## 2 Results and Discussion
### Statistical Analysis
#### 2.3.2 Effect of Reaction Parameters
However, no significant change was observed as the reaction temperature was raised from 30 to 50 \({}^{\circ}\)C. High temperatures for heating large reaction volumes mean a cost for any industrial process. Furthermore, at higher temperatures, the enzyme may be denatured, causing the reduction of enzymatic efficiency. Lipases show the activity of more than 90% in the temperature range of 30 to 50 \({}^{\circ}\)C [76]. Thus, the temperature was not a parameter that influenced the biodiesel conversion in the present study. The authors [PERSON] et al. (2017) [41], reported in their work that a significant change was observed in the biodiesel conversion in a temperature range from 30 to 40 \({}^{\circ}\)C. In the range from 40 to 60 \({}^{\circ}\)C, there was no significant change. While in the range from 60 to 70 \({}^{\circ}\)C there was a drop-in conversion, which may be linked to the denaturation of the enzyme during the process. Temperature was also a parameter studied by the authors [PERSON] et al., 2017 [77], the range adopted was 20 to 40 \({}^{\circ}\)C, the authors observed that there was no relevant change in the biodiesel conversion catalyzed by Novozym(r) 435 using fat from black soldier fly (_Hermetica illucens_) in the presence of methanol. It is possible to observe that the temperature range adopted in the present work and the temperature ranges reported in the literature did not influence the biodiesel conversion. The molar ratio is one of the main parameters in the biodiesel production. It is important to note that the conversion is influenced by the substrate properties and the nature of the catalyst. For the complete conversion of ethanol and free fatty acids or oil to fatty acid ethyl ester, a stoichiometric ratio of three moles of alcohol is required for each mole of the substrate, which may vary in different systems [41]. The molar ratio (free fatty acids/ethanol) was one of the parameters that most influenced the biodiesel conversion in the present work. It was observed that the conversion to ethyl esters was reduced in the mixture of 1:1 (free fatty acids/ethanol) and that the value increased significantly in the 1:15 mixture (free fatty acids/ethanol), Figure 3B. In other studies, reported in the literature using methanol, excess methanol caused the inactivation of Novozym(r) 435. The authors [PERSON] and [PERSON], (2019) [78] showed that the enzyme was deactivated at a 1:15 molar ratio (papaya oil/methanol) reducing the conversion to methyl esters; however, in the proportion of 1:3 and 1:9, the reaction rate was directly favored resulting in better biodiesel yields. A similar result was observed that in the proportion range between 1:3 (sweet basil seed oil/methanol) and 1:12 (sweet basil seed oil/methanol) there was a gradual increase in conversion to methyl ester and between 1:12 (sweet basil seed oil/methanol) and 1:15 (sweet basil seed oil/methanol), a reduction was observed [41]. Even with an excess of 1:15 (fatty acids/ethanol), the biocatalyst continued to present its catalytic activity, as it did not suffer from inhibition of ethanol, unlike methanol, which exerts a greater inhibition in enzymatic activity [79]. For the biodiesel production, the lipase content generally considered is 2 to 10% in relation to the weight of the oil. Increasing the enzyme load causes a greater conversion to ethyl esters [48; 80]. Figure 3C shows the interaction between the biocatalyst contend and the temperature. As well as the temperature, the biocatalyst content (0.05-0.15 g) did not have a significant change in the conversion; however, the greatest conversions were achieved when offered a 0.15 g biocatalyst content. [PERSON] and [PERSON], (2019) [78] reported that the increase in the amount of catalyst from 0.5% to 1% by weight, improved the yield in methyl esters. Though, when a higher load of biocatalyst (1%-1.5% by weight) was offered in a molar ratio, with proportions 1:3 to 1:15 (papaya oil/methanol), the yield decreased due to the formation of a gel and the increase in viscosity in the mixture [81].