Influence of the composition of the medium and the duration of cultivation on the growth of Actinomyces spheroides and their biosynthesis of intracellular proteases and novobiocin

The growth of Actinomyces spheroides 35 under different conditions and the biosynthesis of biomass, proteases and the antibiotic were studied with two types of growth medium: a control one and an optimized one. The rate of biomass accumulation was by 5--7 per cent higher on the latter medium than on the former one. The dynamics of accumulation was studied with novobiocin and proteases which hydrolyzed fibrin and casein. Fibrin hydrolyzing proteases were found to be synthesized under conditions that were unfavourable for the production of novobiocin. If the production of the antibiotic was supressed, the concentration of fibrinolytic proteases in the cultural broth increased almost proportionally. Such a relation has not been found for caseinolytic proteases.     

Plasma-coagulating and fibrinolytic activities of fungi in the genus Aspergillus

The exocellular plasmocoagulating and fibrinolytic activities were studied in 100 cultures of the Aspergillus genus belonging to 29 species during their submerged cultivation in three media. It has been found that 38 cultures can coagulate human plasma, 75 cultures can cause lysis of fibrin platelets, and 22 cultures are capable of dissolving standard plasma clots within 6 hours. The cultures synthesize three types of proteolytic enzymes according to the specificity toward blood proteins: (1) proteases with the predominant fibrinolytic action; (2) proteases which possess both the fibrinolytic and plasmocoagulating activities; (3) proteases manifesting only the plasmocoagulating action. A. ochraceus 19 producing individual plasmocoagulases and fibrinolytic enzymes at a high rate was isolated. The composition of the enzyme complex synthesized by the culture depended on the composition of the medium and on the cultivation conditions.     

Exocellular proteases ofSerratia marcescens and their toxicity to larvae ofGalleria mellonella

Out of 18 strains ofSerratia marcescens producing exocellular proteases the strainSerratia marcescens CCEB 415 was selected according to preliminary experiments. It could be shown that the train exhibits proteolytic activity reaching up to 10 TU per 1 ml of the culture filtrate in a medium with gelatine and peptone. Two proteolytic enzymes could be demonstrated by means of specific inhibitors EDTA and diisopropyfluorophosphate: metaloprotease with optimum activity at pH 7.5 and serine protease with pH optimum of 10.9. The enzymes were purified on Sephadex and DEAE cellulose columns and by means of gel electrophoresis. However, it was not possible to separate them. The optimum temperature for activity of the mixture of the two enzymes was 50 ° C, molecular weight varied around 37000 (according to gel filtration); certain kinetic characteristics of their activity were determined. Excess subtrate (casein) inhibited activity of the enzyme mixture. Toxicity of proteases expressed as LD₅₀ units equals 78. 10⁻³ TU per larva ofGalleria mellonella.     

Regulation of Exocellular Proteases in Neurospora crassa: Role of Neurospora Proteases in Induction

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.     

Use of antibiotics in selecting a nystatin producer]

The lethal and mutagenic effect of streptomycin and nystatin on Act. noursei, strain 408 producing nystatin was studied. The survival of the spores of strain 408 on the medium with streptomycin decreased with an increase in the antibiotic concentration. Streptomycin had a selective effect on the nystatin-producing organism decreasing the frequency of morphologically changed and low active variants and revealing highly active and antibiotic stable variants. The survival of the spores of strain 408 on the medium with nystatin (20,000 units/ml) amounted to 35 per cent. Nystatin had an inhibitory effect on the organism producing it which was evident from delayed growth and significant modification variation of the colonies, as well as from a marked increase in the number of the variants characterized by low antibiotic production.     

Production and characteristics of the exocellular polysaccharide of a mutantMycobacterium strain

Mutant strains ofMycobacterium sp. V-649 producing highly mucous colonies on a solid cultivation medium were prepared after treatment with N-methyl-N′-nitro-N-nitrosoguanidine and production of the exocellular polysaccharide was tested. The strains were cultivated in media with suitable sugar sources under submerged conditions. It was found thatMycobacterium sp. V649/15 produces a maximum of 15–19% polymer after a 5–6-d cultivation. Gas chromatography indicated that the exocellular polysaccharide produced by this strain is of glucan type.     

Relationships between intracellular proteolytic activity and protein turnover in Bacillus megaterium.

