Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of - single strand preferential - deoxyribonuclease activity

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose follow ed by Blue Sepharose. The M-r of the purified enzyme determined by native P AGE was 67 000 and it is a tetramer and each protomer consists of two unide ntical subunits of M-r 21 000 and 13 000. It is an acidic protein with a pi of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate r equirement of divalent cations like Mg2+, Mn2+ and Co2+ for its activity bu t is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperatur e of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg2+ was 40 degrees C, it showed higher optimum temperature (45 degrees C) for dsDNA hydrolysis in presence of Mn2+ and Co2+. Nuclease Rsn was inhibi ted by divalent cations like Zn2+, Cu2+ and Hg2+, inorganic phosphate and p yrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3'- or 5'-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA throu gh a single hit mechanism. The end products of both ssDNA and dsDNA hydroly sis were predominantly oligonucleotides ending in 3'-hydroxyl and 5'-phosph oryl termini. Moreover, the type of metal ion used did not influence the mo de and mechanism of action of the enzyme. (C) 1999 Elsevier Science B.V. Al l rights reserved.