Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis

Abstract

A DNase was isolated from culture supernatants of sporulating B. subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel eletrophoresis. The enzyme differs from other DNase of B. subtilis in MW, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The MW of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12,000.