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.     

Effect of exogenous factors on extracellular alkaline ribonuclease synthesis in Bacillus mesentericus

Bacillus mesentericus was found to assimilate nucleic acids as a source of nitrogen and phosphorus. Nucleic acids added to the medium as a source of nitrogen or phosphorus stimulated synthesis of ribonuclease. When washed bacterial cells were incubated for a short period of time in a fresh nutrient medium containing RNA, synthesis of RNAase was also induced. Synthesis of the enzyme was inhibited by high concentrations of chloramphenicol and actinomycin D, and stimulated by low concentrations of actinomycin D. Therefore, alkaline RNAase is an inducible enzyme which participates in the nutrition processes of bacteria.     

Purification of Bacillus mesentericus proteolytic enzymes by affinity chromatography

By means of affinity chromatography on Ovomucoid-Sepharose two proteinases hydrolyzing casein and elastin were isolated from the supernatant of the Bacillus mesentericus culture medium. The activity yield of proteinases was 100%. The characteristics of the purified enzymes were studied. It is demonstrated that B. mesentericus possesses several proteinases.     

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.     

Extracellular proteolytic activity of bacteria from soda-salt lakes of Transbaikalia

Influence of nitrogen source on proteinases synthesis in aerobic alkalotolerant and halotolerant bacteria from soda-salt lakes of Transbaikalia was studied. Maximal accumulation of proteinases was revealed on medium with peptones. Introduction of various sources of nitrogen in the medium did not result in increase of enzyme activity in cultural liquid. It was indicated that secreting proteinases of the studied bacteria strains possess narrow substrate specificity, hydrolyze proteins and n-nitroanilide substrates have maximal activity during GlpAALpNA hydrolysis. Data of inhibitory analysis and substrate specificity of studied extracellular enzymes indicate that they belong to a class of serine proteinases of subtilisin-like type.     

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.     

Characterization of the pattern of alphas1- and beta-casein breakdown and release of a bioactive peptide by a cell envelope proteinase from Lactobacillus delbrueckii subsp. lactis CRL 581

The cell envelope-associated proteinases (CEPs) of the lactobacilli have key roles in bacterial nutrition and contribute to the development of the organoleptic properties of fermented milk products as well, as they can release bioactive health-beneficial peptides from milk proteins. The influence of the peptide supply, carbohydrate source, and osmolites on the CEP activity of the cheese starter Lactobacillus delbrueckii subsp. lactis CRL 581 was investigated. The CEP activity levels were controlled by the peptide content of the growth medium. The maximum activity was observed in a basal minimal defined medium, whereas in the presence of Casitone, Casamino Acids, or yeast extract, the synthesis of CEP was inhibited 99-, 70-, and 68-fold, respectively. The addition of specific di- or tripeptides containing branched-chain amino acids, such as leucylleucine, prolylleucine, leucylglycylglycine, or leucylproline, to the growth medium negatively affected CEP activity, whereas dipeptides without branched-chain amino acids had no effect on the enzyme's production. The carbon source and osmolites did not affect CEP activity. The CEP of L. delbrueckii subsp. lactis CRL 581 exhibited a mixed-type CEP(I/III) variant caseinolytic specificity. Mass-spectrometric screening of the main peptide peaks isolated by reverse-phase high-pressure liquid chromatography allowed the identification of 33 and 32 peptides in the alpha(s1)- and beta-casein hydrolysates, respectively. By characterizing the peptide sequence in these hydrolysates, a pattern of alpha(s1)- and beta-casein breakdown was defined and is reported herein, this being the first report for a CEP of L. delbrueckii subsp. lactis. In this pattern, a series of potentially bioactive peptides (antihypertensive and phosphopeptides) which are encrypted within the precursor protein could be visualized.     

