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Fibrinolytic agents are essential in treating thrombosis, playing a critical role in improving survival rates in cardiovascular diseases. Microbial fibrinolytic proteases have emerged as promising alternatives due to their affordability, specificity, lower toxicity, and reduced side effects. Consequently, the search for microorganisms capable of producing these enzymes has gained significant economic importance in the pharmaceutical industry. This study reports and characterizes a novel fibrinolytic enzyme produced by Penicillium citrinum CFAM 521, a strain isolated from the Amazon region. The enzyme was purified using a polyethylene glycol (PEG)–phosphate salt aqueous two‐phase system (ATPS). The effects of PEG molecular weight, PEG concentration, and phosphate concentration on the protease partition coefficient (K) were evaluated through a 22 full factorial design. The enzyme exhibited both fibrinolytic and fibrinogenolytic activities. After partitioning in a two‐phase system with 10% (w/w) PEG and 15% (w/w) sodium phosphate, the fibrinolytic proteases were predominantly retained in the salt‐rich bottom phase (K = 0.33). The enzyme has a molecular weight of 34 kDa, with optimal pH and temperature at 9°C and 37°C, respectively. Inhibitory analysis confirmed that it is a serine protease, and its activity was enhanced by the addition of Mn2+. Notably, the enzyme exhibited no hemolytic activity. Therefore, P. citrinum CFAM 521 represents a novel source of fibrinolytic enzymes, highlighting its potential as an alternative for the development of thrombolytic agents. [ABSTRACT FROM AUTHOR]

A novel fibrinolytic enzyme, BSFE1, was isolated from the marine bacterium Bacillus sp. S-3685 (GenBank No.: KJ023685) found in the South China Sea. This enzyme, with a molecular weight of approximately 42 kDa and a specific activity of 736.4 U/mg, exhibited its highest activity at 37 °C in a phosphate buffer at pH 8.0. The fibrinolytic enzyme remained stable over a pH range of 7.5 to 10.0 and retained about 76% of its activity after being incubated at 37 °C for 2 h. The Km and Vmax values of the enzyme at 37 °C were determined to be 2.1 μM and 49.0 μmol min−1 mg−1, respectively. The fibrinolytic activity of BSFE1 was enhanced by Na+, Ba2+, K+, Co2+, Mn2+, Al3+, and Cu2+, while it was inhibited by Fe3+, Ca2+, Mg2+, Zn2+, and Fe2+. These findings indicate that the fibrinolytic enzyme isolated in this study exhibits a strong affinity for fibrin. Moreover, the enzyme we have purified demonstrates thrombolytic enzymatic activity. These characteristics make BSFE1 a promising candidate for thrombolytic therapy. In conclusion, the results obtained from this study suggest that our work holds potential in the development of agents for thrombolytic treatment. [ABSTRACT FROM AUTHOR]

Abstract Cardiovascular diseases, resulting from the deposition of clots in blood vessels, are the leading cause of death worldwide. Fibrinolytic enzymatic activity can catalyze blood clot degradation. Findings show that 36 fungal isolates recovered from Caatinga soils have the potential to produce fibrinolytic protease under submerged conditions. About 58 % of the isolates displayed fibrinolytic activity above 100 U/mL, with Mucor subtilissimus UCP 1262 being the most active. The protease was biochemically and biophysically characterized, showing that the enzyme had a high affinity for SAApNA substrate and was significantly inhibited by fluoride methyl phenyl sulfonyl-C7H7FO2S, suggesting that it is a chymotrypsin-like serine protease. The highest enzyme activity was detected at pH 5.0 and 28 °C. This fibrinolytic protease’s far-UV circular dichroism (CD) showed that its secondary structure was primarily α-helical. The purified fibrinolytic enzyme may represent a novel therapeutic agent for treating thrombosis. At temperatures above 65 °C, the enzyme lost all its secondary structure. Its melting temperature was 58.1 °C, the denaturation enthalpy 85.1 kcal/mol, and the denaturation entropy 0.26 kcal/K∙mol.

