Kinetic and thermodynamic studies on the thermal inactivation of lipase immobilized on glutaraldehyde-activated rice husk silica.

The objective of this study was to obtain sufficient information on the thermal stabilization of a food-grade lipase from Thermomyces lanuginosus (TLL) using the immobilization technique. To do this, a new non-porous support was prepared via the sequential extraction of SiO2 from rice husks, followed by functionalization with (3-aminopropyl) triethoxysilane - 3-APTES (Amino-SiO2), and activation with glutaraldehyde - GA (GA-Amino-SiO2). We evaluated the influence of GA concentration, which varied from 0.25% v v-1 to 4% v v-1, on the immobilization parameters and enzyme thermal stabilization. The thermal inactivation parameters for both biocatalyst forms (soluble or immobilized TLL) were calculated by fitting a non-first-order enzyme inactivation kinetic model to the experimental data. According to the results, TLL was fully immobilized on the external support surface activated with different GA concentrations using an initial protein load of 5 mg g-1. A sharp decrease of hydrolytic activity was observed from 216.6 ± 12.4 U g-1 to 28.6 ± 0.9 U g-1 of after increasing the GA concentration from 0.25% v v-1 to 4.0% v v-1. The support that was prepared using a GA concentration at 0.5% v v-1 provided the highest stabilization of TLL - 31.6-times more stable than its soluble form at 60 °C. The estimations of the thermodynamic parameters, e.g., inactivation energy (Ed), enthalpy (ΔH#), entropy (ΔS#), and the Gibbs energy (ΔG#) values, confirmed the enzyme stabilization on the external support surface at temperatures ranging from 50 to 65 °C. These results show promising applications for this new heterogeneous biocatalyst in industrial processes given the high catalytic activity and thermal stability.

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.

Preparation of ion-exchange supports via activation of epoxy-SiO2 with glycine to immobilize microbial lipase - Use of biocatalysts in hydrolysis and esterification reactions.

Ion-exchange supports have been prepared via sequential functionalization of silica-based materials with (3­Glycidyloxypropyl)trimethoxysilane (GPTMS) (Epx-SiO2) and activation with glycine (Gly-Epx-SiO2) in order to immobilize lipase from Thermomyces lanuginosus (TLL) via adsorption. Rice husk silica (RHS) was selected as support with the aim of comparing its performance with commercial silica (Immobead S60S). Sequential functionalization/activation of SiO2-based supports has been confirmed by AFM, SEM and N2 adsorption-desorption analyses. Maximum TLL adsorption capacities of 14.8 ±â€¯0.1 mg/g and 16.1 ±â€¯0.6 mg/g using RHS and Immobead S60S as supports, respectively, have been reached. The Sips isotherm model has been used which was well fitted to experimental data on TLL adsorption. Catalytic activities of immobilized TLL were assayed by olive oil emulsion hydrolysis and butyl stearate synthesis via an esterification reaction. Hydrolytic activity of the biocatalyst prepared with a commercial support (357.6 ±â€¯11.2 U/g) was slightly higher than that of Gly-Epx-SiO2 prepared with RHS (307.4 ±â€¯7.2 U/g). On the other hand, both biocatalysts presented similar activity (around 90% conversion within 9-10 h of reaction) and reusability after 6 consecutive cycles of butyl stearate synthesis in batch systems.