When incubated in a sporulation medium, the sporogenous strains of Bacillus megaterium degrade proteins at a rate of 4-10% X h-1. The maximal rate of protein turnover is reached after 3-4 hrs at the time of development of forespores and then decreases again. The rate of protein turnover in the asporogenous strain decreases steadily under similar conditions from 3-8% X h-1 at the beginning of incubation to 1% X h-1 after 5-6 hrs in the sporulation medium. The rate of degradation of proteins in vitro in protoplast lysates is similar or higher than the rate of protein turnover. The exocellular, as well as periplasmic proteolytic activity, is suppressed by amino acids more severely than the activity in protoplasts. Mutants devoid of the exocellular proteolytic enzyme contain also less proteolytic activity in the periplasm than in the protoplasts, in contrast to the wild strain. However, their rate of protein turnover, as well as the degradation of abnormal proteins is similar to that in the wild strain. This supports a view that the proteolytic system in protoplasts is involved in intracellular protein catabolism. The periplasmic enzyme can be considered as a kind of the exocellular proteinase.     

Proteolytic complex from Aspergillus terricola (review)

Results are presented of the studies on the proteolytic enzymes produced by the fungus Aspergillus terricola that comprise the antiinflammatory drug Terrilytin. Terrilytin was shown to be a complex of three proteolytic enzymes that contained amylase as well. Two of the proteases (proteases I and II) were of the group of serine proteases by their physico-chemical and enzymatic characteristics and might be considered as two types of the same enzyme. Protease III showed characteristics specific for metalloproteases of the microbial origin, its activity being exhibited in case of the zinc ion presence. Fibrinolytic properties of Terrilytin are associated with the ability of all the three proteases to hydrolyze fibrin and fibrinogen.     

Purification and characterization of proteolytic enzymes from normal and opaque-2Zea mays L. developing endosperms

Purification and characterization of proteases from developing normal maize endosperm and high lysine opaque-2 maize endosperm have been carried out with a view to understand their role in storage protein modification. At day 15, normal maize endosperm had two types of proteolytic enzymes, namely, protease I and protease II, while at day 25 protease n disappeared and in place protease III appeared. However, in opaque-2 maize endosperm at both the stages only one type of enzyme (protease I) was present. These proteases had many properties in common-optimum pH and temperature were respectively, 5.7and 40°C; their activity was inhibited to the extent of 75 –93 % by p-chloromercuribenzoate; trypsin inhibitor inhibited the activity more at early stages of endosperm development; all proteases cleaved synthetic substrates p-tosyl-L-arginine methylesler and N-benzoyl-L-tyrosine ethyl ester and poly-L-glutamic acid. TheKm values of day 15 and 25 normal maize endosperm proteases ranged from 2.73–3.30, while for opaque-2 maize endosperm protease I it was 3.33 mg azocasein per ml assay medium. These enzymes, however, differed with respect to proteolytic activity towards poly-L-lysine. Only normal maize endosperm protease III at day 25 followed by protease II at day 15 showed high activity towards this homopolypeptide suggesting thereby their role in determining the quality of normal maize endosperm protein. Part of Ph.D. thesis submitted by the first author     

Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Summary While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.     

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respeot to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3 – 5 h of incubation. After 7 h 20 – 50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg mg^-1 h^-1. The intracellular proteolytic activity was inhibited to 65 – 88% by EDTA, to 23 – 76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50 – 65% of the activity were localized in protoplasts. Another strain ofBacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60-85%) and the gamma-subunit of nerve growth factor (by 55-93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the gamma-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), (IV), and (VI)], papain [(I), (II), (IV), and (VI)], subtilisin [(V) and (VI)], alpha-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 x 10(-2) M, while, at a concentration of 1 x 10(-3) M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.     

Inhibition of intraperitoneal tumor growth of human ovarian cancer cells by bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Several proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (N-hTIMP-1-chCysWT, N-hTIMP-1-chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.     

Media for the detection of Bacillus larvae spores in honey

Bacillus larvae, the causative agent of American foulbrood in honey bees completes its life cycle of germination, outgrowth and sporulation in young honey bee larvae by killing them and often bringing about the destruction of the entire hive. While B. larvae germinates and outgrows on complex organic media in vitro, the literature suggests, for reasons that are not at all clear, that a relatively large number of spores of B. larvae are required to yield each visible colony (colony forming units, CFU) on media. Various researchers have reported that from 16 to 3,000 or more spores of B. larvae are required to yield a single colony on an agar plate. HANSEN in Denmark designed a useful method of spreading approximately 80 mg of honey directly on the surface of a PETRI plate containing “J” agar medium to determine if B. larvae spores are present in the honey. In the present study, selected media were tested for the ability to recover B. larvae spores in honeys in the form of visible colonies (CFU) using HANSEN's strek method. A modification of a medium (TMYGP) developed by DINGMAN and STAHLY, (T-HCL-YGP agar), recovered considerably more viable B. larvae spores in the form of visible colonies (CFU) than HANSEN's “J” medium. When “J” medium was fortified with 0.1% sodium pyruvate, it was comparable to modified T-HCL-YGP medium in its recovery of B. larvae spores. Brain heart infusion agar (BHIA) with the addition of thiamine recovered more spores in the form of viable colonies than did “J” medium but it was not as efficient as T-HCL-YGP medium. Serial dilution from 100 to 10,000 times of weighed samples of honey with deionized water led to higher spore counts (CFU per g honey) than that indicated by undiluted honeys plated at 80 mg levels directly onto the surface of media by the HANSEN procedure.     

