[The search for strains producing new restriction endonucleases]

Area of Science:

• Microbiology
• Molecular Biology
• Enzymology

Background:

• Restriction enzymes, or restrictases, are crucial tools in molecular biology for DNA manipulation.
• Identifying novel restrictases expands the toolkit for genetic engineering and research.
• Previous screenings have indicated a wide distribution of these enzymes across microbial species.

Purpose of the Study:

• To screen a diverse collection of microbial strains for the presence of novel restriction enzymes (restrictases).
• To identify and characterize newly discovered restrictases and their isoschizomers.
• To assess the prevalence of restrictase production within specific microbial genera, particularly Bacillus.

Main Methods:

• Utilized the rapid toluene assay method for high-throughput screening of microbial strains.
• Detected endonuclease activity by observing the specific fragmentation of bacteriophage lambda DNA in the presence of magnesium ions (Mg2+).
• Identified potential isoschizomers by comparing the activity of newly found restrictases with known restriction-cutting enzymes.

Main Results:

• Screening of 154 strains from 104 species across 32 genera identified 10 strains exhibiting specific DNA endonuclease activity.
• Two novel restrictases, Pae I and Pae II, from Pseudomonas aeruginosa were identified as true isoschizomers of Sph I and Sma I, respectively.
• The study confirmed that restrictase production is widespread among microorganisms belonging to the genus Bacillus.

Conclusions:

• The rapid toluene assay is an effective method for discovering novel restriction enzymes in diverse microbial populations.
• The identification of Pae I and Pae II expands the repertoire of available restriction enzymes for molecular biology applications.
• The genus Bacillus is a significant source of novel restrictases, warranting further investigation for enzyme discovery.