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

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.

Automated summary

Enzymatic transesterification of hydroxytyrosol in an aqueous system in the presence of ethyl acetate yielded a rich extract of 3-hydroxytyrosol acetate. A mutant of the acyltransferase enzyme, immobilized on EziG(2) beads, achieved high hydroxytyrosol conversions of 82-89% in one hour, for at least ten reaction cycles. However, the conversions from olive mill wastewater were lower, ranging from 51% to 62%. A preparative scale reaction resulted in the extraction of 13.8 mg (57%) of 3-hydroxytyrosol acetate from 60 mL of OMWW.