[Thrombolytic activity of Bacillus mesentericus at different conditions of nitrogen nutrition of a culture]. / Tromboliticheskaia aktivnost' u Bacillus mesentericus pri razlichnykh usloviiakh azotnogo pitaniia kul'tury.

The work is concerned with studying the effect exerted by different sources of nitrogen nutrition on the biosynthesis of proteinases with a thrombolytic activity by a variant of Bacillus mesentericus, strain 64, obtained with the aid of analytical selection. Protein substrates taken as a nitrogen source stimulate the synthesis of proteinases by the bacterial culture. These enzymes have a high caseinolytic and thrombolytic activity, and the level of their activity correlates with the amount of a protein substrate added to the medium. Ammonium acetate and succinate are the best stimulants for the formation of proteinases when the salts of mineral and organic acids are used as a source of nitrogen nutrition. In that case, the enzymes have a high thrombolytic activity and a low caseinolytic activity. A semi-synthetic medium with the aforementioned nitrogen-containing compounds as a source of nitrogen nutrition is proposed for the synthesis of thrombolytic proteinase by the variant of B. mesentericus.

[Substrate specificity of enzymes from Bacillus mesentericus]. / Substratnaia spetsivichnost' fermentov Bacillus mesentericus.

The proteolytic enzymes of the sporogenous Bacillus mesentericus strains 64 and 8 were tested for their ability to hydrolyse different protein substrates. The enzymes were isolated using affinity chromatography on bacillichine-silochrome, and eluted with 25% isopropanol in 0.05 M Tris-HCl buffer, pH 8.0-8.4, containing 0.01 M CaCl2. Casein, hemoglobin, elastin, albumin and synthetic peptides, Z-L-Ala-Ala-Leu-pNa and Z-L-Ala-Gly-Leu-pNa, were used as substrates. The activity of esterase was assayed in terms of indophenyl acetate cleavage. The proteinases were compared with terrilytin, a commercial preparation. The proteinase of strain 64 was active in the hydrolysis of casein, hemoglobin and elastin; its specificity was close to that of terrilytin. The proteinase of strain 8 differed from them in a higher thrombolytic and fibrinolytic activity, and had a high esterase activity.

[Effect of inhibitors and cations on the proteolytic activity of Bacillus mesentericus]. / Vliianie ingibitorov i nekotorykh kationov na proteoliticheskuiu aktivnost' Bacillus mesentericus.

The effect of some inhibitors and bivalent metal cations (Mn2+, Ca2+, Fe2+, Zn2+, Mg2+, Co2+ and Cu2+) on the proteolytic activity of two Bacillus mesentericus strains (strain 8 and strain 64 M-variant) was comparatively studied. The both enzymes were shown to be serine proteinases, but the proteinase of strain 64 was also a metal-dependent enzyme. Metal ions exerted no essential effect on the proteinase of strain 8. Ca2+ and Mg2+ ions stimulated the proteinase activity of strain 64 whereas Fe2+ and Zn2+ ions inhibited it in the case of three substrates. Therefore, the two proteinases are different.

[Fibrinolytic activity of natural variants of Bacillus mesentericus]. / O fibrinoliticheskoi aktivnosti estestvennykh variantov Bacillus mesentericus.

The natural variability of the ability to synthesize proteinases by Bacillus mesentericus 64 was studied. The population of this strain was shown to be heterogeneous. Three types of variants (S, M and P) differed in the morphology of their colonies and in the culture characteristics from the typical colonies of the parent strain. The caseinolytic activity of the M variant was three times as high as that of the parent strain, and it also had an elevated fibrinolytic activity and a high rate of blood thrombolysis in experiments in vitro. The rate of proteinase synthesis correlated with the morphological types of sporogenic bacteria.

[A serine proteinase from Thermoactinomyces vulgaris, strain INMI-4a]. / Serinovaia proteinaza Thermoactinomyces vulgaris sht. INMI-4a.

A serine proteinase was isolated from the cultural filtrate of the thermophylic actinomycet Thermoactinomycet vulgaris, strain INMI-4a. The purification procedure included affinity chromatography on bacitracin-Sepharose, ion-exchange separation on aminosilochrome, and gel-filtration on Sephadex G-25, resulting in a 194-fold purification and the 55% yield of the enzyme. The molecular weight of the enzyme as determined by polyacrylamide gel electrophoresis in the presence of Na-SDS as well as by gel-filtration on Sephadex G-75 is equal to 28 000; the amino acid composition is: Lys11, His4, Arg5, Asp33, Thr22, Ser24, Glu16, Pro16, Gly30, Ala38, Cys1-2, Val20, Met1, Ile14, Leu8, Tyr16, The4, Trp6-7. The isoelectric point lies at pH 8--9; the pH optimum for the peptide substrate hydrolysis is Z-L-Ala-L-Ala-L-Leu-pNA is at 8.2. The enzyme is stable at pH 7--9. The temperature optimum of the proteolytic activity lies at 55 degrees; however, the enzyme is stable to heating for 1 h at 37 degrees. The proteinase is completely inactivated by the serine proteinase specific inhibitors--phenylmethylsulphofluoride and the protein inhibitor IT-AjT from Actinomyces, as well as by p-chloromercuribenzoate. The enzyme shows lytic activity against the cells of E. coli, Micrococcus lysodeicticus and of the yeasts. The Thermoactinomyces vulgaris serine proteinase, being definitely different from the serine proteinases from Actinomyces griseus, also reveals specific differences when compared to bacterial serine proteinases, e. g. subtilisins. There are some indications to the enzyme relationship with the family of carboxypeptidase Y-like serine proteinases.

Properties of proteolytic enzymes isolated from a thermophilic strain of Micromonospora vulgaris 42.

Two proteolytic enzymes, protease A and protease B, were isolated in homogeneous state from the cultural broth of the thermophilic actinomycete Micromonospora vulgaris 42. Their physicochemical properties were studied, i.e., molecular weight (50 000 for protease A and 30 000 for protease B), amino acid composition, N-terminal amino acids (phenylalanine for protease A and alanine for protease B). The specificity of the action of these enzymes was assayed by splitting the B chain of oxidized insulin. Both enzymes are neutral proteases of the thermolysine type.

[Factors causing lysis of an Actinomyces rimosus culture, the producer of the antibiotic, oxytetracycline]. / O faktorakh, vyzyvaiushchikh lizis kul'tury Actinomyces rimosus-produtsenta antibiotika oksitetratsiklina

The lysis of Actinomyces rimosus producing oxytetracycline during its mass growth can be caused by two factors which were separated by differential centrifugation. The first factor is phage particles of a temperate phage produced by the culture; they are incapable of growth but may induce the lysis. The phage particles treated with low pH and a temperature of 70 degrees C lose the lytic activity. The second factor is a lytic enzyme produced under the control of the temperate phage during its induction; it seems to consist of at least two enzymes, a lytic enzyme and a proteolytic enzyme.