A novel fibrinolytic enzyme was produced by the liquid fermentation of Coprinus comatus. The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn2+, K+, Ca2+, Mn2+, and Mg2+ while being inhibited by Fe2+, Fe3+, Al2+, and Ba2+. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases. [ABSTRACT FROM AUTHOR]

Background: The fibrinolytic enzymes from Bacillus sp. are proposed as therapeutics in preventing thrombosis. Computational-based analyses of these enzymes’ amino acid composition, basic physiological properties, presence of functional domain and motifs, and secondary and tertiary structure analyses can lead to developing a specific enzyme with improved catalytic activity and other properties that may increase their therapeutic potential. Methods: The nucleotide sequences of fibrinolytic enzymes produced by the genus Bacillus and its corresponding protein sequences were retrieved from the NCBI database and aligned using the PRALINE programme. The varied physiochemical parameters and structural and functional analysis of the enzyme sequences were carried out with the ExPASy-ProtParam tool, MEME server, SOPMA, PDBsum tool, CYS-REC tool, SWISS-MODEL, SAVES servers, TMHMM program, GlobPlot, and peptide cutter software. The assessed in-silico data were compared with the published experimental results for validation. Results: The alignment of sixty fibrinolytic serine protease enzymes (molecular mass 12–86 kDa) sequences showed 49 enzymes possess a conserved domain with a catalytic triad of Asp196, His242, and Ser569. The predicted instability and aliphatic indexes were 1.94–37.77, and 68.9–93.41, respectively, indicating high thermostability. The random coil means value suggested the predominance of this secondary structure in these proteases. A set of 50 amino acid residues representing motif 3 signifies the Peptidase S8/S53 domain that was invariably observed in 56 sequences. Additionally, 28 sequences have transmembrane helices, with two having the most disordered areas, and they pose 25 enzyme cleavage sites. A comparative analysis of the experimental work with the results of in-silico study put forward the characteristics of the enzyme sequences JF739176.1 and MF677779.1 to be considered when creating a potential mutant enzyme as these sequences are stable at high pH with thermostability and to exhibit αβ-fibrinogenase activity in both experimental and in-silico studies.

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Cochliobolus hawaiiensis Alcorn Assiut University Mycological Centre 8606 was chosen from the screened 20 fungal species as the potent producer of fibrinolytic enzyme on skimmed‐milk agar plates. The greatest enzyme yield was attained when the submerged fermentation (SmF) conditions were optimized, and it was around (39.7 U/mg protein). Moreover, upon optimization of fibrinolytic enzyme production under solid‐state fermentation (SSF), the maximum productivity of fibrinolytic enzyme was greatly increased recorded a bout (405 U/mg protein) on sugarcane bagasse, incubation period of 5 days, moisture level of 100%, initial pH of salt basal medium 7.8, incubation temperature at 35°C, and supplementation of the salt basal medium with corn steep liquor (80％, v/v). The yield of fibrinolytic enzyme by C. hawaiiensis under SSF was higher than that of SmF with about 10.20‐fold. The purification procedures of fibrinolytic enzyme by ammonium sulfate (70%), gel filtration, and ion‐exchange columns chromatography caused a great increase in its specific activity to 2581.6 U/mg protein with an overall yield of 55.89%, 6.37 purification fold and molecular weight of 35 kDa. Maximal activity was recorded at pH 7 and 37°C. Significant pH stability was recorded at pH 6.6–7.2, and thermal stability was recorded at 33–41°C. The enzyme showed the highest affinity toward fibrin, with Vmax of 240 U/mL and an apparent Km value of 47.61 mmol. Mg2+ and Ca2+ moderately induced fibrinolytic activity, whereas Cu2+ and Zn2+ greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non‐metalloprotease. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplatelet aggregation activities by completely prevention and dissolution of the blood clot which confirmed by microscopic examination and amelioration of blood coagulation assays. These findings suggested that the produced fibrinolytic enzyme is a promising agent in management of blood coagulation disorders. [ABSTRACT FROM AUTHOR]

