Extracellular nuclease from a thermophile, Streptomyces thermonitrificans: production, purification and partial characterization of – double strand preferential – deoxyribonuclease activity

Abstract

A thermophilic strain of Streptomyces thermonitrificans produced high levels of extracellular nuclease (designated as nuclease Stn β) when grown on nutrient broth glucose medium. Maximal nuclease activity (51 U ml −1) was obtained, in 40 h, when the culture was grown on modified NBG medium at 45 °C. The enzyme was purified to homogeneity with an overall recovery of 5.6% and a specific activity of 10,833. The relative molecular mass of the purified enzyme, determined by gel filtration, was 22.4 kDa and it showed an obligate requirement for Mn 2+ for activity. The optimum pH and temperature of nuclease Stn β were 8.0 and 45 °C, respectively. The enzyme was inhibited by Mg 2+, Co 2+, Cu 2+, Zn 2+ and Hg 2+, inorganic phosphate, pyrophosphate, dithiothreitol, β-mercaptoethanol, EDTA and NaCl. Nuclease Stn β was stable to high concentrations of urea and organic solvents but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Stn β is a multifunctional enzyme with substrate specificity in the order of dsDNA > ssDNA ≫ RNA. The end products of dsDNA hydrolysis were predominantly oligonucleotides (85–90%) and small amounts of 5′ mononucleotides (10–15%) suggesting an endo mode of action.