Secretion of Pseudomonas aeruginosa Lipoxygenase by Pichia pastoris upon Glycerol Feed

Abstract

Pseudomonas aeruginosa lipoxygenase (PaLOX) catalyzes the peroxidation of unsaturated fatty acids. Not only linoleic acid, but also linolenic acid and oleic acid are oxidized. The natural host secretes PaLOX into the periplasmic space. Herein, the aim is to secrete PaLOX to the culture supernatant of Pichia pastoris. Since protein background in the culture supernatant is typically rather low, this strategy allows for almost pure production of PaLOX applicable for the valorization of renewable fatty acids, for example for the production of green leaf volatiles. Using the CAT1 promoter system and the well-established α-factor signal sequence for secretion, methanol- and glycerol-induced secretion are compared and the latter shows four times more LOX activity in the culture supernatant under methanol-free conditions. In addition, secreted PaLOX is purified and the specific activity with enzyme in culture supernatant is compared. Notably, the predominant specific activity is achieved for enzyme in culture supernatant - 11.6 U mg−1 - reaching five times higher specific activity than purified PaLOX.