Recovery of Hydroxytyrosol from Olive Mill Wastewater Using the Promiscuous Hydrolase/Acyltransferase PestE

Abstract

Olive mill wastewater (OMWW) is produced annually during olive oil extraction and contains most of the health-promoting 3-hydroxytyrosol of the olive fruit. To facilitate its recovery, enzymatic transesterification of hydroxytyrosol (HT) was directly performed in an aqueous system in the presence of ethyl acetate, yielding a 3-hydroxytyrosol acetate rich extract. For this, the promiscuous acyltransferase from Pyrobaculum calidifontis VA1 (PestE) was engineered by rational design. The best mutant for the acetylation of hydroxytyrosol (PestE_ I208A_L209F_N288A) was immobilized on EziG 2 beads, resulting in hydroxytyrosol conversions between 82 and 89 % in one hour, for at least ten reaction cycles in a buffered hydroxytyrosol solution. Due to inhibition by other phenols in OMWW the conversions of hydroxytyrosol from this source were between 51 and 62 %. In a preparative scale reaction, 13.8 mg (57 %) of 3-hydroxytyrosol acetate was extracted from 60 mL OMWW.

An Enzyme Cascade Reaction for the Recovery of Hydroxytyrosol Dervatives from Olive Mill Wastewater

“…OMWW samples were collected from different three-phase olive oil mills on Crete (Greece) and prepared as reported previously [11]. Ethyl acetate was purchased from VWR (Darmstadt, Germany) in HPLC grade (99.8+ %).…”

“…The residence time on the column was approximately 10 min and the system was operated at room temperature. The ethyl acetate phase was separated, dried with anhydrous Na 2 SO 4 and analyzed by gas chromatography, as described previously [11].…”

“…While various methods such as chemical oxidation, solvent extraction, membrane systems, or adsorbents have been investigated for phenol removal, open pond treatment is still widely used [4][5][6][7][8][9][10]. Recently, our group developed a system for the lipophilization and recovery of the main phenol 3-hydroxytyrosol (HT) in a biphasic ethyl acetate/ OMWW system using an engineered variant of the promiscuous hydrolase/acyltransferase from the hyperthermophilic archaeum Pyrobaculum calidifontis VA1 (PestE) [11]. In contrast to commonly used lipases, promiscuous hydrolases/acyltransferases such as PestE can perform efficient transesterification reactions in bulk water [12,13].…”

“…Moreover, PestE is a hyperthermostable enzyme that shows no decrease in activity at 100 °C [14] and can even be used in pure organic solvent [15]. Using the PestE variant optimized for HT, PestE_I208A_L209A_N288A, which was immobilized on EziG 2 beads, 265 mg L OMWW -1 HT and hydroxytyrosol acetate (HTA) were recovered from untreated OMWW [11]. It was possible to reuse the immobilized PestE_I208A_L209A_N288A for at least ten rection cycles without loss of activity, emphasizing the high process stability of the promiscuous hydrolase/acyltransferase PestE in this system [11].…”

“…Using the PestE variant optimized for HT, PestE_I208A_L209A_N288A, which was immobilized on EziG 2 beads, 265 mg L OMWW -1 HT and hydroxytyrosol acetate (HTA) were recovered from untreated OMWW [11]. It was possible to reuse the immobilized PestE_I208A_L209A_N288A for at least ten rection cycles without loss of activity, emphasizing the high process stability of the promiscuous hydrolase/acyltransferase PestE in this system [11]. The recovered HT derivatives from 1 L OMWW would be sufficient to fulfill the EU health claim for 1 kg of high-priced health-promoting olive oil, would it be added to any olive oil.…”