## 2 Results and Discussion
### H NMR Analysis  
The \({}^{1}\)H NMR spectra of fused oil, USCO and their derivatives such as FFA, MAE and EMAE are shown in Figure 4. All the samples presented an intense signal at 1.13-1.17 ppm characteristic of hydrogens of terminal methyl groups (-CH\({}_{3}\)). The signals at 1.49-1.58 ppm of hydrogens of the aliphatic methylene groups (-CH\({}_{2}\)-), and at 2.06-2.11 ppm for allylic hydrogens (-CH\({}_{2}\)-CH=CH-), 2.85-2.95 ppm for bis-allylic hydrogens (-CH=CH-CH\({}_{2}\)-CH=CH-) and at 5.24-5.38 ppm of hydrogens linked to sp\({}^{2}\) carbon--olefinic (-CH=CH-) of unsaturated fatty acids are characteristics of USCO and their derivatives (FFA, MAE and EMAE). The signals between 4.10 and 4.40 ppm in the chemical structure of USCO refer to hydrogen atoms of the glycerol moiety that disappeared after hydrolysis step (see FFA spectrum). This confirms the full hydrolysis of USCO (1 st step--see Figure 1), in accordance with the titrimetric method. A characteristic peak at 3.85 ppm in the spectrum of fused oil refers to the methylene hydrogen attached to the hydroxyl group(-CH\({}_{2}\)-OH). The appearance of an intense signal between 4.23 and 4.30 ppm can be observed, one that is characteristic of hydrogen attached to carbon next to the ester group oxygen (-COOCH\({}_{2}\)-), which evidences monoalkyl esters (MAE) production via enzymatic esterification (2 nd step), as shown in Figure 2. After epoxidation (3 rd step--EMAE production), a decrease in intensity of the characteristic signal of olefinic hydrogens (-CH=CH-) at 5.24-5.38 ppm was observed due to partial conversion of unsaturated bonds into epoxy groups. In fact, a slight increase in the intensity of overlapped signals at 1.67 ppm (hydrogen of methylene groups adjacent to epoxy ring: -CH\({}_{2}\)-CHOCH-) and 2.85-3.25 ppm (hydrogens atoms of epoxy groups: -CHOCH-) due to introduction of these epoxy groups in the chemical structure of MAE can also be observed. These results confirm the formation of epoxidized monoalkyl esters (EMAE) via a sequential three-step process. Based on these results, an illustrative scheme of EMAE production by a three-step process was proposed, as shown in Figure 5. This process consists of sequential: (i) enzymatic hydrolysis of USCO, (ii) enzymatic esterification of the produced unsaturated FFA with isoamyl alcohol, the main monoalkyl alcohol present in fused oil's composition, and (iii) partial epoxidation via conversion of unsaturated bonds in the chemical structure of the produced MAE into epoxy groups. \begin{table}
\begin{tabular}{c c c c c c} \hline  & & \multicolumn{2}{c}{**MAE a**} & \multicolumn{2}{c}{**EMAE b**} \\ \cline{3-6}
**Properties** & **Units** & \begin{tabular}{c} **Open** \\ **Reactor** \\ \end{tabular} & \begin{tabular}{c} **Closed** \\ **Reactor** \\ \end{tabular} & \
\begin{tabular}{c} **Open** \\ **Reactor** \\ \end{tabular} &
\begin{tabular}{c} **Closed** \\ **Reactor** \\ \end{tabular} \\ \hline IV c & glz per 100 g & 110.2 \(\pm\) 1.7 & 109.8 \(\pm\) 1.1 & 73.5 \(\pm\) 0.9 & 72.6 \(\pm\) 1.4 \\ X d & \% & - & - & 33.3 & 33.9 \\ AV e & m\({}_{\text{KOH}}\cdot\text{g}^{-1}\) & 5.1 \(\pm\) 0.3 & 5.8 \(\pm\) 0.3 & 7.1 \(\pm\) 0.4 & 7.3 \(\pm\) 0.2 \\ OOC\({}_{\text{theor}}\). f & \% & - & - & 6.50 & 6.47 \\ OOC\({}_{\text{exp}}\). 8 & \% & - & - & 1.75 \(\pm\) 0.01 & 1.78 \(\pm\) 0.03 \\ ROC h & \% & - & - & 26.9 & 27.5 \\ S i & Dimensionless & - & - & 0.81 & 0.81 \\ \hline \end{tabular}  
* monoalkyl esters; b epoxidized monoalkyl esters; c iodine value; d relative conversion percentage; e acid value; f maximum theoretical epoxy oxygen content; g experimental epoxy oxygen content; h relative conversion percentage to oxíner; l selectivity.  
\end{table} 
Table 2: Chemical properties of used soybean cooking oil (USCO), monoalkyl esters (MAE) and epoxidized monoalkyl esters (EMAE) produced in this study. Figure 4: \({}^{1}\)H NMR spectra of fused oil, used soybean cooking oil (USCO) and their derivatives—free fatty acids (FFA), monoalkyl esters (MAE) and epoxidized monoalkyl esters (EMAE).