Produção de um antioxidante lipofílico por esterificação catalisada por lipase imobilizada via adsorção física em suporte hidrofóbico

Abstract

The objective of this project consisted of the enzymatic production of isoamyl gallate, an ester with antioxidantant and antimicrobial properties, via direct esterification reaction of isoamyl alcohol, a renewable alcohol from fusel oil, with gallic acid (3,4,5- trihydroxybenzoic acid) performed in tert-butanol system. Preliminary tests performed in our lab using several crude lipase extracts (soluble or poder extracts) as biocatalysts showed that lipase from Pseudomonas fluorescens (PFL), commercially known as Lipase Amano AK, gave the highest catalytic activity, thus selected to perform the present project. This lipase was used in its immobilized form via physical adsorption by mechanism of interfacial activation on poly(styrene-divinylbenzene) particles. This support has been chosen due to its high capacity to adsorb high lipase amount, an important requisite for preparing active biocatalysts. The heterogeneous biocatalyst (immobilized lipase) was prepared in buffer sodium acetate solution pH 5.0 (5 mM) by 18h of contact time using an initial protein loading of 40 mg per gram of support. High enzyme loaded biocatalyst (immobilized protein concentration around of 33 mg/g of support) was obtained using an initial protein loading of 40 mg g -1 . Its hydrolytic activity was of 121.4 ± 5.5 U/g, which was determined on the hydrolysis of olive oil emulsiona at pH 8.0 (buffer sodium phosphate using an ionic strenght of 100 mM) and 37°C. Subsequently, the effect of relevant factors such as biocatalyst concentration (5% - 25% m/v), reaction temperature (25°C a 70ºC), and acid:alcohol molar ratio on the esterification reactions was evaluated using a central composite rotatable design (CCRD). This set of experiments was performed under fixed conditions: 30 minutes of reaction time, mechanical stirring of 200 rpm, and acid concentration of 250 mM. Acid conversion percentage will be taken as response (dependent variable). Maximum acid conversion of only 37% was observed using crude lipase extract under such experimental conditions. The prepared biocatalyst (immobilized PFL) retained 40% of its original activity after five consecutive esterification batches of 90 minutes each. These resultsshow that a proper of 90 minutes immobilization protocol may improve its catalytic performance and reusability after successive batches of ester production via esterification in a solvent system.