Antibacterial, antifungal and cytotoxic activity of terrestrial cyanobacterial strains from Serbia

Cyanobacteria are known to be a rich source of biologically active compounds some of which can have pharmaceutical importance. In this work we present the screening results of cyanobacterial strains for their antibacterial, antifungal, and cytotoxic activity. Cyanobacterial strains were isolated from various soil types in province of Vojvodina and Central Serbia, Republic of Serbia. The screening included 9 strains of Anabaena and 9 strains of Nostoc. Both, extracellular products (from the culture liquid) and cellular crude lipophilic extracts were tested against 13 bacterial strains and 8 fungal strains. Cytotoxic activity was tested against three human cell lines. Methanol extracts were prepared according to ?stensvik. Antibacterial and antifungal activities were determined measuring inhibition zone, 48 h after inoculation. The cytotoxic activity was determined by sulforhodamine B (SRB) colorimetric assay. Of all cyanobacterial strains tested, 52% showed some antifungal and 41% antibacterial activity. Two out of six tested strains possessed cytotoxic activity. The cytotoxic activity of Anabaena strain S12 was found both in culture liquid and crude cell extract. It occurred specifically between the 21st and 42nd day of cultivation against HeLa and MCF7 cells, but had no activity against cell line derived from a healthy tissue. A high percentage of the active strains among the tested strains justify the effort of screening cyanobacteria that are isolated from terrestrial environments. The most promising strains for the fur- ther study are Anabaena strain S12 which showed strong cytotoxic and antibacterial activity and Ana- baena strain S20 which produces a potent antifungal compound. The future work, besides further screening and chemical identification of the active compounds, should also include the development of culture techniques that would lead to more efficient production of biologically active compounds.     

Herbicidal Activity of Toxin from Fusarium avenaceum GD-2 against Wild Oats (Avena fatua L.)

The herbicidal activity potential of toxin from Fusarium avenaceum GD-2 was evaluated against wild oats (Avena fatua L.) in this study. The toxin was assayed in vitro to evaluate its inhibition against seed germination of A. fatua. The toxin of F. avenaceum GD-2 was shown to have an inhibitory effect of around 77.54% at 5 mg/mL against germination of A. fatua seeds. The inhibitory effect shown by the toxins against radicle had higher activity than plumule under the same concentration. The toxin of F. avenaceum GD-2 significantly diminished the plant length the part on the ground with various treatments when treated with the toxin under greenhouse conditions. However, there were no significantly different reductions in plant length and the weed fresh weight with different treatments. In detached leaf injection bioassay, the toxic metabolite was characterized after the culture filtrates crude extraction with petroleum ether, chloroform, ethyl acetate and n-butanol. The residues left after solvent evaporation were evaluated separately for their toxicity against the target weed. Residue (5 mg/mL) obtained from n butanol fraction showed the highest toxic activity when compared with others. Moreover, a host range experiments on the sensitivity of 10 plant species revealed that barnyard grasses and goosefeet were more sensitive to the toxin of the culture filtrate. Three herbicidal active compounds were isolated and purified from cultural filtrate with the same UV absorption peak. The recent results showed potential for the development of the toxins produced by F. avenaceum GD-2 as a bio herbicidal source to control and eliminate A. fatua weed.     

Characterization and catabolic genes analyses of oil-degrading Pseudomonas aeruginosa 1217

One oil-degrading bacterial strain 1217 isolated from oil contaminated soil could degrade crude oil. It was identified and designated as Pseudomonas aeruginosa by morphology, physiology, biochemical and 16S rDNA sequence analyses. The strain grew well at 5-65 ℃ with the initial pH of 2-10 and NaCl concentrations of 0%-9%. It grew well in the medium containing different organic substrates as sole carbon sources, such as n-dodecane, n-octadecane, benzene, toluene, xylene and naphthaline. The degrading rates for hydrocarbons were 21.57% and 15.15% when it was cultured in minimal medium containing crude oil and different chain alkanes at 30 ℃ and 10 ℃ for 7 days. The bacterium produced biosurfactants with the surface tension reduction from 72.20 mN/m to 35.14 mN/m. The oil degrading related genes such as alkane monooxygenase, toluene dioxygenase, biphenyl dioxygenase, ring hydroxylating dioxygenase and oxidoreductase genes were detected in Pseudomonas aeruginosa 1217. The alkane monooxygenase and ring hydroxylating dioxygenase genes were further cloned and analyzed. The sequence similarities of the two genes with those of Pseudomonas aeruginosa PAO1 were 99.91% and 99.22% respectively. The isolated strain exhibits great potential for the bioremediation of the hydrocarbons contaminated environments.     

