Expression and purification of the thermophilic enzyme, ssopox, in Pseudomonas putida KT2442 and its applications in biotechnology

Thumbnail Image
Ng, Filomena S. W.
Journal Title
Journal ISSN
Volume Title
University of Guelph

This thesis describes the optimization of SsoPox expression and purification using 'Pseudomonas putida' KT2442 as expression host, examines its specificity towards different metal co-factors and substrates, and explores its potential in two biotechnology applications. By expressing and purifying this enzyme in 'P. putida' KT2442 instead of 'Escherichia coli', protein yield increased by 94-fold. This enzyme can catalyze the paraoxonase as well as lactonase reactions, and of the metal cofactors tested, Cd2+ was the optimal divalent cation for both reactions. The specificity of SsoPox towards 'N'-acyl homoserine lactones of varying acyl chain lengths was examined, and the highest specificity constant was obtained with 'N'-3-oxo-decanoyl homoserine lactone. Furthermore, SsoPox was immobilized and tested for the ability to detect paraoxon. The detection limit for this colorimetric assay was 0.2 mM paraoxon. Immobilized SsoPox was also shown to have the ability to inhibit quorum sensing in ' P. aeruginosa' PAO1.

SsoPox, Pseudomonas putida, KT2442, biotechnology, thermophilic enzyme