Thrombolytic activity of Bacillus mesentericus at different conditions of nitrogen nutrition of a culture

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.     

Use of starch-containing media for cultivation of Bacillus mesentericus

When cultivating Bacillus mesentericus to produce proteinases it is advisable to use more available and cheap carbon sources--native maize meal or potato starch--instead of maltose; the products of their complete hydrolysis inhibit the biosynthesis of enzymes.     

Hydrolase biosynthesis by morphological variants of Bacillus mesentericus as dependent on the storage conditions

A method used to prepare the inoculum (a strain of Bacillus mesentericus, a producer of a complex of hydrolytic enzymes) has been studied for its effect on the activity of proteinases and amylases under submerged cultivation in fermenters. Optimal conditions for the culture storage and inoculum cultivation are developed to obtain standard enzymic preparations.     

Effect of inhibitors and cations on the proteolytic activity of 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

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.     

Substrate specificity of enzymes from 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.     

Cloning in Bacillus subtilis cells of the Bacillus mesentericus genes that determine the enzyme synthesis of tryptophan metabolism

In this work, we tried to clone some Bacillus mesentericus genes coding for tryptophan synthesis in Bacillus subtilis cells. We succeeded in obtaining two identical plasmids carrying some genes of Bac. mesentericus and complementing the trpC2 and trpF mutations of Bac. subtilis. The cloned genes of Bac. mesentericus completely replaced the functions of trpC2 and trpF genes.     

Study of catalase and proteolytic activities of different variants of Bacillus mesentericus]

Catalase and proteolytic activity of the culures and morphological variants of Bacillus mesentericus fuscus, Bac. mesentericus vulgatus were studied. The variants were obtained as a result of prolonged cultivation of the stock strains in the potato mash under the layer of vaseline oil. The level of catalase activity varies in different morphological variants of the same culture, changes with age and depends on the storage conditions. The catalase activity in the rough, smooth and papillar variants that were freshly isolated from the potato mash was 1.5=2.5 times lower than that in the variants long kept on the agar medium. The quantitative indexes of the proteolytic activity of different variants also varied.     

Purification and characterization of 3,3-dihydroxyazetidine from culture medium of Bacillus mesentericus and B. subtilis

A method is described for the purification of 3,3-dihydroxyazetidine (DHA), which accelerates the growth of Bifidobacterium spp., from the culture medium of Bacillus mesentericus (BM). Purification involved adsorption to an ion-exchange resin; it required less time and gave a higher yield than a previously reported method. Monitoring the inhibition of E. coli NIHJ JC-2, we searched for other strains that produced 3,3-dihydroxyazetidine. We found that not only B. mesentericus TO-A but also B. subtilis IFO13719 produced the compound of interest.     

Opsonizing activity of the serum of gnotobiotic guinea pigs contaminated with individual representatives of intestinal microflora]

The influence of contamination of germfree guinea pigs with individual representatives of the intestinal microflora (Bac. mesentericus, Bac. subtilis, S. albus, and S. faecalis) on the formation of the serum opsonic activity was studied. An increase of the opsonic activity to all the microorganisms on the 11th day after a corresponding monocontamination and a stimulating influence of the serum on the intracellular digestion of Bac. mesentericus and Bac. subtilis microbes was noted. As to the pathogenic microorganisms (E. coli 055), S. Faecalis only were capable of stimulating the serum opsonic activity. The results indicated the presence of an association between the microflora composition and the opsonic activity of the animal blood serum. The value of this index also depended on the properties of the phagocytosis object.     

Joint culturing of Streptococcus lactis, strain MGU with Proteus vulgaris and Bacillus mesentericus

Nisin synthesis by Streptococcus lactis, strain MGU, grown as a combined culture together with Proteus vulgaris and Bacillus mesentericus under stationary conditions or with stirring does not depend on the quantity of inoculated associated cells. Nisin synthesis in the combined culture drops down by 10-20% at the initial pH 7.5 of the growth medium which is unfavourable for S. lactis producing nisin. The level of nisin biosynthesis does not rise when the pH of the medium is adjusted (either naturally or artificially) to 6.6-6.8 in the presence of glucose and yeast autolysate. S. lactis inhibits the growth of B. mesentericus when grown together with it whereas P. vulgaris inhibits the growth of S. lactis in their combined culture.     

