## 2 Results and Discussion
### Discussion
It was reported by other authors that the use of mechanical stirring for enzymatic esterification using immobilized enzymes generally has a limitation in internal mass transfer that might reduce the rate of ester formation [27,28]. Moreover, the use of ultrasounds allowed the increase in the concentration of substrate that could be used in the reaction. The maximum concentration was improved seven times for Novozym 435 and four times for MCI-CALB. Besides the reduction of the mass transfer limitations, the more homogeneous mixing obtained under ultrasound avoids substrate/product/water gradients inside the porous matrix of the support. Avoiding the formation of water layer surrounding the enzyme, the acid will not be concentrated in the enzyme environment, which could lead to a lower pH, thus decreasing the enzyme stability. In addition, the use of ultrasounds reduced the mechanical stress caused by mechanical stirring, thus Novozym 435 presented better operational stability than MCI-CALB under ultrasound. The higher particle size of Lewatit VP OC 1600 as compared to MCI GEL CHP20P allowed an easier separation and recovery of the biocatalyst, which is important in the reusability. Nevertheless, results strongly support that ultrasound energy improved the activity and the operational stability for both biocatalysts. In the present research, MCI-CALB preparation was quite stable under operational conditions, and it could be reused several cycles without any washings, even \begin{table}
\begin{tabular}{c c c} \hline \hline  & **MCI-CALB** & **Novozym 435** \\ \hline Support & MCI GEL CHP20P & Lewatit VP OC 1600 \\ Matrix & Styrene-divinylbenzene & DVB-crosslinked polymer based on methacrylic esters \\ Particle size & 75–150 μm & 300–800 μm \\ Pore size & 400–600 Å & 150–300 Å \\ Mass transfer & High & Low \\ Hydrophilicity & Low & High \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Summary of the properties of the biocatalysts. under standard stirring techniques. Ultrasound technology improved this operational stability 1.5 times and the activity was twice as high as compared to mechanical stirring. Novozym 435 also improved the same parameters under ultrasonic technology. The effect of this technology was even more significant in the operational stability of Novozym 435, as compared to MCI-CALB, making unnecessary the washings between reaction cycles, whereas it was almost fully inactivated under conventional stirring in only 2 to 3 cycles [8], for the reasons discussed above. Optimal conditions for Novozym 435 under ultrasound, as determined by RSM, were similar to those determined in this work for MCI-CALB: temperature of 46 \({}^{\circ}\)C; substrate molar ratio of 3.6:1 butanol:acetic acid; amount of biocatalyst of 7%; added water of 0.25%. Using 2 M acetic acid, 82% of conversion was reached using Novozym 435 after 2.5 h, whereas conversion of 90% was obtained using MCI-CALB in the same time, even when the amount of protein per mass of dried biocatalyst was about two times higher for Novozym 435 [20]. Therefore, even under ultrasound stirring that greatly improved the Novozym 435 performance, MCI-CALB remained with a better productivity per cycle than Novozym 435 per mass unit, and much higher comparing the productivity per enzyme molecule. Another interesting characteristic associated to ultrasound technology is that it is considered a green technology because of its high efficiency, low instrumental requirements, and significant reduction of the processing time when compared to other techniques [29]. Moreover, is an excellent tool for improvement of chemical, physical and biological processes [12, 28, 30, 31, 32]. Ultrasound has been used in many applications, such as homogenizing, disintegration, sonochemistry, degassing and cleaning, extraction, emulsification, and chemical synthesis [33]. The mechanism of ultrasound is based on the high-energy waves that create cavitation in the liquid solution. The subsequent collapses of the cavitation bubbles release the energy.