CLONING AND EXPRESSION IN BACILLUS-SUBTILIS OF THE NPR GENE FROM BACILLUS-THERMOPROTEOLYTICUS ROKKO CODING FOR THE THERMOSTABLE METALLOPROTEASE THERMOLYSIN

We report the isolation, cloning and expression, in Bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by Bacillus thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other members of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. Further, the thermolysin gene shares a strong homology with two other previously cloned genes from two different strains of Bacillus stearothermophilus. The sequence of the mature secreted protease, inferred from the DNA sequence, is, with two exceptions, identical with the previously published protein sequence of thermolysin [Titani, Hermodson, Ericsson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The exceptions are Asn(37) and Gln(119), originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombinant protein shows that it is indistinguishable from the wild-type thermolysin.