Secretion Ofpseudomonas Aeruginosalipoxygenase Bypichia Pastorisupon Glycerol Feed

Pseudomonas aeruginosalipoxygenase (PaLOX) catalyzes the peroxidation of unsaturated fatty acids. Not only linoleic acid, but also linolenic acid and oleic acid are oxidized. The natural host secretesPaLOX into the periplasmic space. Herein, the aim is to secretePaLOX to the culture supernatant ofPichia pastoris. Since protein background in the culture supernatant is typically rather low, this strategy allows for almost pure production ofPaLOX applicable for the valorization of renewable fatty acids, for example for the production of green leaf volatiles. Using theCAT1promoter system and the well-established alpha-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, secretedPaLOX 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 purifiedPaLOX.