Background: Fibrinolytic enzyme is one of the cardiovascular disease therapies. Rhizopus microsporus var. oligosporus is microorganism that has been evaluated to produce fibrinolytic protease by fermentation. This study conducted fermentation of helianthi annui semen (sunflower seed) by Rhizopus microsporus var. oligosporus to produce fibrinolytic enzyme. Objective: This study aims to determine the effect of Initial pH and incubation temperature and its optimization in the production of fibrinolytic protease by Rhizopus microsporus var. oligosporus FNCC 6010 in solid-state fermentation on helianthi annui semen (sunflower seed) substrate. Optimum condition was determined by maximum protease and fibrinolytic activity. Method: A crude enzyme of protease fibrinolytic was obtained from the supernatant extract of fermented sunflower seed. Protease activity was measured by the skimmed milk agar (SMA) plate method, and fibrinolytic activity was determined by the fibrin agar plate method. Result: It was found that the starting pH affects both the proteolytic and fibrinolytic activity of enzymes that are produced in fermentation. The starting pH of 5.0 showed higher fibrinolytic and proteolytic activity values compared to the starting pH of 7.0. The incubation temperature 33±1 °C had the higher activity compared to 28±1 °C or 37±1 °C. Conclusion: Initial pH and incubation temperature affect the proteolytic and fibrinolytic activity of crude enzyme extracted from fermented sunflower seed by Rhizopus microsporus var. oligosporus. The optimum condition for producing fibrinolytic protease in the state fermentation method was an initial pH of 5.0 and an incubation temperature of 33±1°C. [ABSTRACT FROM AUTHOR]

A novel fibrinolytic enzyme, BSFE1, was isolated from the marine bacterium Bacillus sp. S-3685 (GenBank No.: KJ023685) found in the South China Sea. This enzyme, with a molecular weight of approximately 42 kDa and a specific activity of 736.4 U/mg, exhibited its highest activity at 37 °C in a phosphate buffer at pH 8.0. The fibrinolytic enzyme remained stable over a pH range of 7.5 to 10.0 and retained about 76% of its activity after being incubated at 37 °C for 2 h. The Km and Vmax values of the enzyme at 37 °C were determined to be 2.1 μM and 49.0 μmol min−1 mg−1, respectively. The fibrinolytic activity of BSFE1 was enhanced by Na+, Ba2+, K+, Co2+, Mn2+, Al3+, and Cu2+, while it was inhibited by Fe3+, Ca2+, Mg2+, Zn2+, and Fe2+. These findings indicate that the fibrinolytic enzyme isolated in this study exhibits a strong affinity for fibrin. Moreover, the enzyme we have purified demonstrates thrombolytic enzymatic activity. These characteristics make BSFE1 a promising candidate for thrombolytic therapy. In conclusion, the results obtained from this study suggest that our work holds potential in the development of agents for thrombolytic treatment.

Objective: To investigate the protective effect and mechanism of Lyophyllum ulmarium fibrinolytic enzyme （LUFE） on rats with hyperlipidemia. Methods: Hyperlipidemia models were established by feeding with a high-fat diet, and the model rats were gavaged with different doses of LUFE. HE stain was used to analyze the morphological changes of liver.The biochemical indexes of rat plasma were detected by colorimetric method.WB analyses the expression level of related proteins. Results: LUFE could alleviate the liver tissue injury of hyperlipidemia rats and reduce the plasma lipid level and IL-6, TNF-α，MCP-1，ALT，AST. LUFE can down regulate TLR4，MyD88，phosphorylated PI3K，phosphorylated Akt and phosphorylated NF-кB protein level. Conclusion: LUFE protects against hyperlipidemia-induced liver injury in rats by regulating PI3K/Akt/NF-кB and TLR4/MyD88/NF-кB signaling pathways.

