RESUMEN
n-Octyl oleate was synthetized by enzymatic esterification reaction of oleic acid and n-octanol. Lipases from porcine pancreatic (PPL), Mucor javanicus (MJL), Candida sp. (CALA), Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL) were immobilized via interfacial activation on poly-methacrylate particles (PMA) and tested as biocatalysts. Their catalytic properties were determined in the hydrolysis of olive oil emulsion. Among them, TLL-PMA was the biocatalyst that yielded the highest hydrolytic activity (217.8±1.1 IU/g) and immobilized protein loading (37.5±0.4mg/g). This biocatalyst was also the most active in n-octyl oleate synthesis, thus selected for further studies. Maximum conversion percentage of 95.1±1.3% was observed after 60min of reaction at 45°C, 10% m/v of TLL-PMA, and molar ratio oleic acid:n-octanol of 1:1.5 in a solvent-free system. The biocatalyst fully retained its original activity after twelve cycles of reaction of 60min each. The product was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy analysis and their physico-chemical properties were determined according to ASTM standard methods. These results show that the immobilization of an alkalophilic and thermostable lipase (TLL) on PMA particles allowed the preparation of a highly active biocatalyst in hydrolysis and esterification reactions.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Lipasa/metabolismo , Lubricantes/farmacología , Animales , Biocatálisis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Enzimas Inmovilizadas/metabolismo , Esterificación/efectos de los fármacos , Hidrólisis , Cinética , Ácido Oléico/metabolismo , Ácidos Polimetacrílicos/química , Espectroscopía Infrarroja por Transformada de Fourier , Sus scrofa , TemperaturaRESUMEN
Lipase from Thermomyces lanuginosus (TLL) was immobilized on mesoporous hydrophobic poly-methacrylate (PMA) particles via physical adsorption (interfacial activation of the enzyme on the support). The influence of initial protein loading (5-200mg/g of support) on the catalytic properties of the biocatalysts was determined in the hydrolysis of olive oil emulsion and synthesis of isoamyl oleate (biolubricant) by esterification reaction. Maximum adsorbed protein loading and hydrolytic activity were respectively ≈100mg/g and ≈650 IU/g using protein loading of 150mg/g of support. The adsorption process followed the Langmuir isotherm model (R(2)=0.9743). Maximum ester conversion around 85% was reached after 30min of reaction under continuous agitation (200rpm) using 2500mM of each reactant in a solvent-free system, 45°C, 20%m/v of the biocatalyst prepared using 100mg of protein/g of support. Apparent thermodynamic parameters of the esterification reaction were also determined. Under optimal experimental conditions, reusability tests of the biocatalyst (TLL-PMA) after thirty successive cycles of reaction were performed. TLL-PMA fully retained its initial activity up to twenty two cycles of reaction, followed by a slight decrease around 8.6%. The nature of the product (isoamyl oleate) was confirmed by attenuated total reflection Fourier transform infrared (ATR-FTIR), proton ((1)H NMR) and carbon ((13)C NMR) nuclear magnetic resonance spectroscopy analyses.
Asunto(s)
Ascomicetos/enzimología , Proteínas Fúngicas/metabolismo , Lipasa/metabolismo , Adsorción , Biocatálisis , Enzimas Inmovilizadas/metabolismo , Esterificación , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Lubricantes/metabolismo , Ácidos Oléicos/metabolismo , Ácidos Polimetacrílicos , SolventesRESUMEN
Isoamyl butyrate (banana flavor) was synthesized by esterification reaction of isoamyl alcohol and butyric acid in heptane medium. Immobilized Thermomyces lanuginosus lipase (TLL) prepared via physical adsorption on mesoporous poly-methacrylate particles (PMA) was used as biocatalyst. The factors that affect the esterification reaction were optimized by response surface methodology (RSM). Under optimal experimental conditions, maximum ester conversion percentage of 96.1 and 73.6% was reached after 50 and 90 min, respectively, for esterification reaction performed at equimolar ratio alcohol:acid at 500 and 2000 mM of each substrate. Under these experimental conditions, the esterification reaction was not controlled by external and intra-particle mass transfer effects. The product (isoamyl butyrate) was confirmed by proton nuclear magnetic resonance ((1)H NMR) spectroscopy. Reusability tests showed that the biocatalyst retained around 96 and 31% of its initial activity after eight successive esterification cycles performed at 500 and 2000 mM, respectively. The application of the biocatalyst prepared showed to be a promising strategy to catalyze flavor ester synthesis in a non-aqueous medium.