Three metaltolerance Halobacillus sp. isolated from salt lakes in Shaanxi, China

Three Gram positive, spore forming, halophilic bacterial strains were isolated from a salt lake in shanxi China and subjected to a polyphasic taxonomic study. Strains A375, A381 and A389 grew in the presence of 0~24% (w/v) NaCl in complex medium. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains A375, A381 and A389 were located in the genus Halobacillus. Levels of 16S rRNA gene sequence similarity between the isolated strains and the type trains of Halobacillus species were in range 91.6%~98.9%. On the basis of phenotypic and chemotaxonomic properties and phylogenetic analysis, strains A375, A381 and A389 should be placed in the Halobacillus species. Metaltolerance test verify that all strains can tolerate different levels of metal salts.     

The Role of dinB in the Appearing of Antibiotics Resistance in E.coli

The role of dinB gene in the appearing of antibiotics resistance was studied. Plasmid containing multi-copy dinB gene was transfected into E. coli to create an overexpression. The strains carrying multi-copies of dinB gene demonstrate a significant survival advantage over the wild strain. In vitro experiment, the dinB-overexpressed strain evolved resistance within 8 hours, while wild strain could not.In vivo experiment with mice model infected with dinB-overexpressed strain, resistant clones emerged significantly earlier and demonstrated significant higher level of resistance than those infected with the wild control strain. The results showed that dinB gene made a contribution in the appearing of the antibiotics resistance and has a potential as a target for prevention from the appearing of antibiotic resistance.     

Production, Purification and Characterization of Alkaline β-Glucosidase Isolated from Agrobacterium sp. PJD-1-1

In order to screen novel β-glucosidase producing strains from environment, one targeted novel strain PJD-1-1 producing β-glucosidase were isolated from putrefied sugarcane leaves with screening and spreading plate. 16S rDNA analysis revealed it was a novel Agrobacterium sp. When the strain was incubated at initial pH 7.0, 20 ℃ with lactose as carbon and NaNO₃ as nitrogen sources, the maximum enzyme activity was 3.92 U/mg. β-glucosidase from this strain was purified using (NH₄)₂SO₄ precipitation followed by dextran gel filtration chromatography and ion exchange chromatography. A purifying fold of 4.85 with gaining rate of 8.0% was obtained. SDA-PAGE analysis of the purified enzyme showed that it was a clear and pure band with molecular mass of ca. 40 kDa. The most optimum activity of the enzyme was at 50 ℃ and pH at 8.0. The enzyme could maintain stability under the conditions below 50 ℃. Hg²⁺ and Ag⁺ heavily inhibited the enzyme activity suggesting that the active catalytic sites of the enzymes might possess thiol radical. Ba²⁺, Ca²⁺, Pb²⁺, Co²⁺, Zn²⁺, Mn²⁺, Na⁺, K⁺, EDTA, and urea had no obvious effects on the enzyme activity. It is concluded that the novel strain Agrobacterium sp. PJD-1-1 producing β-glucosidase was successfully screened from putrefied sugar cane leaves. The produced enzyme had thermal stability, alkaline feature and metal ions tolerance made it useful in the food and broad potential applications in other fields.     

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.     

