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.
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 characterizationLipase 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.3.
4. Characterization of free and mGO-CLEAs lipase3.4.
1. Effect of different temperatures and pHs on the lipase activity As shown in Fig. 5A, the maximum activity of both forms of enzyme was obtained at pH 8.0 and 9.0, respectively.
Moreover, relative lipase activity of mGO-CLEAs-lipase was faintly lower than free enzyme in acidic pH, but marginally greater than in basic pH. Hence, the immobilization process seems to grow the lipase stability in strict basic environments. Lipase activity in diverse temperatures were 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 mGO-CLEAs lipaseImmobilization system is one of the most promising strategies to improve catalytic activity for the applied application. To explore the thermal stability, free and mGO-CLEAs lipases were preserved in 100 mM phosphate buffer (pH 7.5) for 3h at 60 °C.
Next, the remaining lipase activities were dignified in 100 mM phosphate buffer (pH 7.5) with pNPP as substrate. The lipase activity of both forms of lipase was highest up to 45 min of incubation at 60 °C. The lasting activity of the free lipase is 50 % while the mGO-CLEAs 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 tolerance.3.4.
3. Determination of Km and Vmax Kinetic factors of free and mGO-lipase were investigated by calculating initial reaction speed with different substrate concentrations. As shown in Fig. 6B and Table 1, Vmax values of mGO-CLEA-lipase was slightly upper 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 better lipase affinity for the pNPP substrate, about 2.25 folds. It approves that conformational changes by the 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 of mGO-CLEAs lipase Reusability of mGO-CLEAs lipase is a dominant factor for its commercial application 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 decreased over 5 cycles (Fig. 7a). Protein leaking was also investigated throughout reusability tests 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 tolerant of both free and mGO-CLEAs lipase were also examined by storing them at 4 °C and checking the lipase activity. Results displayed mGO-CLEAs-lipase reserved about 75 % of its original activity after 30 days of incubation, wherein free enzyme missed its preliminary activity at the similar 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 prevents protein leaking from mGO-CLEAs nanomaterials (Yong et al.
, 2008). 3.5. Biodiesel production from non-edibleNowadays, non-edible oil resources as a favorable source for biodiesel synthesis has 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 dispersed. The highest biodiesel synthesis (26 %) from R.
communis oil was gained at room temperature after 24 h of incubation by Entrobacter Lipase MG10 (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 MG10 lipase such as methanol-tolerant, and short time reaction make it capable as a latent enzyme for biodiesel creation from non-edible oils.Remarkably, mGO-CLEAs lipase formed the highest biodiesel construction (78 %) from R. communis oil after 24 h (Fig.
5). Besides, the immobilized MG10 lipase enriched 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 fabrication by consuming 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 affiliate 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. ConclusionLipase MG10 is a high potent lipase (thermostable, inducible, high methanol-tolerant, and short time reaction rate) which was isolated from Gehver hot spring. The CLEA of lipase MG10 was immobilized on the mGO. The lipase immobilization considerably established the thermal tolerant and the lipase reusability. In addition, the obtained nanocomposite displayed a shift to acidic pH, which is outstanding possessions for biodiesel construction. Biodiesel fabrication was also attained by 75% recovery from R.
communis oil as non-edible oil feedstock which would have prospective in green and clean construction methods.