A producing-fibrinolytic enzyme strain was isolated with high yield. The strain was identified as Bacillus amyloliquefaciens. B. amyloliquefaciens GUTU06 fibrinolytic enzyme was purified by acetone precipitation and reverse micelle. Acetone precipitation condition and reverse micelle condition were examined. Results showed that the total reverse micelle extraction efficiency was 64.49 % ± 1.6 %. The purification fold of the entire process reached 13.38. The optimum pH of purified enzyme is 5, and the optimum temperature is 45 °C. Fe3+ and K+ can enhance the fibrinolytic activity of the enzyme. Compared to commercial fibrinolytic enzymes such as urokinase and lumbrukinase, GUTU06 fibrinolytic enzymes have a lower pH optimal range and higher temperature stability. The molecular weight of the enzyme was approximately 28 kDa. Reverse micelle extraction with cetyl trimethylammonium bromide as a surfactant combined with acetone precipitation is suitable for separating and purifying fibrinolytic enzymes and a promising technique for obtaining active proteins.

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FIBRINOLYTIC enzyme production from Scopulariopsis brevicaulis OS 3456 isolated from a local soil sample was studied. The enzyme was purified by ammonium sulfate precipitation and gel filtration chromatography using Sephadex G-100, increasing its specific activity to 370 U/mg with a yield of 1.5% and a purification fold of 3.4. The molecular weight of the purified enzyme was 61.5 kDa determined by SDS-PAGE analysis. The optimum temperature of the enzyme was 37oC, and it was stable over a pH range of 5.0-9.0 with maximum stability at pH 7.0. The activity was increased in the presence of ß-mercaptoethanol, Mn2+, Ba2+, triton X-100, and xylene by 137.1, 51.6, 41.4, 37.5, and 23%, respectively. Furthermore, the enzyme activity was inhibited by Cd2+, Al3+, EDTA, PMSF, and acetone. The in vitro thrombolytic activity of the undiluted purified enzyme (370 U/mg) was found to be 100%. Meanwhile, in the cases of 185, 92.5, 46.25, 23.125, and 11.562 U/mg, the clot lysis percentage was 76.8, 67.4, 57.8, 39.5, and 28%, respectively. A carrageenan-induced tail thrombosis model was applied to test the in vivo thrombolytic activity of the enzyme. The result indicated no obvious thrombus in the tails of mice treated with the tested enzyme (370 U/mg). However, when the enzyme was diluted, its thrombolytic activity decreased gradually. All these results explore the promising thrombolytic activity of the extracted fibrinolytic enzyme. Hence, more purification steps and more experimental animal studies are required in the future for its use as a commercial drug. [ABSTRACT FROM AUTHOR]

A novel fibrinolytic enzyme was produced by the liquid fermentation of Coprinus comatus. The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn2+, K+, Ca2+, Mn2+, and Mg2+ while being inhibited by Fe2+, Fe3+, Al2+, and Ba2+. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases.

The antioxidative proteolytic fraction, MA-1, was partially purified from Mycoleptodonoides aitchisonii. MA-1 was purified to homogeneity using a two-step procedure, which resulted in an 89-fold increase in specific activity and 42.5% recovery. SDS-PAGE revealed two proteins with a molecular weight of 48 kDa. The zymography results revealed proteolytic activity based on the MA-1 band. MA-1 was found to be stable in the presence of Na+, Ca2+, Fe3+, K+, and Mg2+. MA-1 was also stable in methanol, ethanol, and acetone, and its enzyme activity increased by 15% in SDS. MA-1 was inhibited by ethylenediaminetetra-acetic acid or ethylene glycol tetraacetic acid and exerted the highest specificity for the substrate, MeO-Suc-Arg-Pro-Tyr-pNA, for chymotrypsin. Accordingly, MA-1 belongs to the family of chymotrypsin-like metalloproteins. The optimum temperature was 40 °C and stability was stable in the range of 20 to 35 °C. The optimum pH and stability were pH 5.5 and pH 4–11. MA-1 exhibited stronger fibrinolytic activity than plasmin. MA-1 hydrolyzed the Aα, Bβ, and γ chains of fibrinogen within 2 h. MA-1 exhibited an antithrombotic effect in animal models. MA-1 was devoid of hemorrhagic activity at a dose of 80,000 U/kg. Overall, our results show that M. aitchisonii produces an acid-tolerant and antioxidative chymotrypsin-like fibrinolytic enzyme, and M. aitchisonii containing MA-1 could be a beneficial functional material for the prevention of cardiovascular diseases and possible complications. [ABSTRACT FROM AUTHOR]