Plasmid in Bacillus pumilus and the Enhanced Sporulation of Plasmid-Negative Variants

Approximately 3% of the deoxyribonucleic acid (DNA) of Bacillus pumilus NRS576 can be isolated as covalently closed, circular duplex molecules of homogeneous size. The mol wt of the 576 plasmid is approximately 30 million. NRS576 (plasmid(+)) is oligosporogenic; less than 1% of the cells form spores during incubation in liquid AK sporulation medium. Variants that form spores at a high frequency (greater than 50% spores in 24 h) occur spontaneously at a frequency of approximately 10(-5). More than 25 such variants have been examined and all lack detectable plasmid DNA. A relationship appears to exist between the oligosporogenic properties of NRS576 and the presence of the 576 plasmid.     

Dependence of activities of polysaccharide hydrolases and oxidases from Cerrena unicolor on the source of carbon and aromatic acids in culture media

The activities of carboxymethylcellulase and xylanase in the higher basidial fungus Cerrena unicolor grown in avicel-containing medium reached 1.95 and 1.50 units per mg protein, respectively, whereas in mannitol-containing medium they ranged from 0.02 to 0.05 units per mg protein. The activity of fungal beta-glucosidase depended on the carbon source in the culture medium and ranged from 2.1 units per mg protein in the presence of mannitol to 17.3 units per mg protein in the presence of avicel. In contrast to polysaccharides, easily metabolizable substrates (cellobiose, mannitol, and glucose) provided the highest rates of secretion of laccase (52.7-123.5 ncat per mg protein) and ligninase (22-106 units per mg protein). The addition of tangerine pomace, a substrate enriched with aromatic compounds, to the culture medium caused an increase in the rate of bio-synthesis of laccase and ligninase to 862 ncat per ml and 557 units per ml, respectively. Aromatic compounds such as p-xylidine and veratric aldehyde increased the laccase activity of C. unicolor IBB 62 from 7.9 to 23.6 and 18.3 ncat per mg protein, respectively. Veratryl alcohol caused a sevenfold increase in the activity of Mn-dependent peroxidase in the culture medium.     

Multiple genetic changes determine ribose utilization by Novikoff hepatoma cell variants

In variants of the Novikoff hepatoma cell line, the ability to use D-ribose as a carbon source appeared to be due to changes in the expression of ribokinase. Examination of ribokinase activity was prompted by the finding that uptake of radiolabeled ribose was linear for 30 min in six variants but became saturated within 2 min in nine other variants. The linear uptake of ribose was due to a high rate of phosphorylation by ribokinase. Variants which showed linear uptake kinetics had ribokinase levels of 6.8 +/- 1.7 nm/min per mg protein as compared to the parental levels of 0.90 +/- 0.25 nm/min per mg protein. The nine variants which showed saturable uptake kinetics had low parenteal levels of ribokinase. However, these variants showed a change in the subcellular location of that activity. The enzyme was predominantly membrane-associated in both parental cells and high ribokinase variants. In contrast, the low ribokinase variants had a cytoplasmic form of the enzyme. A more general membrane change probably occurred in these variants, since they showed an increased sensitivity to the unrelated membrane reactive compounds, phytohemagglutinin and ouabain.     

Secretion of Proteinases with Fibrinolytic Activity by Micromycetes of the Genus <Emphasis Type="Italic">Aspergillus</Emphasis>

Proteolytic activity of extracellular enzymes of 11 strains of different Aspergillus species was studied. Comparison of the enzymatic indices of strains grown on agar medium containing either casein or fibrin allowed the selection of the strain Aspergillus terreus 2 as a promising producer of fibrinolytic proteases. It was found that A. terreus 2 proteinases demonstrated maximum activity at pH 8.0. The highest values of fibrinolytic and total proteolytic activities expressed in U_Tyr (amount of micromoles of tyrosine released from fibrin or casein for 1 min) were 34.0 and 358.3, respectively. Maximum activities were detected when growing the producer on a medium containing only amine nitrogen sources (fish flour hydrolysate and peptone); however, the amount of extracellular protein and the specific fibrinolytic and total proteolytic activities were greater in the medium containing both mineral and amine nitrogen sources (fish flour hydrolysate and sodium nitrate) than in the medium containing only fish flour hydrolysate and peptone as nitrogen sources.