Production and characterization of a new diamine oxidase from Yarrowia lipolytica

Abstract

A putative diamine oxidase (DAO) from Yarrowia lipolytica PO1f (DAO-1) was homologously recombinantly integrated into the genome of Y. lipolytica PO1f using the CRISPR-Cas9 system for the subsequent DAO production in a bioreactor. Thereby, it was proven that the DAO-1 produced was indeed a functional DAO. The cultivation yielded 2343 ± 98 nkat/Lculture with a specific DAO activity of 1301 ± 54.2 nkat/gprotein, which was a 93-fold increase of specific DAO activity compared to the native Y. lipolytica PO1f DAO-1 production. The DAO-1 showed a broad substrate selectivity with tyramine, histamine, putrescine and cadaverine being the most favored substrates. It was most active at 40 °C, pH 7.2 in Tris-HCl buffer (50 mM) (with histamine as substrate), which is comparable to human and porcine DAOs. The affinity of DAO-1 towards histamine was lower compared to mammalian DAOs (Km = 2.3 ± 0.2 mM). Nevertheless, DAO-1 degraded around 75% of the histamine used in a bioconversion experiment with a food-relevant concentration of 150 mg/L. With its broad selectivity for the most relevant biogenic amines in foods, DAO-1 from Y. lipolytica PO1f is an interesting enzyme for application in the food industry for the degradation of biogenic amines.