2

2.8.2. Thermal stability investigation of immobilized lipase MG10
To test the irreversible thermal inactivation of both form of lipase, the enzyme solution was incubated at revealed temperature for 3 h. At different time interval, aliquots were picked up and examined for remaining activity.
Storage stabilities of the free and coated-MGO-magnetic CLEAs enzyme were also examined by placing enzyme solutions in phosphate buffer (100 mM, pH 7.5) without substrate at 4 °C. Every 2 days, cMGO-CLEAs lipase was picked up by a magnetic and washed by distilled water. After that, the activity of both forms of enzymes was measured as described formerly. The residual enzyme activities were dignified by calculating the original lipase activity as 100%.

2.8.3. Investigation of Kinetic parameters
Kinetic factors of both free and coated-MGO-magnetic CLEAs lipase were examined using diverse concentrations of substrate in 100 mM phosphate buffer (pH 7.0) at 45 °C. In both forms, 2 mg of lipase was used in assay reaction. The amounts of Vmax, Km factors for free and coated-MGO-magnetic CLEAs lipase were considered from line Waver-Burk plot of the initial reaction rates equivalent to different substrate concentrations.

2.9. Biodiesel construction
Enzymatic transesterification reactions were carried out by free and cMGO-CLEAs lipase and maintained for 48 h with a stirring speed of 160 rpm. The reaction consists of 0.4 g oil (oil from Ricinus communis), methanol (1:3 molar ratio between R. communis oil and methanol) and 0.2% enzyme (free or correspond lipase on support) (w/w, based on the oil weight, g). At diverse time intervals (6, 12, 24 and 28 h), 100 µl of reaction blend was picked up and diluted with the same volume of n-hexane solvent. Afterward, the sample was gathered and the upper layer (10 µL) was performed to gas chromatography (GC) investigation for biodiesel measurement (Ji et al., 2010; Wang et al., 2017; Malekabadi et al., 2018).
3. Results and discussion
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 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.