Use of membrane filters for soil algal bioassays

A culture technique for eucaryotic and procaryotic soil algae involving the use of membrane filters superimposed on the surface of inorganic nutrient agar is described. Nostoc commune grew exponentially when cultured in this manner. No significant differences in biomass production or nitrogenase activity were detected among culture subsets within replicate experiments run under standard conditions. Estimates of daily growth rates (0.340), culture doubling time (48.9 h), and nitrogenase activity (14.54 nM C₂H₂ reduced μg⁻¹ chl a h⁻¹) were consistent with laboratory and field estimates reported for several planktonic species of Anabaena and strains of Nostoc commune isolated from diverse terrestrial habitats. Therefore, the filter-culture technique is an alternative which may be superior to traditional liquid culture methodology for studies involving certain soil procaryotic and eucaryotic algae.     

猪原始生殖细胞的分离、培养与鉴定

Embryonic germ cells (EG cells) are pluripotential undifferentiated stem cells isolated from cultured primordial germ cells (PGCs). Like ES cells, EG cells are of importance for gene targeting, therapeutical cloning and organ trans-plantation. The aim of this study was to isolate and characterize EG cells from porcine PGCs. The genital ridges from 24- 26 days old porcine embryos were treated in 0.02% EDTA for 20 min and pricked with a needle to release PGCs. The isolated PGCs were cultured on a SNL feeder layer in an EG cell medium. The EG cell medium consisted of Dulbecco‘‘s modified Eagle‘‘ s medium (DMEM) supplemented with 20 % Buffalo rat lever (BRL) cell-conditioned medium, 15 % fe-tal bovine serum, 1 mmol L-glutamine, 0.1 mol nonessential amino acids, 10 μmol β-mercaptoethanol and antibiotics.The freshly isolated PGCs were positive for alkaline phosphatase activity and Periodic acid-Schiff‘‘ s staining. Under this culture regime, PGCs could be maintained in an undifferentiated state and used for further cultures. One strain of the cul-tured PGCs was cultured 8 times, and alkaline phosphatase activity was detected in the colony formed from this strain.These cultured PGCs could spontaneously differentiate into fibroblast-like cells. These data suggested that we had success-fully isolated EG-like cells from oorcine PGCs.     

Staphylococcus aureus adherence to nasal epithelial cells in a physiological in vitro model

Summary Nasal carriage of Staphylococcus aureus represents a risk factor for subsequent invasive infections and interpatient transmission of strains. No physiological in vitro model of nasal epithelial cells is available to study both patient- and bacteria-related characteristics and their interaction, leading to adherence and colonization. Starting with tissues from human nasal polyps, a confluent, squamous, nonkeratinized epithelium in collagen-coated 96-well microtiter plates was obtained after 14 d. This in vitro cell-layer was characterized histologically, ultrastructurally, and immunohistochemically and showed features that were indistinguishable from those observed in the squamous nonkeratinized epithelium found in the posterior part of the vestibulum nasi. Adherence experiments were performed with four different ³H-thymidine-labeled Staphylococcus aureus strains. The effect of bacterial inoculum size, temperature of incubation, and incubation medium were studied. The adherence results were found to be reproducible, reliable and sensitive, allowing detection of small quantitative differences in adherence between the Staphylococcus aureus strains. There was no significant difference in adherence at 23° C and 37° C, nor between the incubation medium M199 and phosphate-buffered saline. Plastic adherence could be reduced and standardized with use of siliconized tips and a constant bacterial inoculum volume of 100 μl/well. This physiological and reliable in vitro cell-culture model offers a unique opportunity to study Staphylococcus aureus adherence to squamous, nonkeratinized nasal epithelial cells and both patient and bacterial characteristics involved in this interaction.     

Fermentation Kinetics of Media Optimization for the Production of Alpha Amylase by a New Isolate of Aspergillus Oryzae

The present study is concerned with the isolation and screening of different strains of Aspergillus oryzae for the production of alpha amylase. Ninety strains were isolated from soil and tested for the production of alpha amylase in shake flasks. Of all the strains tested, Aspergillus oryzae GCB-32 and Aspergillus oryzae GCB-35 gave maximum production of alpha amylase. Different culture media were screened for the production of alpha amylase by these two strains. M1 medium containing starch, yeast extract, NH₄Cl, MgSO₄·7H₂O and CaCl₂ gave the maximum production of alpha amylase by both the strains Aspergillus oryzae GCB-32 and Aspergillus oryzae GCB-35.Kinetic analysis revealed that the values of product yield coefficient(Y_p/x) and specific product yield coefficient(q_p) were found highly significant(p≤0.05) when medium M1 was used for the enzyme production.     

