## 2 Results and Discussion
### Effect of Process Parameters and Optimal Conditions  
The obtained results can only describe the reaction within the range of variable values investigated in the experimental design. The linear effects of the four variables were: temperature (39.9), substrate molar ratio (2.5), amount of biocatalyst (94.5), and added water (\(-\)4.5). Only substrate molar ratio was not statistically significant at a 95% confidence level, in contrast with results seen when using Novozym 435 [19]. Reaction temperature and amount of biocatalyst presented the highest effects in the ultrasound-assisted butyl acetate synthesis catalyzed by MCI-CALB. The increase in both variables leads to an increase in the initial reaction rate. However, added water presented a negative effect on the initial reaction rate, which means that increasing the initial water content decreases the reaction rate. The tendency of the effect of these three variables was similar to that found using Novozym 435, although optimal values were slightly different. Thus, some differences could be found in this preliminary study comparing Novozym 435 and MCI-CALB [19]. The optimal conditions for the ultrasound-assisted butyl acetate synthesis catalyzed by MCI-CALB were found using the software Statistica 7.0, and were: temperature, 48.8 \({}^{\circ}\)C; substrate molar ratio, 3.46:1 alcohol:acid; amount of biocatalyst, 7.5%; and added water 0.28%, both by substrate mass. This could be better observed through the contour plots in Figure 1a, where it is possible to obtain an initial reaction rate over 250 mmol:L^-1h^-1. These conditions were similar to those found when Novozym 435 was used as catalyst under ultrasound stirring [19], but it was quite different from the optimal conditions for MCI-CALB when the mechanical stirring was used [20]. However, using our previous experience, we fixed substrate molar ratio and water content at their lowest level (\(-\)2), since substrate molar ratio was not statistically significant and added water showed a negative effect, and a new curve was plotted. This curve was represented in Figure 1b and it is possible to observe a new optimal condition: temperature, 60 \({}^{\circ}\)C; substrate molar ratio, 1:1 alcohol:acid; amount of biocatalyst, 5%; and added water 0%, both by substrate mass. This optimal condition differs from the previous regarding the two most important variables, temperature and amount of biocatalyst. In the optimal condition I, a lower temperature was used at the expense of higher amount of biocatalyst, whereas at the optimal condition II (see Figure 1b), it was possible to use lower amounts of biocatalyst and increase the temperature. For model validation, experiments under both optimal conditions were performed, and the results of time courses for the ultrasound assisted butyl acetate catalyzed by MCI-CALB are shown in Figure 2, as well as the comparison with the previous results. The predicted values by the model were 276.2 and 248.4 mmol\(\cdot\)L\({}^{-1}\)h\({}^{-1}\), whereas the observed values were 278.7 and 256.5 mmol\(\cdot\)L\({}^{-1}\)h\({}^{-1}\) for conditions I and II, respectively, showing a good correlation of the experimental results with the statistical predicted by the model. As it can be seen in Figure 2, the reaction courses were quite similar under both conditions, _i.e._, it is possible to reduce the enzyme content while increasing the reaction temperature and reaching the same conversion. Higher than 90% of conversion was obtained after 1 h, which is much better than the results obtained using mechanical stirring (50% in 1 h, and over 90% in 2 h) [20], or when using Novoym 435 under ultrasound-assisted conditions (less than 80% in 1 h, and over 90% in 1.5 h) [19]. In conclusion, MCI-CALB used under ultrasonic energy was two times faster than when used under mechanical stirring [20] It was also 2.5 and 1.5 times more rapid than Novoym 435 under mechanical stirring and ultrasonic energy, respectively [8, 19].