Tubular and Spherical SiO₂ Obtained by Sol Gel Method for Lipase Immobilization and Enzymatic Activity

Abstract

A wide range of hybrid biomaterials has been designed in order to sustain bioremediation processes by associating sol-gel SiO₂ matrices with various biologically active compounds (enzymes, antibodies). SiO₂ is a widespread, chemically stable and non-toxic material; thus, the immobilization of enzymes on silica may lead to improving the efficiency of biocatalysts in terms of endurance and economic costs. Our present work explores the potential of different hybrid morphologies, based on hollow tubes and solid spheres of amorphous SiO₂, for enzyme immobilization and the development of competitive biocatalysts. The synthesis protocol and structural characterization of spherical and tubular SiO₂ obtained by the sol gel method were fully investigated in connection with the subsequent immobilization of lipase from Rhizopus orizae. The immobilization is conducted at pH 6, lower than the isoelectric point of lipase and higher than the isoelectric point of silica, which is meant to sustain the physical interactions of the enzyme with the SiO₂ matrix. The morphological, textural and surface properties of spherical and tubular SiO₂ were investigated by SEM, nitrogen sorption, and electrokinetic potential measurements, while the formation and characterization of hybrid organic-inorganic complexes were studied by UV-VIS, FTIR-ATR and fluorescence spectroscopy. The highest degree of enzyme immobilization (as depicted from total organic carbon) was achieved for tubular morphology and the hydrolysis of p-nitrophenyl acetate was used as an enzymatic model reaction conducted in the presence of hybrid lipase⁻SiO₂ complex.

Keywords: SiO2; enzymatic catalysis; lipase immobilization; tubular and spherical morphology.

Figure 1

(a–c) SEM micrographs at different magnifications and (d) EDX spectrum of SiO₂-t.

Figure 2

(a,b) SEM micrographs of SiO₂-T at different magnifications, showing the hollow tubes structure.

Figure 3

(a,b) SEM micrographs of SiO₂-S at different magnifications showing the spherical morphology.

Figure 4

N₂ adsorption-desorption isotherms (a) and the pore size distribution obtained from the desorption branch (b) for the SiO₂-t samples before lipase immobilization; (c,d)—the same curves recorded after lipase immobilization.

Figure 5

N₂ adsorption-desorption isotherms (a) and the pore size distribution obtained from the desorption branch (b) for the SiO₂-T samples before lipase immobilization; (c,d)—the same curves recorded after lipase immobilization.

Figure 6

N₂ adsorption-desorption isotherms (a) and the pore size distribution obtained from the adsorption branch (b) for the SiO₂-S samples before enzymatic immobilization.

Figure 7

Comparative FTIR spectra of SiO₂ samples, (prior to lipase immobilization) with different morphology: thin tubes (SiO₂-t), bigger tubes (SiO₂-T) and spherical particles (SiO₂-S). Inset shows in detail the shoulder appearing at ~960 cm⁻¹.

Figure 8

FTIR-ATR spectra for lipase and SiO₂ samples in K₂HPO₄ buffer solution after immobilization: (a) recorded from supernatant—with two Gaussian signals deconvolution (Peakfit program) revealing the 3271 cm⁻¹ peak, and (b) from solid powder.

Figure 9

Normalized fluorescence of (о) free and (□) nanoparticle-bound lipase of the samples (a) SiO₂-T, (b) SiO₂-S and (c) SiO₂-t, for λ_exc = 270 nm. (d) Fluorescence spectrum registered for SiO₂-t bound lipase for λ_exc = 320 nm.

Figure 10

Soluble TOC values normalized to initial lipase concentration in buffer solution, registered for triplicate samples (three specimens for each morphology).

Figure 11

The different quantitative lipase loading (including the error bars of each sample) on SiO₂ matrices, obtained for a triplicate set of samples.

Figure 12

The electrokinetic potential of the SiO₂ matrices and their derivative hybrid systems, registered for a triplicate set of samples.

Figure 13

p-nitrophenol acetate (p-NPA) hydrolysis product after 1 h of incubation; Inset: calibration of p-NP concentration from UV-VIS.

Figure 14

p-NP concentration (μM) resulted from p-NPA hydrolysis reaction conducted in the presence of immobilized lipase on SiO₂ samples. The reaction was performed in triplicate, error bars representing the deviation for three catalytic independent runs.

Figure 15

Specific activity (μM/μg) of immobilized lipase on SiO₂ samples for p-NPA hydrolysis reaction. Inset: Biological activity of loaded lipase expressed in Units; 1 lipase unit (U) represents the amount (mg) of enzyme liberating 1 μmol p-NP per minute. All experiments were done in triplicate.