Extracellular serine proteinases produced by two taxonomically remote microorganisms - B. thuringiensis and T. vulgaris were shown to share common structural and functional features. Both enzymes contain cysteine residue apparently essential for their activity. Their N-terminal sequences are clearly homologous (10 coinciding residues among 14 compared), whereas only marginal extent of homology could be found when the N-terminal sequences of these enzymes were aligned with those of subtilisins. It is suggested that within the family of evolutionary related bacterial serine proteinases exists a subfamily of SH-containing serine proteinases.

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN′!, produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.

Intracellular serine proteases of Bacillus licheniformis and Bacillus subtilis are closely related.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.

Intracellular serine protease was isolated in a pure state from sporulating Bacillus subtilis A-50. The enzyme has the molecular weight about 30.000 daltons and pI 4.3, is completely inhibited with phenylmethylsulfonyl fluoride and EDTA, and possesses a rather low activity against protein substrates, but high specific activity with subtilisin chromogenic substrates. Amino acid composition shows higher level of Glx, Lys and Phe residues and lower content of Val residues in comparison with known subtilisins. N-terminal sequence of the enzyme, being two residues shorter from N-terminus than subtilisin BPN', is NH2-Ser-Leu-Pro-Glu-Gly-Ile-X-Val-Ile-Lys-Ala-Pro-Glu-Leu-Gln-Ala-Gln-Gly-Phe-Lys-(residues identical with BPN' are underlined). This clearly indicates the presence of two homologous structural genes, for intra- and extracellular serine proteases, in B.subtilis genome.