communis oil about 3.1 folds at diverse time of incubation, compare to free lipase (Fig. 5). De los Ríos reported 42% of biodiesel production by using immobilized lipase of C. antarctica (De los Ríos et al., 2011).
As mentioned formerly, construction of several links between lipase and support, could reserve protein in open conformation and improved the enzyme rigidity with affiliated making of a protected micro-environment. Furthermore, it made a further active lipase cross-linking in mCLEAs lipase which evades enzyme leaking from composite and shield it against methanol solvent and the other by products (Talekar et al., 2012; Aytar and Bakir, 2008; Sangeetha and Abraham, 2008).

4. Conclusion
Lipase MG40 is a high potent lipase (thermostable, inducible, high methanol-tolerant, and short time reaction rate) which was isolated from local oil contaminated soils. Entrobacter lipase MG40 was immobilized on the magnetic graphene oxide nanocomposites. This nanobiocatalyst was characterized and employed for the production of biodiesel from non-edible oil feedstocks such as R. communis oil. The immobilization of lipase significantly increased the storage stability, the thermal stability and the reusability of the enzyme. Remarkably, lipase nanocomposite showed a shift to low temperatures and acidic pH, which is excellent properties for biodiesel production. Lipase-graphene nanocomposite was totally active after 5 cycle of enzyme activity. Biodiesel production was also achieved by 75% recovery from oil feedstock which would have potential in green and clean production processes.