Maria Carolina Cucatti Corradini

Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system

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.

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system

ABSTRACT The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL–PHB), absence of molecular sieves, agitation of 234 rpm, and 1,000 mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105 min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120 min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45 min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.

Optimization of Enzymatic Synthesis of n-Propyl Acetate (Fruit Flavor Ester) – Effect of the Support on the Properties of Biocatalysts

The present study deals with the optimization of the enzymatic synthesis of n-propyl acetate using response surface methodology (RSM). The biocatalysts were prepared by physical adsorption of lipase from Thermomyces lanuginosus (TLL) on mesoporous hydrophobic supports – poly-hydroxybutyrate (PHB) and poly-methacrylate (PMA) particles. Their catalytic properties were also assayed in the hydrolysis of olive oil emulsion. The biocatalyst TLL–PMA presented the highest immobilization yield (IY, 90.6 ± 2.8%), immobilized protein concentration (36.5 ± 0.4 mg/g of PMA), and catalytic activity in esterification reaction. TLL–PHB was more active in the hydrolysis of olive oil emulsion by a four-fold factor (1240.5 ± 29.2 IU/g of PHB). Maximum acid consumption percentage of 84.3% was observed after 50 min of reaction catalyzed by TLL–PMA using 2000 mM of each reactant (n-propanol and acetic acid) in heptane medium. The purified ester was confirmed by gas chromatography mass spectrometry (GC–MS) analysis. After six consecutive cycles of esterification reaction, the biocatalyst retained around 50% of its initial activity. The results show that PMA may be an interesting support to prepare active biocatalysts in the synthesis of fruit flavor ester by esterification reaction.