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3.1. Screening and identification of bacterial producing 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 this isolate was a strain which displayed the maximum pink area 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 MG10 isolate and the other members of the Enterobacter genus with a extreme sequence homology (99%) to Enterobacter cloacae. The phylogenetic tree (Fig. 1) designated that the strain MG10 was associated with Enterobacter species and used for the following study.

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3.2. Purification and immobilization of the lipase
Cell free supernatant of MG10 stain was exposed to ammonium sulfate precipitation (85% saturation) and Q-sepharose chromatography. Lipase MG10 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 MG10 lipase was analogous to the lipase of S. maltophilia (33.9%) (Li et al., 2013) and lower than lipase from P. aeruginosa PseA (51.6%) (Gaur et al., 2008), but greater than lipase of B. licheniformis (8.4 %) (Sharma and Kanwar, 2017). SDS–PAGE analysis of the MG10 lipase shown that it has a single band about 33 kDa, which it is dissimilar with 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 determination of protein loading on these nanomaterials shown that, immobilization efficiency was achieved about 73%. mGO-CLEAs lipase was spread in phosphate buffer. After a magnet was positioned sidewise, mGO-CLEAs Lipase showed fast response (60 seconds) to the peripheral magnetic field. It incomes that the magnetic CLEAs-Lip particles were shown suitable magnetic concern even though layers of CLEAs-Lipase were covered on their surfaces, wherein it is significant in term of lipase immobilization.

3.3. Analytical characterization
Lipase MG10 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 earlier reports (Wang et al. 2015; Dwivedee et al. 2017), given that a bulky specific surface zone of the nanomaterials. Results of SEM image in Fig. 2b shown that lipase immobilization seem to diminish 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 containing 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 graphene and lipase immobilization 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), representing the presence of Fe3O4 in the graphene oxide which focused 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 carboxyl and carbonyl functional 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 surface of mGO (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 coated 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 nanoparticle 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 owing to the lipase immobilization were discovered, which specified that the enzyme was covalently bounded to the mGO nanocomposites via amide links.

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