The influence of low-frequency ultrasound (40 kHz) in the esterification reaction between acetic acid and butanol for flavor ester synthesis catalyzed by the commercial immobilized lipase B from Candida antarctica (Novozym 435) was evaluated. A central composite design and the response surface methodology were used to analyze the effects of the reaction parameters (temperature, substrate molar ratio, enzyme content and added water) and their response (yields of conversion in 2.5 h of reaction). The reaction was carried out using n-hexane as solvent. The optimal conditions for ultrasound-assisted butyl acetate synthesis were found to be: temperature of 46 °C; substrate molar ratio of 3.6:1 butanol:acetic acid; enzyme content of 7%; added water of 0.25%, conditions that are slightly different from those found using mechanical mixing. Over 94% of conversion was obtained in 2.5h under these conditions. The optimal acid concentration for the reaction was determined to be 2.0 M, compared to 0.3 M without ultrasound treatment. Enzyme productivity was significantly improved to around 7.5-fold for each batch when comparing ultrasound and standard mechanical agitation. The biocatalyst could be directly reused for 14 reactions cycles keeping around 70% of its original activity, while activity was virtually zeroed in the third cycle using the standard mixing system. Thus, compared to the traditional mechanical agitation, ultrasound technology not only improves the process productivity, but also enhances enzyme recycling and stability in the presence of acetic acid, being a powerful tool to improve biocatalyst performance in this type of reaction.

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BACKGROUND: In this work two immobilized preparations of lipase (EC 3.1.1.3) B from Candida antarctica (CALB) were compared as biocatalysts in the synthesis of ethyl butyrate, a short-chain esters with fruity notes. Commercial Novozym 435 and CALB immobilized on styrene-divinylbenzene beads (MCI-CALB) were tested for esterification reactions. Central composite design and response surface methodology were used to optimize the reaction temperature, substrate molar ratio, enzyme content, and the added water. RESULTS: The two enzymatic preparations presented different optimal conditions concerning ethyl butyrate production, with higher yields of conversion around 85% in 1.5 h being achieved. However, MCI-CALB presented productivities 1.6 times higher than Novozym 435. The main difference between the biocatalysts was in relation to operational stability during batch reuse experiments, in which MCI-CALB retained 80% of its initial activity after eight batches, while Novozym 435 retained only 20% under the same conditions. CONCLUSION: It was verified that variations in the protocols for enzyme immobilization causes different optimal conditions for the esterification reaction. These are very interesting results because reaction times were short, producing high conversion yields and productivities considering the mass of biocatalyst used.

Butyl butyrate is an ester present in pineapple flavor, which is very important for the food and beverages industries. In this work, the optimization of the reaction of butyl butyrate synthesis catalyzed by the immobilized lipase Lipozyme TL-IM was performed. n-Hexane was selected as the most appropriate solvent. Other reaction parameters such as temperature, substrate molar ratio, biocatalyst content and added water, and their responses measured as yield, were evaluated using a fractional factorial design, followed by a central composite design (CCD) and response surface methodology. In the fractional design 2(4-1) , the four variables were tested and temperature and biocatalyst content were statistically significant and then used for optimization on CCD. The optimal conditions for butyl butyrate synthesis were found to be 48°C; substrate molar ratio 3:1 (butanol:butyric acid); biocatalyst content of 40% of acid mass. Under these conditions, over 90% of yield was obtained in 2 h. Enzyme reuse was tested by washing the biocatalyst with n-hexane or by direct reuse. The direct reuse produced a rapid decrease on enzyme activity, while washing with n-hexane allowed reusing the enzyme for five reactions cycles keeping approximately 85% of its activity.