Green Synthesized Magnetic Nanoparticles as Effective Nanosupport for the Immobilization of Lipase: Application for the Synthesis of Lipophenols

Abstract

In this work, hybrid zinc oxide-iron oxide (ZnOFe) magnetic nanoparticles were synthesized employing Olea europaea leaf aqueous extract as a reducing/chelating and capping medium. The resulting magnetic nanoparticles were characterized by basic spectroscopic and microscopic techniques, namely, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), fourier-transform infrared (FTIR) and atomic force microscopy (AFM), exhibiting a spherical shape, average size of 15-17 nm, and a functionalized surface. Lipase from Thermomyces lanuginosus (TLL) was efficiently immobilized on the surface of ZnOFe nanoparticles through physical absorption. The activity of immobilized lipase was found to directly depend on the enzyme to support the mass ratio, and also demonstrated improved pH and temperature activity range compared to free lipase. Furthermore, the novel magnetic nanobiocatalyst (ZnOFe-TLL) was applied to the preparation of hydroxytyrosyl fatty acid esters, including derivatives with omega-3 fatty acids, in non-aqueous media. Conversion yields up to 90% were observed in non-polar solvents, including hydrophobic ionic liquids. Different factors affecting the biocatalyst performance were studied. ZnOFe-TLL was reutilized for eight subsequent cycles, exhibiting 90% remaining esterification activity (720 h of total operation at 50 °C). The green synthesized magnetic nanoparticles, reported here for the first time, are excellent candidates as nanosupports for the immobilization of enzymes with industrial interest, giving rise to nanobiocatalysts with elevated features.

Keywords: Olea europaea; bioactive lipophenols; biocatalysis; green synthesis; hydroxytyrosol; immobilization; lipase; magnetic nanoparticles.

Scheme 1

Schematic representation of ZnOFe biosynthesis from Olea europaea leaf extract (OLE). Phenols and flavonoids are the major constituents of aqueous OLE extract. The abundant medium served as a reducing/chelating agent for the biosynthesis of ZnOFe nanoparticles. After the bioreduction of metal ions (Zn²⁺, Fe²⁺) and growth of the resulted nanoparticles, specific compounds from the phytoconstituent-rich extract with different terminal groups (X,Y,Z: CO, COC, COOH, OH, C–C groups) act as stabilizers or capping agents, covering nanoparticles surfaces in order to prevent further agglomeration (red ring around nanoparticles).

Figure 1

The XRD pattern of ZnOFe nanoparticles.

Figure 2

Representative atomic force microscopy (AFM) height images (a) and cross section analysis profile (b) of ZnOFe nanoparticles deposited on Si-wafers by drop-casting from aqueous dispersions.

Figure 3

AFM height (a,b) and cross sectional analysis (c,d) images of ZnOFe-enzyme nanoparticles deposited on Si-wafers by drop-casting from aqueous dispersions.

Figure 4

Representative elemental mapping of ZnOFe nanoparticles (Zn, Fe, O, C), (a) scanning electron microscopy micrograph of sample in the same region, (b) EDS analysis.

Figure 5

XPS spectrum of (a): Fe2p, (b): Zn2p, (c): C1s Zn–iron oxide nanoparticles. (d): Comparative C1s spectrum of Zn–iron oxide nanoparticles after the enzyme immobilization.

Figure 6

FTIR spectra of ZnOFe-TLL compared to: pristine ZnOFe NPs and soluble TLL.

Figure 7

Effect of enzyme to nanosupport mass ratio on immobilized TLL activity (●) and immobilization yield (▪). One unit of enzymatic activity (U) was defined as the amount of lipase that liberates 1 μmol of p-NP per minute per mL of reaction at 40 °C.

Figure 8

(a) Effect of temperature on the hydrolytic activity of free (●) and immobilized TLL (▲) at pH 7.5; (b) Effect of pH on the hydrolytic activity of free (●) and immobilized TLL (▲) at 40 °C. The activity of the free enzyme on the optimal temperature and the optimal pH was taken as 100%.

Figure 9

Effect of solvent and reaction time on the bioconversion of hydroxytyrosol with oleic acid catalyzed by ZnOFe-TLL at 50 °C and 180 rpm.

Figure 10

Operational stability of ZnOFe-TLL towards the consistent synthesis of hydroxytyrosyl-oleate in MTBE (15 mg mL⁻¹, 72 h per cycle). The nanobiocatalytic system was retrieved after its cycle by applying a commercial magnet.