Thermosensitive, extracellular neutral proteases in Bacillus subtilis: isolation, characterization, and genetics

Abstract

Two mutants (NT02 and NT17), each producing a thermosensitive neutral protease, were isolated from Bacillus subtilis NP58, a transformant which acquired the property of hyperproduction of neutral protease from Bacillus natto IAM 1212. The neutral proteases produced by these two mutants were partially purified and enzymologically characterized. The two mutant neutral proteases displayed increased thermosensitivity as well as altered pH optima compared with those of the NP58 enzyme. In addition, the hydrolytic activity of the thermosensitive neutral proteases on synthetic peptide substrates was found to be extremely different. These results strongly suggest that the site of mutation in each of the temperature-sensitive strains is located within the structural gene for neutral protease (nprE). Previous studies indicated the existence of a specific regulator gene (nprR) in addition to the structural gene for neutral protease. Phage PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation studies with the parental and mutant strains suggest that the chromosomal order of these genes is recA-pyrA-nprR-nprE-fruB-metC. Moreover, the results of these genetic analyses imply that the mutations to thermosensitivity are located proximate to each other within the nprE gene.