## 2 Results and Discussion
### Discussion  
As part of a continued research effort studying esterification catalyzed by lipases, we improved the results reported in the literature by developing a fast and efficient procedure for the synthesis of butyl acetate catalyzed by Novozym 435 [8]. Furthermore, biocatalyst activities and operational stabilities were enhanced by the development of an immobilized system on styrene-divinylbenzene beads [20]. Novozym 435 was then used as a catalyst under the same reaction conditions, but using ultrasonic energy, allowing the use of seven times higher substrate concentration, thus obtaining improved activity and much higher stability of the biocatalyst [19]. Based on these facts, we raised the hypothesis that the use of MCI-CALB under ultrasonic energy could enhance enzyme activities and stabilities, when compared to previous researches [8,19,20]. However, there is a complexity in this apparently simple process that requires explanation. This complexity starts with the differences between the biocatalysts used. A summary of the properties of both biocatalysts is presented in Table 2.  
Novozym 435 is the commercial preparation of Novozymes for immobilized CALB. The support used is Lewatit VP OC 1600, which is a macroporous, DVB-crosslinked polymer in spherical bead form, based on methacrylic esters, according to the supplier. Lewatit support has a particle size of 300-800 \(\upmu\)m, which is adequate for filtration recovery for multiple reuses [24]. MCI-CALB was prepared by the immobilization through interfacial activation of CALB on the MCI GEL CHP20P [22]. This support has a styrene-divinylbenzene matrix and it has a smaller particle diameter than Lewatit VP OC 1600, 75-150 \(\upmu\)m [24]. The structural differences of both supports, in terms of matrix and particle size, could affect the mechanical resistance, thus the operational stabilities. Comparing both preparations under mechanical stirring, MCI-CALB presented higher activity and operational stability  
Figure 4: Time course of ultrasound-assisted butyl acetate synthesis catalyzed by MCI-CALB under condition I (temperature, 48.8 \({}^{\circ}\)C; substrate molar ratio, 3.46:1 alcohol:acid; amount of biocatalyst, 7.5%; and added water 0.28%) at (\(\Circle\)) 2.0 M and (\(\blacksquare\)) 0.3M acetic acid. than Novozym 435. The better performance of MCI-CALB can be explained by the following facts. MCI GEL CHP20P has higher pore diameter than Lewatit VP OC 1600. The mean pore size of MCI GEL CHP20P is around 400-600 A [22], whereas for Lewatit VP OC 1600 it is around 150-300 A [24,25]. The higher pore size eases the substrates/products mass transfer through the support and may explain the higher activity of MCI-CALB. Additionally, Lewatit VP OC 1600 is by far more hydrophilic than styrene-divinylbenzene beads of MCI GEL CHP20P. This might facilitate the accumulation of water, acid, and alcohol in the enzyme environment, reducing enzyme operational stability. This accumulation of substrates and products in the enzyme environment may be strongly reduced using ultrasound stirring technology. Moreover, Lewatit VP OC 1600 has shown to have a relatively low mechanical resistance [24]. In stirred-reactor systems, fragmentation of the carrier can be observed, resulting in decreased enzymatic activity by failure in enzyme recovery [26]. Thus, the lower operational stability of Novozym 435 under mechanical stirring could also be attributed to fragmentation, whereas the ultrasound stirring does not promote such negative effect on the bead integrity. Nevertheless, it was observed that MCI-CALB did not present any significant advantage over Novozym 435 for the esterification reaction under ultrasound energy. There are also some possible reasons for this. Although MCI-CALB has higher pore size than Novozym 435, and this was an advantage under mechanical stirring, the use of ultrasound allowed better mixing in the reaction mixture, reducing the mass transfer limitations.