Enzymatic production of wax esters by esterification using lipase immobilized via physical adsorption on functionalized rice husk silica as biocatalyst.

The present study consists of developing an enzymatic process for the production of wax esters (lauryl stearate and cetyl stearate) by esterification in a heptane medium. Lipase from Thermomyces lanuginosus (TLL) immobilized via interfacial activation on silica particles from rice husks functionalized with triethoxy(octyl)silane (TLL-Octyl-SiO2 ) was used as biocatalyst. Maximum immobilized protein loading of around 22 mg g-1 (that corresponds to an immobilization yield of ≈55%) of support was observed using an initial protein loading of 40 mg g-1 of Octyl-SiO2 . Its hydrolytic activity (olive oil emulsion hydrolysis) was of 620 U g-1 of biocatalyst. The effect of certain factors on the cetyl estearate production was evaluated using a central composite rotatable design (CCDR). Under optimal conditions (64°C, 21% of mass of biocatalyst per volume of reaction mixture, 170 rpm, and stoichiometric acid:alcohol molar ratio 1 mol L-1 of each reactant), maximum acid conversion percentage of 91% was observed after 60 min of reaction. Lauryl stearate was also produced under such conditions, and an acid conversion of 93% after 60 min of reaction was also achieved. Free lipase exhibited acid conversion of only 15%-20% for both reaction mixtures. After nine successive esterification batches, TLL-Octyl-SiO2 retained 85%-90% of its original activity. These results show the promising use of the prepared biocatalyst in wax esters production due to its high catalytic activity and reusability.

Preparation, functionalization and characterization of rice husk silica for lipase immobilization via adsorption.

Silica has been extracted from rice husks via a simple hydrothermal process and functionalized with triethoxy(octyl)silane -OCTES (Octyl-SiO2) and (3-aminopropyl)triethoxysilane - 3-APTES (Amino-SiO2), with the aim of using it as support to immobilize lipase from Thermomyces lanuginosus (TLL) via adsorption. The supports have been characterized by particle size distribution and elemental analyses, XRD, TGA, SEM, AFM and N2 physisorption so as to confirm their functionalization. Effect of pH, temperature, initial protein loading and contact time on the adsorption process has been systematically evaluated. Maximum immobilized protein loading of 12.3 ± 0.1 mg/g for Amino-SiO2 (5 mM buffer sodium acetate at pH 4.0, 25 °C and initial protein loading of 20 mg/g) and 21.9 ± 0.1 mg/g for Octyl-SiO2 (5 mM buffer sodium acetate at pH 5.0, 25 °C and initial protein loading of 30 mg/g) was observed. However, these biocatalysts presented similar catalytic activity in olive oil emulsion hydrolysis (between 630 and 645 U/g). TLL adsorption was a spontaneous process involving physisorption. Experimental data on Octyl-SiO2 and Amino-SiO2 adsorption were well-fitted to the Langmuir isotherm model. It was also investigated whether these biocatalysts could synthesize cetyl esters via esterification reaction. Thus, it was found that cetyl stearate synthesis required 100-110 min of reaction time to attain maximum conversion percentage (around 94%). Ester productivity of immobilized TLL on Amino-SiO2 was 1.3-3.1 times higher than Octyl-SiO2.