Fermentation Kinetics of Media Optimization for the Production of Alpha Amylase by a New Isolate of Aspergillus Oryzae

The present study is concerned with the isolation and screening of different strains of Aspergillus oryzae for the production of alpha amylase. Ninety strains were isolated from soil and tested for the production of alpha amylase in shake flasks. Of all the strains tested, Aspergillus oryzae GCB-32 and Aspergillus oryzae GCB-35 gave maximum production of alpha amylase. Different culture media were screened for the production of alpha amylase by these two strains. M1 medium containing starch, yeast extract, NH₄Cl, MgSO₄·7H₂O and CaCl₂ gave the maximum production of alpha amylase by both the strains Aspergillus oryzae GCB-32 and Aspergillus oryzae GCB-35.Kinetic analysis revealed that the values of product yield coefficient(Y_p/x) and specific product yield coefficient(q_p) were found highly significant(p≤0.05) when medium M1 was used for the enzyme production.     

Fumonisins production by Fusarium proliferatum strains isolated from asparagus crown

The present work deals with the capability for producing fumonisin by Fusarium proliferatum strains isolated from asparagus in China. Fifty of F. proliferatum strains were randomly selected and incubated on cultures of maize grain and asparagus spear, respectively. Fumonisin levels (FB₁ and FB₂) were determined by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that all 50 strains produced fumonisins in maize culture within a wide range of concentrations, 10–11,499 μg/g and 2–6,598 μg/g for FB₁ and FB₂, respectively. On culture of asparagus spear,48 strains (96%) produced fumonisins in the range 0.2–781.6 μg/g and no detected to 40.3 μg/g for FB₁ and FB₂, respectively. All of F. proliferatum strains produced much higher levels of FB₁, FB₂ and total fumonisins (FB₁ + FB₂) in maize grain culture than in asparagus spear culture. Meanwhile, fumonisin B₃ (FB₃) was identified in all maize culture extracts and most of asparagus spear culture extracts. This is the first study carried out the fumonisin-producing ability of F. proliferatum strains isolated from asparagus in China. The information obtained is useful for assessing the risk of fumonisins contamination in asparagus spear. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

A rhizobia strain isolated from root nodule of gymnosperm Podocarpus macrophyllus

Symbiotic nitrogen fixation of rhizobia and leguminous plants is considered as the most important biologic nitrogen fixation system on earth. Symbiotic nodulation of gymnosperm Podocarpus macro-phyllus and rhizobia has never been reported. In this study, 11 endophytic bacteria strains were isolated from root nodules of P. macrophyllus and its variation P. macrophyllus var. maki. The plant infection tests on these strains indicated that the isolated strains could be nodulated on P. macrophyllus plants, and weak nitrogenase activity of nodules was found in acetylene reduction method. According to the physiological and biochemical characteristics of the 11 strains, GXLO 02 was selected as the representative strain. 16S rDNA full-length sequence analysis of GXLO 02 confirmed that the representative strain GXLO 02 belongs to Rhizobium sp.     

Phylogenetic assignment and mechanism of action of a crop growth promoting <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> strain used as a biofertiliser on graminaceous crops in Russia

