Development and Validation of a Three-Step Screening Strategy for Extracellular Salt-Tolerant Nucleases From Marine Bacteria

Abstract

This study reports the development of a 3-step strategy that is both cost-effective and quick to screen marine organisms and validate the presence of extracellular nucleases. The assay plates (M9 or Luria Broth [LB] media with 500 mM salt) were overlaid with a thin layer of top agar containing Toluidine Blue as the indicator and salmon sperm DNA as the substrate. Primary screening of halophiles was based on their zone of clearance. Secondary screening of the isolates involved assaying the supernatants using a well-diffusion assay. The isolates were further screened and validated by ammonium sulfate fractionation of the cell-free supernatants to enrich the secreted nuclease. The three-step method narrowed down nine potential isolates from ∼500 bacterial colonies, of which SH1 demonstrated nuclease activity, discernibly due to a secreted extracellular enzyme(s). Further characterization of this enriched nuclease(s) showed that it is likely made up of multiple peptides/subunits, acts as an endo- as well as exonuclease, degrades both DNA and RNA, is Mg⁺² dependent, has a wide range of salt tolerance from 80–1500 mM, is optimally active at 37°C, and is stable against reducing agents. This validates the screening strategy thus opening doors to further bioengineering of novel nucleases from other extremophiles.