A Novel Polyamine Oxidase from Kluyveromyces marxianus with Potential for Total Polyamine Determination

Polyamines and their acetylated derivatives are promising biomarkers for noninvasive cancer diagnostics, creating demand for robust enzymatic tools for their detection. In this study, we screened several phylogenetically diverse yeast polyamine oxidases and identified a new enzyme from Kluyveromyces marxianus (KmaPAO) as a promising candidate for analytical development. Among the tested candidates, only enzymes from K. marxianus and Lachancea thermotolerans were obtained in soluble active form, while only KmaPAO could be purified and characterized in detail. KmaPAO was produced in soluble active form in Escherichia coli at approximately 250 ± 40 mg of active enzyme per liter of culture, purified to near homogeneity in a single IMAC step, and showed a melting temperature of 66.6 ± 0.5 °C. The enzyme preferred acetylated polyamines and spermine, while showing lower activity toward spermidine. Kinetic analysis revealed sub-micromolar or low-micromolar Michaelis constants for several substrates and the highest catalytic efficiency toward spermine, 2.2 × 10⁷ M^-1 s^-1. Due to its favorable expression level, stability, and substrate profile, KmaPAO represents a promising basis for the development of enzymatic assays for total polyamine determination.