Lipase purification process Added bulking/stabilizing agents Activity screening assay Biodiesel-oriented tests Immobilization procedure Cell extract (LipT6 M _CE) Heat treated CE (LipT6 M _HT) Pure lipase T6 M (LipT6 M ) Skimmed milk addition (LipT6 M +SM-1-30) E. coli proteins addition (LipT6 M +lysate) Sol-gel immobilization in a ternary organosilane system Aromatic matrix TEOS:PTEOS:DMDEOS Aliphatic matrix TEOS:OTEOS:DMDEOS Esterification reaction towards butyl-laurate formation in hexane Choosing the most active immobilized lipase Biodiesel production (soybean and waste chicken oils) Recyclability (butyl-laurate formation) Figure 1: Work scheme of lipase T6 M immobilization into sol-gel matrices as a biodiesel catalyst 50 ° C for 15min Affinity purification

M Figure 2: SDS-PAGE visualization of different immobilized fractions. The triple mutant was used throughout. (M) protein marker in kDa, (1) LipT6 M _CE cell extract, (2) LipT6 M _HT cell extract after heat treatment, (3) LipT6 M purified lipase (5 mg/mL), (4) lysate - cell lysate without lipase, (5) LipT6 M _3% soluble purified lipase T6 M at 3% (w/w) in buffer (6) LipT6 M +lysate - re-combined mixture of lysate and 3% lipase T6 M (7) TLL_3% soluble TLL at 3% concentration in buffer, (8) TLL+lysate - re-combined mixture of lysate and 3% lipase TLL, (9) CaLB_3% soluble lipase CaLB at 3% concentration in buffer (10) CaLB+lysate - re- combined mixture of lysate and 3% CaLB. 45 μg protein samples were loaded into 15% acrylamide gel and stained with Coomassie Blue. LipaseT kDa TLL 31.80kDa CaLB 35.52kDa

M Figure 3: SDS-PAGE visualization of pure lipase T6 M with and without skimmed milk powder. (M) protein marker in kDa, (1) LipT6 M (2 mg/mL), (2) LipT6 M +SM-15 (1:15 lipase:skimmed milk), (3) LipT6 M +SM-30, (4) and (5) SM-15 and SM-30 control, respectively, comprising SM suspended in buffer without enzyme. 45 μg protein samples were loaded into 15% acrylamide gel and stained with Coomassie Blue.

Figure 4: Esterification activity [BLU gr -1 ] of sol-gel immobilized lipase T6 M in two matrices and different ratios of skimmed milk addition. The reaction was conducted in 45°C with n-butanol and lauric acid in n-hexane. Dry sol-gel particles were added to the systems and samples were collected every 15 min and analyzed by GCMS.

Figure 6: Relative effect of E. coli lysate on different immobilized lipases in the aromatic sol-gel matrix. The values were calculated by dividing the activity value (BLU gr -1 ) of immobilized lipase + lysate with the activity value of immobilized lipase without lysate (buffer only, 3% w/w enzyme). Results are mean ± SD (n=3), while SD were lower than 0.3.

Figure 7: Successive utilization of sol-gel immobilized LipT6 M _CE for lauric acid esterification. The 2 h reactions were conducted in 45°C with n-butanol and lauric acid as substrates in hexane. 200 mg of dry beads were used. Results are mean ± SD (n=4). Total sol-gel beads mass loss was less than 4% w/w.

Figure 8: FAME biosynthesis from soybean and waste chicken oil using immobilized LipT6 M _CE in an aliphatic and aromatic sol-gel matrix. Reaction conditions: 2 g oil, 5% water, 4.5:1 methanol to oil molar ratio, and 50 mg dry immobilized lipase (2.5% based on the oil weight), 1350 rpm, 45°C. The results represent triplicates. aro=aromatic; ali=aliphatic.