The taxonomic position of “Agrobacterium radiobacter strain 204,” used in Russia as a cereal crop growth promoting inoculant, was derived by a polyphasic approach. The phenotypic analyses gave very similar biochemical profiles for strain 204, Rhizobium radiobacter NCIMB 9042 (formerly the A. radiobacter type strain) and R. radiobacter NCIMB 13307 (formerly the Agrobacterium␣tumefaciens type strain). High percentage similarities, above the species separation level, were observed between the 16S rRNA, fusA and rpoB housekeeping gene sequences of these three strains, and the genomic DNA–DNA hybridisation of strain 204 against the type strain of R. radiobacter NCIMB 9042 was over 70%. Strain 204 is not phytopathogenic and it does not fix atmospheric N₂ or form a physical association with the roots of barley. Strain 204 culture and culture supernatant stimulated the rate of mobilisation of seed reserves of barley in darkness and promoted its shoot growth in the light. Gibberellic acid (GA) concentration was 1.3 μM but indole acetic acid was undetectable (<50 nM) in cultures of strain 204. It is concluded that strain 204 is phenotypically and genotypically very similar to the current R. radiobacter type strain and that the mechanism of its effect on growth of cereals is via the production of plant growth promoting substances. GA is likely to play an important role in the strain 204 stimulation of early growth of barley.     

Mechanism of cyanide and thiocyanate decomposition by an association of Pseudomonas putida and Pseudomonas stutzeri strains

The intermediate and terminal products of cyanide and thiocyanate decomposition by individual strains of the genus Pseudomonas, P. putida strain 21 and P. stutzeri strain 18, and by their association were analyzed. The activity of the enzymes of nitrogen and sulfur metabolism in these strains was compared with that of the collection strains P. putida VKM B-2187^T and P. stutzeri VKM B-975^T. Upon the introduction of CN⁻ and SCN⁻ into cell suspensions of strains 18 and 21 in phosphate buffer (pH 8.8), the production of NH ₄ ⁺ was observed. Due to the high rate of their utilization, NH₃, NH ₄ ⁺ , and CNO⁻ were absent from the culture liquids of P. putida strain 21 and P. stutzeri strain 18 grown with CN⁻ or SCN⁻. Both Pseudomonas strains decomposed SCN⁻ via cyanate production. The cyanase activity was 0.75 μmol/(min mg protein) for P. putida strain 21 and 1.26 μmol/(min mg protein) for P. stutzeri strain 18. The cyanase activity was present in the cells grown with SCN⁻ but absent in cells grown with NH ₄ ⁺ . Strain 21 of P. putida was a more active CN⁻ decomposer than strain 18 of P. stutzeri. Ammonium and CO₂ were the terminal nitrogen and carbon products of CN⁻ and SCN⁻ decomposition. The terminal sulfur products of SCN⁻ decomposition by P. stutzeri strain 18 and P. putida strain 21 were thiosulfate and tetrathionate, respectively. The strains utilized the toxic compounds in the anabolism only, as sources of nitrogen (CN⁻ and SCN⁻) and sulfur (SCN⁻). The pathway of thiocyanate decomposition by the association of bacteria of the genus Pseudomonas is proposed based on the results obtained. Original Russian Text ? N.V. Grigor’eva, T.F. Kondrat’eva, E.N. Krasil’nikova, G.I. Karavaiko, 2006, published in Mikrobiologiya, 2006, Vol. 75, No. 3, pp. 320–328.     

Chemical composition and antimicrobial activity of <Emphasis Type="Italic">Thymus pannonicus</Emphasis> All. (<Emphasis Type="Italic">Lamiaceae</Emphasis>) essential oil

This paper presents the results of a study on chemical composition and antimicrobial activity of Thymus pannonicus All. (Lamiaceae) essential oil from Vojvodina province (north of Serbia). The investigated oil was hydrodistilled from a flowering plant and analysed by GC and GC-MS. Fifty-three constituents were identified (>97% of total oil), with geranial (41.42%, w/w) and neral (29.61%, w/w) as the most prominent. The antimicrobial activity of the oil was evaluated using agar disc diffusion and broth microdilution method against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, two strains of Klebsiella pneumoniae and two strains of Candida albicans. The essential oil exhibited antimicrobial activity to varying degrees against all tested strains. The maximum activity of T. Pannonicus oil was observed against E. coli, S. aureus and both tested strains of C. Albicans (MIC = 50 μ/ml, each). Moderate activity was observed against P. aeruginosa and one of the tested strains of K. Pneumoniae (MIC = 200 μ/ml), while E. faecalis and the other strain of K. Pneumoniae expressed a higher degree of resistance (MIC > 200 μ/ml). This study confirms that essential oil of T. pannonicus possesses remarkable in vitro antimicrobial activity against several medicinally important pathogens. This is attributable to lemon-scented citral, a mixture of geranial and neral, which has well-documented antimicrobial activity against a range of bacteria and fungi.     

