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).
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.