Enzymatic synthesis of Hydroxytyrosol from Oleuropein for valorization of an agricultural waste

Abstract

Oleuropein (OP) is an appreciated compound present not only in fruits but also in leaves of olive trees, which can be transformed into hydroxytyrosol (HT), a substance with high antioxidant activity. In this work, the transformation of an agricultural residue containing OP (olive leaves or wastewater from mills) to the high added value compound HT is accomplished through different enzymatic strategies. Different enzymes were used, immobilized on various supports by diverse binding forces: beta-glucosidase encapsulated in siliceous material, esterases and lipases immobilized on hydrophobic supports (octyl-functionalized amorphous silica and periodic mesoporous organosilica), and esterase immobilized on amine-functionalized ordered mesoporous silica. All these biocatalysts were tested for oleuropein hydrolysis through two different reaction approaches: a) split of glucosidic bond catalyzed by beta-glucosidase (β-glu), followed by hydrolysis of the aglycon and further ester hydrolysis. 5 mg·mL^-1 of β-glu fully hydrolyzed 5 mM OP at pH 7 and 50°C in 7 days, and further enzymatic hydrolysis of the aglycon yielded near to 0.5 mM HT in the best conditions tested. b) via direct hydrolysis of the ester bond to produce hydroxytyrosol in a one-step reaction using esterases or lipases. The latter reaction pathway catalyzed by lipase from Penicillium camemberti immobilized on octyl-silica (4 mg·mL^-1) at 35°C and pH 6 directly produced 6.8 mM HT (1 mg·mL^-1), transforming in 12 days near to 30% of the initial 25 mM OP from a commercial olive leaves extract.

Keywords: Oleuropein; enzymatic transformation; hydroxytyrosol; immobilized enzymes; waste valorization.

Graphical abstract

Scheme 1.

Enzyme catalyzed reaction pathways tested in this work for the production of hydroxytyrosol from oleuropein.

Figure 1.

Enzyme leaching from biocatalysts through a 48 h period at pH 6.0 and temperature of 35°C. Symbols correspond to experimental data. Lines are a guide to the eye.

Figure 2.

Oleuropein conversion during hydrolysis under different conditions. a) hydrolysis of 20 mM OP solutions at 40°C and pH 5.0 (squares), 6.0 (circles), 6.5 (stars) and 7.0 (triangles) using a biocatalyst ratio of 5 (full symbols) and 10 mg·mL⁻¹ (open symbols). b) hydrolysis in solutions with OP concentration of 20 (triangles), 5 (circles) and 2.5 mM (squares) at 40°C and pH 7.0 using 5 mg·mL⁻¹ biocatalyst ratio. c) hydrolysis of 5 mM OP solutions using 5 mg·mL⁻¹ biocatalyst ratio at pH 7.0 (triangles) and 6.5 (stars) and a temperature of 40 (full symbols) and 50°C (open symbols). Symbols correspond to experimental data. Lines are a guide to the eye.

Figure 3.

HT obtained with various lipases immobilized on OAS. HT concentration in the OP solution is shown for each biocatalyst before and after 24 h incubation. Control: OP solution without enzyme. Conditions were room temperature, pH 7.0, 12 mM OP and 2 mg·mL⁻¹ biocatalyst.

Figure 4.

HT obtention using biocatalysts prepared with OAS or PMO supports. (a) Lipase G, 0.09 mg of enzyme per mL of suspension. (b) Lipase CaLB, 0.2 mg of enzyme per mL of suspension (* 0.09 mg of enzyme per mL of suspension). Control: OP solution without enzyme. Conditions were 40°C, pH 7, and 12 mM OP solution. Lines are a guide to the eye.

Figure 5.

HT obtention at different temperatures using biocatalysts prepared with OAS support. (a) Lipase G. (b) Lipase CaLB. Conditions were pH 7, 12 mM OP, 2 mg·mL⁻¹ biocatalyst and temperatures of 30°C (squares), 35°C (circles) and 40°C (triangles). Lines are a guide to the eye.

Figure 6.

HT obtention with different concentrations of OP and biocatalysts prepared with OAS support. (a) Lipase G. (b) Lipase CaLB. Conditions were pH7, temperature of 35°C, 12 mM (circles) or 25 mM OP (triangles) and 2 mg·mL⁻¹ (full symbols) or 4 mg·mL⁻¹ biocatalyst (open symbols). Lines are a guide to the eye.

Figure 7.

OP degradation in control solutions at different pH values. Conditions were temperature of 35°C, 12 mM OP, no biocatalysts added and pH of 6 (squares), 7 (circles) or 8 (triangles). Lines are a guide to the eye.

Figure 8.

OP conversion and HT production in enzymatic reactions under best performing conditions through a 288 h incubation period using biocatalysts prepared with OAS and PMO supports. (a) Esterase E/OAS (rhombus). (b) Lipases G (squares) and CaLB (triangles) on PMO support. (c) Lipases G (squares) and CaLB (triangles) on OAS support. Conditions were pH 6, temperature of 35°C, 25 mM OP and 4 mg·mL⁻¹ biocatalyst. Results of control test (without biocatalyst addition) are shown as open circles and broken lines. Lines are a guide to the eye.