Interactive effects of nitrate and phosphate salts, sucrose, and inoculum culture age on growth and sesquiterpene production in Artemisia annua hairy root cultures

Summary We analyzed four factors (phosphate and nitrate salts, sucrose, and culture inoculum age), simultaneously at three levels using a fractional factorial design method to determine the most suitable conditions for maximizing both root biomass and terpenoid production in transformed Artemisia annua root cultures. Optimal growth conditions were determined to be: nitrate (15 mM), phosphate (1.0 mM), sucrose content (5% wt/vol), and inoculum age (8 d-old). Determination of optimal conditions for sesquiterpene production was more complicated than for biomass production. For most experiments artemisinic acid was undetectable especially in experiments where phosphate was greater than 0.5 mM and for nearly all culture inoculum ages of 14 d. Artemisinic acid was also never detected whenever arteannuin B was present. Arteannuin B was the major artemisinic compound detected in these experiments, sometimes at levels exceeding 300 μg/g fresh weight. When the sum of artemisinin and its three precursors is analyzed, three factors (sucrose, nitrate, and inoculum age) are heavily dependent on one another, and in conjunction with possible degradation of artemisinin by peroxidase, the current analysis does not provide a clear picture regarding the most effective conditions for maximizing the production of artemisinin. Abbreviations: AA, artemisinic acid; AB, arteannuin B; AT, artemisitene; AN, artemisinin; FW, fresh weight; DW, dry weight; S, sucrose; N, potassium nitrate; P, sodium phosphate; A, inoculum culture age.     

Detection of chromosomally located and plasmid-borne genes on 20 kb DNA fragments in parasporal crystals from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The association of 20 kb heterologous DNA fragments with the parasporal crystals from native and recombinant Bacillus thuringiensis strains was analyzed, respectively. The cry2Aa10 gene cloned in plasmid pHC39 was transformed into B. thuringiensis subsp. kurstaki strains CryˉB and HD73, producing recombinant strains CryˉB(pHC39) and HD73(pHC39). SDS-PAGE and scanning electron microscopy analyses demonstrated that the recombinant CryˉB(pHC39) produced cuboidal crystals of Cry2Aa10 protoxin, while recombinant HD73(pHC39) produced both bipyramidal crystals of Cry1Ac1 protoxin and cuboidal crystals of Cry2Aa10 protoxin. Bioassay results proved that recombinant HD73(pHC39) showed higher insecticidal activity to Helicoverpa armigera than CryˉB(pHC39). It was found that 20 kb DNA fragments were present in bipyramidal and cuboidal crystals from both native and recombinant strains, and the 20 kb heterologous DNAs contained chromosome-specific and resident large plasmid-borne DNA fragments, suggesting the 20 kb heterologous DNA fragment embodied in crystals came randomly from the bacterial chromosomal and plasmid genome. This was the first investigation devoted exclusively on the origin of 20 kb DNA fragments in the parasporal crystals of B. thuringiensis. The data provides a basis for further investigation of the origin of 20 kb DNAs in the crystals and the interaction of DNA and protoxins.     

Prokaryotic expression, purification and characterization of nattokinase

Nattokinase is a fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. In this study, nattokinase was purified from fermentation broth of a Bacillus subtilis strain by ammonium sulfate salting-out, gel filtration chromatography, and hydrophobic interaction chromatography with a purification fold of 5.2 and at a yield of 46.3%. The purified enzyme has molecular mass of 28 kDa and fibrinolytic activity of 4 580 U/mg. Since the concentration of nattokinase on fermentation broth was quite low, we cloned nattokinase gene from B. subtilis and expressed it in E. coli BL21 (DE3). Nattokinase was actively expressed in the recombinant strain. The yield of nattokinase was increased significantly, but the activity of the protein produced by recombinant strain was low.