Preparação e caracterização de um suporte heterofuncional à base de quitosana e seu uso na imobilização de lipase

Abstract

In this study, a new mixed heterofunctional support (Chit–GA–Gly) has been prepared by sequential activation of chitosan hydrogel (Chit) with glutaraldehyde (GA) and further functionalization with glycine (Gly). The immobilization of the lipase from Thermomyces lanuginosus (TLL) on this support was compared with that on GA-activated Chit hydrogel (Chit–GA). The supports have been characterized by FT–IR, zeta potential and TG analyses to confirm the introduction of functional groups on the support surfaces. Initially, the effect of immobilization pH on TLL immobilization process has been evaluated for the different prepared supports using a low protein loading (5 mg/g). These tests have been performed at low ionic strength (5 mM) using a volume ratio support:lipase solution of 1:20, 18 h of contact and mechanical agitation in an orbital shaker at 200 rpm. Complete immobilization of TLL on the new support (Ghit-GA-Gly) has been observed at acidic medium (pH range from 3.0 to 6.0). On the other hand, TLL was fully immobilized on Chit–GA under a broad pH range varying from 4.0 to 10.0. The immobilization of TLL on non-chemically modified chiotsan hydrogel (Chit) was not possible at acidic medium (pH 4.0) due to a partial solubilization of the support and maximum immobilized protein concentration of 1.5 mg/g of hydrogel was obtained at pH 7.0. The effect of offered protein loading varying from 5 to 70 mg/g was also studied and a similar maximum lipase loading of 53-55 mg per gram of support has been obtained for both supports (Chit-GA and Chit-GA-Gly). Langmuir isotherm model obtained better fit to the experimental data obtained by both prepared supports The adsorption process of TLL on the new prepared support was better fitted to the Langmuir isotherm model on account of achieving higher correlation coefficient (R

2 = 0.9545). This model assumes monolayer adsorption of TLL molecules on a support surface and the adsorption of each lipase molecule on the adsorbent surface has the same adsorption activation energy. The catalytic activity (olive oil emulsion hydrolysis) of the prepared heterogeneous biocatalyst

using Chit-GA-Gly as support was 4-times higher than TLL immobilized on classical Chit- GA – 1000 U/g and 265.1 ± 14.4 U/g, respectively. Both biocatalysts retained ≈40% of their

initial activity after 48 h of incubation at 50°C in apolar organic solvents (heptane, toluene or isooctane). Desorption tests performed under different conditions (NaCl + Triton X-100 at different concentrations) revealed that TLL was preferentially immobilized on Chit-GA via covalent attachment (95%). On the other hand, TLL was immobilized on Chit-GA-Gly via ionic interactions (65%), followed by interfacial activation (hydrophobic interactions (20%) and covalent attchment (15%). The catalytic activity of the prepared biocatalysts on the esterification of palmitic acid with several alcohols (isoamyl alcohol, hexanol, 2-ethyl-1- hexanol and decanol) in isooctane medium, apolar organic solvent chosen in stability tests, was also performed. The esterification reactions were conducted under fixed experimental conditions: 50 oC, stoichiometric acid:alcohol molar ratio (500 mM of each reactant) at 70 min of reaction under mechanical agitation in an orbital shaker at 240 rpm. TLL immobilized on Chit–GA–Gly was ≈4-times more active than when immobilized on Chit–GA in both olive oil emulsion hydrolysis and alkyl palmitate synthesis via esterification. Maximum acid conversion of 80.3 ± 0.6% and 23.7 ± 2.5% using TLL immobilized on Chit-GA-Gly and Chit-GA, respectively, was achieved for isoamyl alcohol. In That way, isoamyl palmitate synthesis in iso-octane at 50 oC using this new biocatalyst gave a maximum acid conversion of 85% after 90 min of reaction. After nine consecutive esterification batches, the biocatalyst retained around 40% of its initial activity. These results show clearly that the new biocatalyst prepared in this study is promising to catalyze reactions of industrial interest in both aqueous (olive oil emulsion hydrolysis) and organic (lubricant ester production) media.