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 wh ich is produced by Bacillus thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other m embers of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. F urther, the thermolysin gene shares a strong homology with two other p reviously cloned genes from two different strains of Bacillus stearoth ermophilus. The sequence of the mature secreted protease, inferred fro m the DNA sequence, is, with two exceptions, identical with the previo usly published protein sequence of thermolysin [Titani, Hermodson, Eri csson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The excep tions are Asn(37) and Gln(119), originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombi nant protein shows that it is indistinguishable from the wild-type the rmolysin.