3.2. Methods

All experiments were performed in duplicate. The results were expressed as an average ± standard deviation (σ). Analyses of variance between averages were performed by Tukey test at 5% significance.

The oils were physically-chemically characterized in terms of acidity index [82] and saponification index [83]. Water content was determined by Karl Fisher titrimetric analysis [84] in a Titrino 907 titrator (Metrohm, Herisau, Switzerland).

The immobilization of Eversa was carried out according to the methodology described previously [26]. A solution containing 21 mL of a PEI solution (50 mg/mL), 32 mL of liquid Eversa (32.75 ± 1.75 mg protein/mL and 127.63 ± 12.88 U_est/mL), and 17 mL of sodium phosphate buffer (5 mM and pH 7.0) was prepared. This solution was incubated at 25 °C under stirring at 150 rpm for 60 min. Subsequently, 0.8 mL of a suspension of SMNPs (75 mg/mL), 0.128 g of starch, 1.85 mL of 5 mM sodium phosphate buffer (pH 7.0), and 1.35 mL of the enzyme solution previously treated with PEI (14.97 mg protein/mL and 58.34 U_est/mL) were added in a Falcon tube, followed by precipitation with 12 mL of anhydrous ethanol (volumetric ratio enzyme suspension/ethanol of 1:3) in an ice bath. The suspension was stirred at 150 rpm, 4 °C in an orbital shaker (Model MA830, Marconi, Piracicaba, SP, Brazil). After 30 min, 1.51 mL of glutaraldehyde solution (50%, v/v, in water) were added and the cross-linking proceeded at 4 °C, with stirring at 150 rpm for 2.5 h. Then, the CLEAs were recovered by magnetic separation (using a neodymium magnet, 50 × 20 × 20 mm, the CLEA separation was very fast), washed once with 12 mL of sodium phosphate buffer (100 mM and pH 7.0), and resuspended in 4 mL of sodium phosphate buffer (5 mM and pH 7.0). 50 µL of α-amylase (BAN 480 L) and 50 µL of amyloglucosidase (AMG 300 L) were added to the suspension and incubated at 25 °C for 4 h to hydrolyze the starch present in the structure of the CLEAs. After, the CLEAs were recovered by magnetic separation, washed twice with 4 mL of sodium phosphate buffer (5.0 mM and pH 7.0). Then the Eversa-mCLEAs were washed twice with 4 mL of tert-butanol and kept overnight in the refrigerator for dehydration. A total of 48 tubes were prepared under the same conditions described above. The total enzyme offered to the immobilization (970.06 mg protein and 3780.43 U_est) yielded 11.57 g of Eversa-mCLEAs with a specific activity of 106 U_est/g biocatalyst, that is, 1226.42 U_est, giving a recovered activity of 32.4%.

The esterification activity was measured in terms of the synthesis of butyl butyrate following the methodology described in [85]. Free or immobilized Eversa (50 μL of liquid enzyme or 50 mg of dried CLEAs) was added to the reaction medium containing 7.5 mL of heptane, butanol (0.1 M), butyric acid (0.1 M), and molecular sieves (0.1 g). The reaction was carried out in closed glass bottles at 37 °C under 250 rpm stirring in an orbital shaker (Model MA832, Marconi, Piracicaba, SP, Brazil). After 60 min of reaction, 5 mL of ethanol were added to quench the reaction, and the acid concentration was measured by titration in a Titrino 907 titrator (Metrohm, Herisau, Switzerland) using a 20 mM KOH solution. One unit of esterification (U_est) was defined as the initial rate of production of butyl butyrate (in μmol min⁻¹) under the assay conditions.

The transesterification activities of the immobilized Eversa were assessed using the reaction between WCO and different alcohols (methanol, ethanol, octanol, and isoamyl alcohol). The reaction was carried out in closed glass bottles at 40 °C and 250 rpm stirring in an orbital shaker for 2 h. The standard transesterification conditions were oil/alcohol molar ratio of 1:6 and enzyme load of 2.13 U_est/g oil for Eversa-mCLEA. Samples of 0.5 mL were withdrawn from the reaction mixtures every 30 min up to 2 h of reaction, and the fatty acid alkyl esters were quantified by gas chromatography. The samples were previously pretreated using phase separation, where the oil phase (esters and unreacted acylglycerols) was recovered, washed with hot distilled water (using the same volume of sample), and centrifuged (three washing steps), followed by drying overnight in an oven at 60 °C. One unit of transesterification activity (U) was defined as the initial rate of esters production (g of esters/g of sample per minute) under the conditions described previously.

