Cloning, heterologous expression, and molecular characterization of a highly active and stable non-specific endonuclease from Pseudomonas fluorescens

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 ( Pf Nuc) with high activities for both DNA and RNA was successfully cloned and expressed in Escherichia coli . The production of Pf Nuc in flask scale could be achieved to 1.73 × 10 6 U/L and 4.82 × 10 6 U/L for DNA and RNA by investigation of the culture and induction conditions. The characterization of Pf Nuc indicated that it was Mg 2+ -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 Mg 2+ . The specific activity of Pf Nuc for DNA was 1.44 × 10 5 U/mg at pH 8.0 and 40 °C, and 3.93 × 10 5 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 × 10 5 U/mg and 1.11 × 10 6 U/mg for DNA and RNA, respectively. There was no observed activity loss when Pf Nuc was stored at 4 °C and − 20 °C after 28 days or 10 repeated freeze–thaw cycles at − 80 °C. Molecular docking revealed that Pf Nuc 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 Pf Nuc, suggesting its great potential applications in the industry and clinic.