BACKGROUND The objective of this work was to evaluate the activation of Candida antarctica lipase B (CALB) in pressurized CO2 or liquefied petroleum gas (LPG) and, thereafter, to evaluate the synthesis of esters using activated CALB as a biocatalyst. Before activation, CALB was immobilized in polyurethane. For the esterification reactions, mechanical or ultrasonic agitations were studied. The stability of CALB after reuse cycles was also evaluated. RESULTS Before activation, the enzymatic activity of immobilized CALB was 789 U g‐1. After activation in pressurized CO2 and LPG, the activities increased to 2486 U g‐1 and 1413 U g‐1, respectively. Consequently, the residual activities of CALB after activation in pressurized CO2 and LPG were 315% and 179%, respectively. For the best condition of reaction under ultrasonic agitation, oleic acid conversion was 51.2% when using non‐activated CALB. Otherwise, oleic acid conversions were 66.5% and 77.1% when using CALB activated in LPG and CO2, respectively. This biocatalyst maintained more than 80% of its residual activity when reused for up to 13 cycles. CONCLUSION Activation of CALB in pressurized CO2 provided enzymatic activities 1.76 times higher than activation in pressurized LPG did. Immobilized CALB had good performance as a stable biocatalyst for esterification reactions under ultrasonic agitation. © 2017 Society of Chemical Industry

Synthesis of isoamyl acetate by mechanical and ultrasonic solvent‐free agitations using Candida antarctica lipase B (CALB) as biocatalyst immobilized in polyurethane was studied. Temperature (24.8–75.2 °C), ultrasonic power (35–123 W; only in ultrasonic system) and molar ratio of substrates (1:3.64–1:10.36) were the parameters evaluated. The responses taken into account were the acetic acid conversion in the esterification reaction (21–95%) and the residual conversion of substrates after successive reuse cycles of CALB. Ultrasonic system was advantageous because the substrate conversions into isoamyl acetate were reached six times earlier than in mechanical system. An increase in the temperature and ultrasonic power and a reduction in acetic acid : isoamyl alcohol ratio favored an increase in conversion yields. CALB used in ultrasonic reactions maintained more than 60% of its residual activity after 27 reuse cycles, which was more than thrice (eight reuse cycles) when using CALB in mechanical reactions. Practical applications The practical uses of isoamyl acetate are mainly as a food additive and as a solvent in the aircraft industry. In food‐related areas, this ester is used to confer characteristics such as banana (or pear) smell and flavor in foods and beverages. It has a pleasant odor and low toxicity, which encourages its widespread use in different receipts, including in the formulation of beers.

The Candida antarctica lipase B immobilized on polyurethane catalyzed of esterification butanoic acid with ethyl alcohol in mechanical and ultrasonic system in a system solventfree, was studied. The maximum of ethyl butanoate esterification obtained 666.40 and 585.84 U.g -1 , whit 6 cycles de reuse in mechanical agitation (160 rpm) and mechanical ultrasound system (maximum power 1800 A, US 40 KHz, US 132 W) after 180 minutes of reaction time. The process was considered efficient with significant reduction of the reaction time, low instrumental requirements and improve the bioprocess performance. Until now, there were no studies available in the open literature in relation to the ester synthesis catalyzed by immobilized lipase in polyurethane as support in the ultrasound system. In addition, considered an environmentally correct and economically viable technology, it can be used in cosmetics, pharmaceuticals and food industry.

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RESUMO -As lipases podem ser eficientemente aprisionadas nos poros de silicatos hidrofóbicos obtidos pelo processo sol-gel. Este método é simples e barato, em que uma mistura de um precursor da sílica é hidrolisada por iniciadores da reação de polimerização na presença da enzima. Aditivos, tais como polietilenoglicol (PEG), aumentam a eficiência deste tipo de imobilização da lipase. A principal área de aplicação destes biocatalisadores heterogêneos diz respeito esterificação ou transesterificação em solventes orgânicos. Para a imobilização, o percursor da sílica tetraetilortosilicato (TEOS) foi dissolvido em álcool etílico absoluto, e testados os três diferentes iniciadores da reação de polimerização: ácido (HCl), básico (HBr) e nucleofílico (NH 4 OH). As determinações das atividades de esterificação foram realizadas na síntese de oleato de etila na solução enzimática (enzima livre) e nos xerogéis imobilizados armazenados em temperatura ambiente e refrigeração. Os resultados dos ensaios de estabilidade confirmaram a tendência do processo de imobilização de enzimas, ou seja, a imobilização conferiu uma maior estabilidade da enzima imobilizada comparada com a enzima livre. No estudo foi possível verificar, também, que a presença de aditivo PEG influenciou no aumento do número de ciclos e os suportes mantiveram-se estáveis por mais tempo armazenada em refrigerador quando se utilizou os iniciadores da reação de polimerização nucleofílico e básico. INTRODUÇÃOA técnica de imobilização apresenta-se como uma alternativa para contornar limitações reacionais, pois permite a separação da enzima e, consequentemente, a sua utilização mais que uma vez, além de possibilitar uma maior estabilidade à enzima (Sabbani et al., 2006; Wang e Hsieh, 2008).No processo de imobilização, além das questões econômicas, devem ser consideradas as questões operacionais, como as condições experimentais empregadas na síntese, à facilidade de produção, o tempo envolvido no processo e quantidade de enzima incorporada. Dentre os diferentes métodos destaca-se a imobilização em matrizes obtidas pela técnica sol-gel (Pinheiro et al., 2008;Souza et al., 2013).

The Candida antarctica Lipase B immobilized on polyurethane catalysed esterification of acetic acid with isoamyl alcohol in mechanical and ultrasonic system in a system solvent-free, was studied. The maximum esterification of isoamyl acetate used immobilized catalyst was the highest esterification, 666.05 U/g with 19 cycles of reuse after 360 minutes of reaction time in mechanical system and 1319.48 U/g was observed and 14 cycles de reuse after 60 minutes of reaction time in ultrasound system. Thus, the process was considered efficient with significant reduction of the reaction time, low instrumental requirements and improve the bioprocess performance. Until now, there were no studies available in the open literature in relation to the ester synthesis catalyzed by immobilized Lipase in polyurethane as support in the ultrasound system. In addition, considered an environmentally correct and economically viable technology, it can be used in cosmetics, pharmaceuticals and food industry.