Cardiovascular disease (CVD) has become the leading cause of death, and it is critical to develop new functional foods to prevent intravascular thrombosis, the key cause of CVD. Fermented soy-based food is a good choice because of its native fibrinolytic enzyme (FE) activity. In this study, a strain that can produce a new type of fibrinolytic enzyme was selected from Chinese Douchi and identified as Bacillus licheniformis SFD-Y5 by molecular biology experiments and physiological and biochemical experiments. Single factor experiments combined with statistical experiments, including Plackett–Burman experiment, steepest ascent experiment and RSM (Box–Behnken design), were used to optimize the fermentation of FE by B. licheniformis SFD-Y5. The final FE activity was 2434.45 ± 28.49 IU/mL under optimal conditions, which is the highest FE activity produced by wild B. licheniformis so far. Further studies showed that Y5 FE is a serine metalloproteinase with good stability at alkaline pHs (pH 8.0–11.0). The results of our study could lay a foundation for the future production, molecular modification and further application in functional foods of Y5 FE. [ABSTRACT FROM AUTHOR]

Fibrinolytic enzymes are a kind of proteolytic enzymes that can hydrolyze fibrin and dissolve blood clots. They could be used as a therapeutic agent for treating thrombosis. It is important for the treatment of cardiovascular disease to find and develop new thrombolytic drugs. In order to explore new fibrinolytic enzymes, a strain named 214L-11 with protease and fibrinolytic enzyme activity, which was isolated from the Flaming Mountain of Xinjiang Province, was screened using the skimmed milk plate, the blood powder agarose plate and the fibrin plate methods. Phylogenetic analyses showed that strain 214L-11 shared the highest similarity with Streptomyces fumanus NBRC 13042T (98.88%), which indicated that it represented a potential novel species in the Streptomyces genus. The fibrinolytic enzyme produced by 214L-11 displayed thrombolytic and anticoagulant activities, and it could degrade a single specific protein in the thrombus, thereby destroying the thrombus structure. The fermentation medium optimized through response surface methodology was 15 g/L soluble starch, g/L KNO3 0.58, 0.43 g/L peptone, 0.01 g/L FeSO4·7H2O, 0.5 g/L MgSO4·7H2O, 0.2 g/L Mn2+, 0.5 g/L NaCl and 1 L distilled water, pH 8, and the maximum amount of fibrinolytic enzyme produced by strain 214L-11 in the optimal fermentation medium was 1255.3 FU/mL. Overall, the fibrinolytic enzyme-producing strain was screened from the Flaming Mountain of Xinjiang for the first time, which provided a basis for further research and the development of new efficient and safe hemolytic drugs. [ABSTRACT FROM AUTHOR]

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This study was performed to investigate the fibrinolytic enzyme-producing potentials of locally isolated soil bacteria and to purify and characterize the fibrinolytic enzyme of the most potent bacterial isolate. Among 40 isolates, the isolate V4 was found to have the highest potential to produce the fibrinolytic enzyme. According to 16 S rRNA sequence analysis, the isolate V4 was identified as Bacillus atrophaeus (GenBank number: OL662991). The optimal parameters for fibrinolytic enzyme production from B. atrophaeus were determined as a skim milk powder concentration of 15 g/L, an initial pH of 7.0, a temperature of 35 °C and an incubation time of 72 h. The molecular weight of the purified enzyme was calculated as 36 kDa. After ammonium sulphate precipitation, ion-exchange chromatography and gel filtration chromotography-based processes, the specific activity of the fibrinolytic enzyme was determined as 6414.7 U/mg. The purified enzyme showed the maximum activity at pH 7.0 and 35 °C. The enzyme was inhibited by PMSF and EDTA. The substrate specifity of the enzyme was in the following order; fibrin (62.3 U/mg) > fibrinogen (46.2 U/mg) > casein (42.1 U/mg) > serum albumin (6.8 U/mg). This is the first report on the fibrinolytic enzyme production potential of the species B. atrophaeus. [ABSTRACT FROM AUTHOR]

