Secretion of Pseudomonas Aeruginosa Lipoxygenase by Pichia Pastoris upon Glycerol Feed

Chiam Hashem, Holly Stolterfoht, Claudia Rinnofner, Stefan Steinberger, Margit Winkler, Harald Pichler*

*Corresponding author for this work

Research output: Contribution to journalArticle

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.

Original languageEnglish
Article number2000089
JournalBiotechnology Journal
Volume15
Issue number11
Early online date4 Aug 2020
DOIs
Publication statusPublished - Nov 2020

Keywords

  • lipoxygenase
  • methanol-free protein expression
  • Pichia pastoris
  • protein secretion
  • Pseudomonas aeruginosa

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Molecular Medicine

Fields of Expertise

  • Human- & Biotechnology

Cooperations

  • NAWI Graz
  • BioTechMed-Graz

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