Extracellular nuclease of Bacillus mesentericus]

Bacillus mesentericus is found to secrete three type of nucleases: alkaline ribonuclease (EC 2.7.7.17), acidic ribonuclease (EC 2.7.7.17) and Ca2+-activated exonucleease (EC 3.1.4.7). These nucleases are purified and characterized. They are similar to those from Bac. subtilis in main biochemical and physico-chemical properties and in their chromatographical behaviour. Studying physiological functions of Bac. mesentericus extracellular nucleases, it is shown that bacteria, which are capable to produce extracellular nucleases, utilize exogenous RNAs and a bit worse, DNAs as a single and additional source of nitrogen or phosphorus. In view of this it is believed that extracellular nucleases participate in bacteria nutrition.     

Fibrinolytic activity of Bacillus mesentericus strains

Two Bacillus mesentericus strains with a high activity of proteolytic enzymes having the thrombolytic action were selected from a group of its collection strains. The effect of different carbon sources on the synthesis of proteases was studied. A growth medium containing potato broth (10%), peptone (0.5%) and lactose (0.5%) allowed one to obtain a cultural broth dissolving human blood clots within 2.5 to 3 hours in experiments in vitro.     

Characteristics of hydrolase biosynthesis in Bacillus mesentericus grown on various media

The dynamics of the consumption of major carbon and nitrogen sources and the biosynthesis of hydrolytic enzymes were studied in Bacillus mesentericus grown on semisynthetic media. Conditions were chosen that provide the obtaining of the culture liquid with predominantly proteolytic or amylolytic activity. The replacement of maltose with native starch resulted in more intensive accumulation of the biomass and hydrolytic enzymes, and in more rapid (by 3-5 hr) transformation from the logarithmic to the stationary growth phase.     

Optimization of the nutrient medium composition for directed biosynthesis of Bacillus mesentericus lectins

The medium has been optimized in order to increase the lectin-producing activity of Bacillus mesentericus. When a producer grew on the optimized medium, a 4-8-fold increase of the hemagglutination test titres and a 5.6-fold increase (as compared with the initial medium) of the specific lectin activity of the culture liquid was attained.     

金鱼不同组织器官及胚胎发育不同时期蛋白水解酶的种类和活性变化

采用复性电泳方法研究了金鱼组织器官蛋白水解酶及个体发生过程中蛋白水解酶的种类和活性变化,主要结果表明：⑴金鱼各组织器官蛋白水解酶种类差异不大,大多数组织器官都具有１１３、６９、２０、１６ｋＤ四条带,但不同组织器官常具有其特异性蛋白水解酶;肠道蛋白水解酶种类最多、活性最强。⑵蛋白水解酶的活性受ｐＨ值影响和制约,大多数组织器官蛋白水解酶活性最适ｐＨ值为８．５。⑶在金鱼胚胎发育早期（从卵裂到心跳期）多数     

A new factor from Bacillus mesentericus which promotes the growth of Bifidobacterium

It was reported previously that supernatants of cultures of Bacillus mesentericus TO-A promote the growth of Bifidobacterium species. In this study, a new growth-promoting factor, BM-1, was purified from the supernatant of such a culture and its chemical structure was determined. BM-1 was identified as 3,3-dihydroxyazetidine, and it promoted the growth of several strains of Bifidobacterium.     

Various physico-chemical properties of the amylolytic complex from Bacillus mesentericus]

Some physico-chemical properties of the Bacillus mesentericus amylolytic complex were studied, and optimal conditions of starch hydrolysis (pH 7.5-8.0; 45 degrees C) were found. The half-life of amylases at 50 degrees was 75 min. The heat stability of the enzymes increased in the presence of Ca2+ ions. Amylase was stable at pH 7-9 and readily inactivated at pH below 6.0. By physical and chemical characteristics the complex was close to analogous preparations from Bacillus alkalophilic strains. Isoelectrofocusing revealed that the complex consisted at least of two amylolytic enzymes.     

Genome polymorphism in dissociative variants of Bacillus subtilis (mesentericus) 76. Identification of dissociants using genome fingerprinting

DNA fingerprinting procedure with M13 repeat probe as we have shown earlier makes it possible to apply a new approach in theoretical and applied fields of microbiology and bacteriology. In this work, using the method described we have revealed genomic polymorphism of dissociative variants of Bac. subtilis (mesentericus) 76. The data obtained may be referred as strong evidence that bacterial dissociation do has genetic nature.