Tauco is one kind of traditional fermented food from Indonesia. Tauco is processed from soybean paste with a salty taste and commonly has yellow and black color. Generally, tauco is used as spice or seasoning in various daily dishes. The study aims to find and identify the bacteria that produced fibrinolytic enzymes in tauco. The sample used in this research is tauco from six different manufacturers. Two tests have to do in this study: a proteolytic test of the Skim Milk Aga and a fibrin plate test. Both tests used to gain the bacteria-producing fibrinolytic enzyme are Bacteria identified macroscopically, gram staining, and 16S rRNA sequencing. In the proteolytic test, the sample was diluted with 0.9% to 10-7 NaCl and then inoculated on Agar Milk Skm medium, incubated at 37ºC for 24 samples have a proteolytic activity that shown by the formation of clear zones around bacteria. 17 isolates of proteolytic bacteria were incubated at 37ºC for 24 hours and then used for the fibrin plates. All proteolytic bacteria produce fibrinolytic activity, the most extensive fibrinolytic index in TC4a isolate of 3,46. Furthermore, TC4a bacteria were genotypically identified by sequencing the 16S rRNA gene. According to the result of tauco identification, it is found that the bacteria that strongly produce fibrinolytic enzymes in tauco sample TC4a is similar to Bacillus cereus TC4a. [ABSTRACT FROM AUTHOR]

Purpose This study aimed to evaluate the effect of different additives on chemical composition, fermentation characteristics, and gas production parameters of tomato pomace and pumpkin waste silages.Method Treatments were: tomato pomace silage, pumpkin waste silage, tomato pomace and pumpkin waste silage mix (50:50), tomato pomace and pumpkin waste silage mix treated with the fibrinolytic enzyme (E), tomato pomace and pumpkin waste silage mix treated with LAB made inoculants (LMI), and tomato pomace and pumpkin waste silage mix treated with E+ LMI. Representatives of samples were packed manually into laboratory silos and allowed to ensile for 1, 3, 7, 21, 45, and 90 days.Results The results showed a significant difference between the experimental treatments in chemical composition (p

A novel thrombolytic enzyme was produced by food grade microorganism Neurospora crassa using agro-industrial by-products as substrates. Process parameters were optimized using Plackett–Berman and Box–Benhken design. Under the optimized fermentation conditions, high fibrinolytic activity of 403.59 U/mL was obtained. It was purified with a specific activity of 3572.4 U/mg by ammonium sulfate precipitation and SP Sepharose chromatography. The molecular weight of the enzyme was approximately 32 kDa. It exhibited maximum activity at 40 °C and pH 7.4. Its activity was enhanced by Cu2+, Na+, Zn2+, and completely inhibited by phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, which indicates it could be a serine protease. The enzyme could degrade fibrin clot directly without the need of plasminogen activator, and effectively cleaved Aα, Bβ, γ chains of fibrinogen. It could inhibit the formation of blood clots in vitro and acts as an anticoagulant. Compared to heparin the purified enzyme showed extended anticoagulant activity. Blood clots were dissolved effectively and dissolution rate was increased with time. Based on these results, this novel enzyme has the potential to be developed as a thrombolytic agent. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa was isolated from injuries of patients' wounds and burns, and to ensure that the isolate was belonging to P. aeruginosa, several tests were performed, such as staining techniques, a biochemical test, morphological test, Vitek 2 system, and sensitivity test. The results of the gram stain test showed rod pink gram-negative bacteria, demonstrating that the isolate belonged to P. aeruginosa. Growth optimization of bacterial was performed by assessing different combinations of pH and temperatures. It is revealed that the best conditions for increasing the number of bacteria were achieved at 37°C with the bacterial number of 5.53×108 and pH 6 with the bacterial number of 5.87×108. Fibrinolytic enzyme is an agent that lysis fibrin clots. This fibrinolytic factor has prospective use to treat cardiovascular diseases, such as stroke and heart attack. Cardiovascular diseases have attracted worldwide attention for their elevation morbidity and mortality. Fibrinolytic enzyme was extracted by centrifugation at 10000 × g at 4°C for 10 min, the supernatant was kept and the pellet having bacterial cells was discarded. Purification of the fibrinolytic enzyme was achieved using salt precipitation, ion exchange, and gel filtration chromatographic techniques. The results showed that the gel filtration chromatography had optimal specific activity and purification fold at 562.6 U/ml, and the final specific activity of the purified enzyme increased 4.1 times. The molecular weight of the fibrinolytic enzyme was determined at26 kDa by gel filtration chromatography. The purified fibrinolytic enzyme had optimum activity atpH 7 and40°C. The pH stability for the enzyme activity was found in pH 6-7 and the range of 10-40°C.