The influence of cultural medium composition on the proteolytic enzyme secretion of fungus <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

It was shown that change of medium growth composition of photopathogenic fungus Rhizoctonia solani Kühn, especially accessible sources of nutrition, leads to change of both quantity of produced proteinases and their action specificity. The mineral source of nitrogen suppressed the fungus proteinase secretion on cultivatiin medium containing potato thermostable proteins but an organic source of nitrogen accelerated mycelium growth and increased proteinase secretion. On the basis of an analysis of a fungus extracellular proteinase substrate-specificity, it is established that the presence of thermostable proteins of a potato in the cultural liquid induces the secretion of trypsin-like proteinases mainly, and the addition of yeast extract to this growth medium induces the secretion of subtilisin-like ones, thus suppressing the trypsin-like enzymes production. This fact can indicate that mycelium of fungus R. solani loses pathogenic properties and becomes saprophytes when the growth medium was enriched by an organic source of nitrogen.     

Screening for Milk-clotting Enzyme from Basidiomycetes

Screening tests for aspartic proteinases with milk-clotting activity were done on basidiomycetes. Crude enzymes from 6 strains had a high ratio of milk-clotting activity to caseinolytic activity. These enzymes showed acidic pH optimum for proteolytic activity and were inhibited considerably by pepstatin, a specific aspartic proteinase inhibitor. Among them, the crude enzyme from Laetiporus sulphureus was more heat-labile than the other enzymes.     

Isolation and thermal stability studies of two novel serine proteinases from the fungus Tritirachium album Limber.

A number of serine proteinases are secreted into the culture medium when Tritirachium album Limber is supplied with protein as the only nitrogen source. From this population of proteinases, we have isolated two novel proteolytic enzymes, designated as proteinase R and T. We have compared the thermal stability of these two proteinases with that of subtilisin BPN' and proteinase K. Both of these proteinases were thermally stable in the absence of detergents in buffers of low (4.0) and high (10.0) pH. The thermal stability of proteinase T was not affected by the presence of 1.0% SDS either at pH 8.0 or 10.0 in contrast to proteinase R which became heat labile. At low pH, the presence of SDS was detrimental to the stability of all the proteinases.     

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.     

Proteinase activities of Saccharomyces cerevisiae during sporulation.

Sporulation in Saccharomyces cerevisiae occurs in the absence of a exogenous nitrogen source. Thus, the internal amino acid pool and the supply of nitrogen compounds from protein and nucleic acid turnover must be sufficient for new protein synthesis. Since sporulation involves an increased rate of protein turnover, an investigation was conducted of the changes in the specific activity of various proteinases. A minimum of 30% of the vegetative proteins was turned over during the course of sporulation. There was a 10- to 25-fold increase in specific activity of various proteinases, with a maximum activity around 20 h after transfer into the sporulation medium. The increase in activities was due to de novo synthesis since inhibition of protein synthesis by cycloheximide blocks both an increase in proteinase activities and sporulation. There was no increase observed in proteinase activities of nonsporogenic cultures (a and alpha/alpha strains) inoculated into the sporulation medium, suggesting that the increase in proteinase activities is "sporulation specific" and not a consequence of step-down conditions. The elution patterns through diethylaminoethyl-Sephadex chromatography of various proteinases extracted from T0 and T18 cells were similar, and no new species was observed.     

Effect of various sources of nitrogen and carbon on the biosynthesis of proteolytic enzymes in a culture of Aspergillus awamori 21/96

Effects of various conditions of nitrogen and carbon nutrition on the biosynthesis of proteolytic enzymes in a selected culture of Aspergillus awamori 21/96 were studied. This strain was shown to produce proteolytic enzymes constitutively. In the presence of mineral sources of nitrogen, the synthesis of the enzymes under study was not induced by proteinaceous substrates. Optimum conditions of the enzyme biosynthesis were achieved with casein as a source of nitrogen and starch or dulcitol as a source of carbon (which increased the production of the enzymes 1.7 and 8 times, respectively). When the cells were grown on starch, their specific activity exceeded control levels 18 times.     

