The heterologous production of enzymes has been a central focus of modern biotechnology for several decades, driven by the increasing demand for industrial enzymes due to their ecological and economic advantages. Lipoxygenases (LOX) catalyse the peroxidation of unsaturated fatty acids into fatty acid hydroperoxides, which are of particular interest due to their ability to be readily transformed into various valuable compounds. These compounds can be used across a broad spectrum of applications, ranging from the production of oleochemicals to the creation of flavour compounds, the synthesis of signalling compounds, and their use as food additives. Although lipoxygenases are present in almost every living organism, they differentiate by the type of substrate they utilise. Furthermore, bacterial LOXs may possess improved thermostability and activity over a broader pH range compared to their eukaryotic counterparts [1]. Pseudomonas aeruginosa lipoxygenase (PaLOX) is a resilient enzyme that exhibits considerable activity at elevated temperatures and can peroxidize not only linoleic and linolenic acids but also arachidonic acid and oleic acid, demonstrating a diverse range of substrates [2]. This versatility highlights its potential as an eco-friendly biocatalyst for a wide range of industrial applications.
The aim of this study is to produce an active recombinant P. aeruginosa lipoxygenase employing the yeast Pichia pastoris as the expression system. The palox gene was fused with a C- or N-terminal histidine tag to facilitate purification by immobilised metal affinity chromatography (IMAC) and cloned into the pPic9K shuttle vector. The construct ensures methanol-induced protein expression and secretion. The recombinant plasmid was then introduced into P. pastoris GS115. Transformed cells were screened and cultivated in BMMY medium to induce expression. SDS-PAGE and lipoxygenase activity assay were used to verify the successful expression of PaLOX. The detailed presentation of the findings will be showcased at the poster session.