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 marginally greater than in basic pH. Therefore, the immobilization process seems to expand the stability of the lipase in strict basic environments. Lipase activity in different 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 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 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 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 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-edible
Nowadays, 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).
Lipase 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 developed the thermal tolerant, storage stability 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.
The authors express their gratitude to the Research Council of the Shahid Bahonar University of Kerman, Kerman (Iran) for financial support during the course of this project.