Síntese enzimática de um biossurfactante por esterificação em meio orgânico - otimização, caracterização estrutural e avaliação de sua atividade antimicrobiana

Abstract

The present study aimed the preparation of heterogeneous biocatalysts by interfacial activation of three different lipases – Thermomyces lanuginosus (TLL), Candida rugosa type VII (CRL), and Pseudomonas fluorescens (PFL), using rice husk silica functionalized with triethoxy(octyl)silane (Octyl–SiO2). Immobilization parameters were determined in the hydrolysis of olive oil emulsion and concentration of adsorbed protein. The prepared biocatalysts were used in the esterification of palmitic acid and DL-Isopropylidenoglycerol (Solketal) in organic medium for the synthesis of solketila palmitate, an ester with emulsifying properties. According to the results, maximum immobilized protein loading of 20–22 mg of lipase/g of support was obtained, independently of the lipase source. TLL–Octyl–SiO2 was the most active biocatalyst in oil hydrolysis (656 ± 24 U/g) and ester synthesis (productivity of 7 mmol/min.gbiocat), and it has been chosen for further ester optimization. The effect of some important parameters such as biocatalyst concentration, reaction temperature and acid:alcohol molar ratio on ester synthesis was evaluated using a Central Composite Rotatable Design (CCRD), keeping mechanical stirring (240 rpm) and reaction time (15 min) fixed. Subsequently, the effect of reactants concentration and molecular sieve concentration was also examined. Under optimal reaction conditions (56 °C, absence of molecular sieve, acid:alcohol molar ratio of 1:3 with initial palmitic acid concentration of 1 mol/L and 800 units of hydrolytic activity (U), that corresponds to 20% wt. of TLL–Octyl–SiO2 per volume of reaction mixture), maximum acid conversion of 83% was obtained after 150 min of reaction. The biocatalyst was highly stable, retaining 87% of its initial activity after seven successive reaction batches. The product (solketila palmitate) was identified by nuclear magnetic resonance (NMR) analysis. Antimicrobial activity studies showed that the synthesized ester demonstrated antifungal activity against Candida albicans and Candida parapsilosis, with a minimum inhibitory concentration (MIC) between 200 and 400 µg/mL, and bacteriostatic/fungistatic action – minimum microbicidal concentration (MMC) >400 µg/mL.