The performance of liquid and immobilized Eversa was evaluated in the esterification reaction of oleic acid with octanol and isoamyl alcohol at the following conditions: enzyme load of 2.13 U_est/g acid, oleic acid:alcohol molar ratio of 1:2, 40 °C, and stirring of 250 rpm in an orbital shaker for 24 h. In this case, molecular sieve was used to capture the water produced during esterification.

The transesterification reaction was used in order to evaluate the performance of the immobilized Eversa in the production of biolubricants using refined soybean oil and WCO as acyl donors and isoamyl alcohol. The experimental conditions were enzyme load of 2.13 U_est/g oil, acyl donor/alcohol molar ratio of 1:6, 40 °C, and stirring of 250 rpm in an orbital shaker for 24 h. After evaluation of the different acyl donors, the effect of several WCO/alcohol molar ratios (1:3, 1:4.5, 1:6, 1:9, and 1:12) on the isoamyl ester mass yield was evaluated. In this set of experiments, an enzyme load of 2.13 U_est/g oil was used. All reactions were carried out in closed glass bottles at 40 °C and stirring at 250 rpm. The experiments to evaluate the effect of enzyme load (2.13 and 12 U_est/g oil) on the isoamyl ester mass yield were performed in a vortex-type batch reactor (radius ratio of 0.24, aspect ratio of 6.72) operated at 40 °C and 1500–2000 rpm stirring for 72 h. In this case, liquid Eversa was also used for comparison purpose. Samples were withdrawn to analyze glycerides (monoglycerides (MAGs), diglycerides (DAGs), and triglycerides (TAGs)), and isoamyl fatty acid esters by liquid and gas chromatography, respectively (Section 3.2.5). The FFAs contents in the biolubricant were determined according to the AOCS Official Method Ca 5a-40 [82], but modified by Rukunudin et al. [86].

Analyses of fatty acid esters were performed according to the ASTM D6751 and EN14103 methods [87], adapted by [26], using a 7890A Agilent chromatograph (Santa Clara, CA, USA) equipped with FID detector and Rtx-Wax capillary column (30 m × 0.25 mm × 0.25 μm, Restek Corporation, Bellefonte, PA, USA). The injector and detector were set at 250 °C. Methyl heptadecanoate was used as an internal standard. 50 mg of the washed and dried sample was dissolved in 1 mL of internal standard solution (10 mg mL⁻¹, in heptane) and 1 μL was injected into the equipment. The ester mass yield (wt.%) was calculated according to Equation (1):

where ΣA is the total peak area of fatty acid esters C14:0 to C24:0, A_SI is the peak area of the internal standard (methyl heptadecanoate, C17), C_SI is the concentration of the internal standard (10 mg·mL⁻¹), V_SI is the volume of the internal standard solution (1 mL), and m is the mass of sample (50 mg).

The analysis and quantification for samples containing TAGs, DAGs, and MAGs (in wt.%) were performed according to the methods described by Holcapek et al. [88]. The E-2695 Waters chromatograph (Waters, Millford, CA, USA) equipped with a UV detector (set to 205 nm) and Ascentis^®Express C18 reverse-phase column (10 cm × 46 mm × 2.7 μm, Sigma-Aldrich, St. Louis, MO, USA) was used in the analysis. The mobile phase was composed of water (Phase A), acetonitrile (Phase B), and isopropanol:hexane (5:4, v/v) (Phase C) with a flow rate of 1 mL·min⁻¹. 50 mg of the washed and dried sample was diluted 1650 times in a 2-propanol-hexane solution (5:4, v/v) and 20 μL was injected into the equipment.

In order to check the transesterification reaction, a Fourier Transform Infrared spectrophotometer (ATR-FTIR) (model Vertex 70, Bruker, Germany) with a diamond crystal single bounce accessory was used. The samples were directly applied to the crystal cell. ATR-FTIR spectra for isoamyl ester were acquired after 32 scans between 4000 and 400 cm⁻¹ with a spectral resolution of 4 cm⁻¹. Thermal analyses were carried out using a TGA Q500 thermogravimetric analyzer (TA Instruments) in a nitrogen atmosphere from room temperature up to 600 °C using a heating rate of 10 °C·min⁻¹.