Thermolysin is a well-known protease which exhibits its specificity against hydrophobic amino acid residues such as L-leucine, L-phenylalanine, etc. whose amino groups donate the susceptible peptide bonds (amino-endopeptidase). The present study was undertaken to investigate the effects of neighboring residues surrounding the sensitive amino acid residues at the amino-side in peptide substrates. For the purpose, a kinetic study was made using various synthetic oligopeptides such as Z-A-(Gly),-i;Leu-Ala or Z-Gly+eu-(Gly),-B (A or B = various I)-or L-amino acid residues; n = 0, 1 and/or 2; the arrow shows the bond split) as substrates. Other kinetic or inhibition studies were also made. These studies indicated that the specificity is affected by a t least three amino acid residues on the N-terminal side and by two amino acid residues on C-terminal side from the sensitive amino acid residue (at amino-side) in peptide substrates. The effect of each of the five neighboring amino acid residues for appearance of the specifiicity was similar with that of the corresponding one which had been observed in a neutral protease of Bacillus subtilis, but that was not completely the same.Thermolysin is a thermo-stable protease produced by Bacillus thermopotwlyticus. It was discovered by Endo El], and its enzymatic specificity was first studied by Matsubara et al.[2], using beef cytochrome c as substrate, who assumed that the enzyme is specific for peptide bonds containing the amino group of hydrophobic amino acid residues such as L-leucine, L-isoleucine, L-phenylalanine, etc. This assumption was supported by a further study by the same authors using insulin using Z-Gly-X-NH, (X = various amino acid residues of D-or L-configuration; the arrow shows the bond split) as substrates indicated that the highest activity was obtained when X was L-leucine, or L-phenylalanine, well reflecting the specificity against large moleular peptides or proteins. These characteristics of specificity have since been found to be Unueuul Abbreviations. DFP, diisopropyl phosphofluoridate ; Z, benzyloxyoarbonyl.A 181. The specificities of various neutral proteases from bacterial origin, including thermolysin, were shown [15] to be affected by at least the next five residues adjacent to the sensitive amino acid residue in peptide substrates. A further study of B. subtilis neutral protease [16,17] with various synthetic peptides somewhat clarified the contribution to specificity of each of these five amino acid residues in peptide substrates. The present kinetic study of thermolysin with synthetic peptides was undertaken to compare the influences of neighboring residues on the specificity against the amino acid residue donating the amino group to be hydrolyzed with those found with the B. subtilis enzyme.

Morihara, Kazuyuki (Shionogi Research Laboratory, Osaka, Japan). Production of elastase and proteinase by Pseudomonas aeruginosa . J. Bacteriol. 88: 745–757. 1964.—Some strains of Pseudomonas aeruginosa produced elastase, but the others did not. The elastase-positive strains produced two proteinases (fractions II and III), and the elastase-negative strains produced one proteinase (fraction III). Moreover, one proteinase (fraction I) was produced by both elastase-positive and elastase-negative strains, but the activity was small and negligible. The three proteinases were separated by column chromatography on diethylaminoethyl-cellulose, and elastolytic activity corresponded to fraction II. The effect of components of the medium was slight for production of fraction II; fraction III was produced only in synthetic medium containing Ca ion and not in natural medium. The optimal pH of fractions I, II, and III for casein was 6.5, 8.0, and 10.0, respectively; that of fraction II for elastin was 7.5 to 8.0. The other characters of the three proteinases also differed. Both the proteolytic and the elastolytic activities of fraction II showed a similar behavior for various treatments, except the inhibition test by NaCl and serum. Fraction III proteinases from various strains were identical in their enzymatic or other characters, showing that various strains were related regardless of the difference of their elastolytic activity. The reason that the elastolytic activity of P. aeruginosa differed according to the origin of the strain is discussed on the basis of a taxonomic study; results indicate that the ability to produce elastolytic activity (fraction II) may be a dissociative character of the species.

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