Cloning, heterologous expression, and molecular characterization of a highly active and stable non-specific...

Non-specific endonucleases can be used for the digestion of nucleic acids because they hydrolyze DNA/RNA into 3-5 base pairs (bp) length oligonucleotide fragments without strict selectivity. In this work, a novel non-specific endonuclease from Pseudomonas fluorescens (PfNuc) with high activities for both DNA and RNA was successfully cloned and expressed in Escherichia coli. The production of PfNuc in flask scale could be achieved to 1.73 × 10⁶ U/L and 4.82 × 10⁶ U/L for DNA and RNA by investigation of the culture and induction conditions. The characterization of PfNuc indicated that it was Mg²⁺-dependent and the catalytic activity was enhanced by 3.74 folds for DNA and 1.06 folds for RNA in the presence of 5 mM Mg²⁺. The specific activity of PfNuc for DNA was 1.44 × 10⁵ U/mg at pH 8.0 and 40 °C, and 3.93 × 10⁵ U/mg for RNA at pH 8.5 and 45 °C. The K_m of the enzyme for both DNA and RNA was close to 43 µM. The V_max was 6.40 × 10⁵ U/mg and 1.11 × 10⁶ U/mg for DNA and RNA, respectively. There was no observed activity loss when PfNuc was stored at 4 °C and - 20 °C after 28 days or 10 repeated freeze-thaw cycles at - 80 °C. Molecular docking revealed that PfNuc formed 17 and 19 hydrogen bonds with single-stranded RNA and double-stranded DNA, respectively. These results could explain the high activity and stability of PfNuc, suggesting its great potential applications in the industry and clinic.