Effect of Various Sources of Nitrogen and Carbon on the Biosynthesis of Proteolytic Enzymes in a Culture of Aspergillus awamori 21/96

Effects of various conditions of nitrogen and carbon nutrition on the biosynthesis of proteolytic enzymes in a selected culture of Aspergillus awamori 21/96 were studied. This strain was shown to produce proteolytic enzymes constitutively. In the presence of mineral sources of nitrogen, the synthesis of the enzymes under study was not induced by proteinaceous substrates. Optimum conditions of the enzyme biosynthesis were achieved with casein as a source of nitrogen and starch or dulcitol as a source of carbon (which increased the production of the enzymes by 1.7 and 8 times, respectively). When the cells were grown on starch, their specific activity exceeded control levels by 18 times.     

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 and collagenolytic activity of extracellular proteinases of the strains of micromycetes <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis> L-1 and <Emphasis Type="Italic">Aspergillus ustus</Emphasis> 1

It was shown that extracellular proteinases produced by the strains of micromycetes A. ochraceus L-1 and A. ustus 1 differ by the activity at various pH as well as by the intensity of the effect on fibrillar proteins. It was revealed that the proteinases of A. ochraceus L-1 demonstrated maximum activity during the growth of the producer in the nitrate-free growth medium (the pH of enzyme reaction was 8.0), whereas those of A. ustus 1 showed maximal activity during the growth of the micromycete in the medium containing sodium nitrate (the pH of enzyme reaction was 6.0). Values of specific fibrinolytic and collagenolytic activities of A. ochraceus L-1 were 2.2 and 1.6 times higher than those of A. ustus 1. At the same time, A. ustus 1 showed very low values of total proteolytic (caseinolytic) activity and had a high ratio of fibrinolytic activity to total proteolytic (caseinolytic) activity (6.92). It makes the strain a promising producer of proteinases, which hydrolyze fibrin and collagen.     

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.     

Isolation and properties of extracellular proteinases from Sporothrix schenckii.

Sporothrix schenckii, mainly in the yeast form of the organism, produced extracellular proteinases when cultivated in liquid media containing albumin or collagen as a nitrogen source, but did not do so in brain heart infusion medium. Isolation of two extracellular proteinases from albumin-containing medium was performed by chromatography on DEAE-Sepharose CL-6B and Sephacryl S-200. Proteinase I had a molecular weight of 36,500, an optimal pH at 6.0, and a pI at 4.8. Despite its activities in weakly acidic conditions, proteinase I demonstrated chymotrypsinlike characteristics, these being indicated by strong inhibitory activity by phenylmethylsulfonyl fluoride and chymostatin and good kinetic constants for a synthetic chymotrypsin substrate, Suc-Ala-Ala-Pro-Phe-MCA. Proteinase II had a molecular weight of 39,000, an optimal pH at 3.5, and a pI at 3.8. Proteinase II showed cathepsin D-like characteristics, these being indicated by strong inhibitory activity by pepstatin, an acidic optimal pH, and good kinetic constants for hemoglobin. These two enzymes hydrolyzed natural substrates such as stratum corneum, type I collagen, and elastin although not type IV collagen. Proteinase production and cell growth in collagen-containing medium and the enzymatic digestion of skin constituents by isolated proteinases suggested that these two proteinases cooperatively enable the organism to invade skin and to obtain peptides from insoluble proteins.     

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.     

Specific control over the synthesis of plasmin-like and plasminogen-activating proteinases in marine bacteria

The biosynthesis of proteinases with various substrate specificities was studied in Bacillus firmus 44b and Bacillus oligonitrophilus 21p as influenced by the growth conditions and growth phases of the bacteria. The period of the maximum synthesis of plasmin-like enzymes was observed 6 h later than the period of the maximum growth rate period of B. firmus 44b, and 3 h as compared to the growth rate of B. oligonitrophilus 21p. The periods of the maximum accumulation of activating enzymes were delayed 9 and 12 h, respectively, as compared to the rapid growth periods of these two bacteria. Catabolite repression of proteinase synthesis and stimulation of the latter with substrate proteins were insignificant. The production of both plasmin-like and plasminogen-activating enzymes was most sensitive to repression by nitrogen deficiency. The production of plasminogen-activating proteinases was less dependent on the carbon source than the production of plasmin-like enzymes.