The antioxidative proteolytic fraction, MA-1, was partially purified from Mycoleptodonoides aitchisonii. MA-1 was purified to homogeneity using a two-step procedure, which resulted in an 89-fold increase in specific activity and 42.5% recovery. SDS-PAGE revealed two proteins with a molecular weight of 48 kDa. The zymography results revealed proteolytic activity based on the MA-1 band. MA-1 was found to be stable in the presence of Na+, Ca2+, Fe3+, K+, and Mg2+. MA-1 was also stable in methanol, ethanol, and acetone, and its enzyme activity increased by 15% in SDS. MA-1 was inhibited by ethylenediaminetetra-acetic acid or ethylene glycol tetraacetic acid and exerted the highest specificity for the substrate, MeO-Suc-Arg-Pro-Tyr-pNA, for chymotrypsin. Accordingly, MA-1 belongs to the family of chymotrypsin-like metalloproteins. The optimum temperature was 40 °C and stability was stable in the range of 20 to 35 °C. The optimum pH and stability were pH 5.5 and pH 4–11. MA-1 exhibited stronger fibrinolytic activity than plasmin. MA-1 hydrolyzed the Aα, Bβ, and γ chains of fibrinogen within 2 h. MA-1 exhibited an antithrombotic effect in animal models. MA-1 was devoid of hemorrhagic activity at a dose of 80,000 U/kg. Overall, our results show that M. aitchisonii produces an acid-tolerant and antioxidative chymotrypsin-like fibrinolytic enzyme, and M. aitchisonii containing MA-1 could be a beneficial functional material for the prevention of cardiovascular diseases and possible complications.

Cardiovascular disease (CVD) has become the leading cause of death, and it is critical to develop new functional foods to prevent intravascular thrombosis, the key cause of CVD. Fermented soy-based food is a good choice because of its native fibrinolytic enzyme (FE) activity. In this study, a strain that can produce a new type of fibrinolytic enzyme was selected from Chinese Douchi and identified as Bacillus licheniformis SFD-Y5 by molecular biology experiments and physiological and biochemical experiments. Single factor experiments combined with statistical experiments, including Plackett–Burman experiment, steepest ascent experiment and RSM (Box–Behnken design), were used to optimize the fermentation of FE by B. licheniformis SFD-Y5. The final FE activity was 2434.45 ± 28.49 IU/mL under optimal conditions, which is the highest FE activity produced by wild B. licheniformis so far. Further studies showed that Y5 FE is a serine metalloproteinase with good stability at alkaline pHs (pH 8.0–11.0). The results of our study could lay a foundation for the future production, molecular modification and further application in functional foods of Y5 FE.

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With the dramatic increase in mortality of cardiovascular diseases (CVDs) caused by thrombus, this has sparked an interest in seeking more effective thrombolytic drugs or dietary nutriments. The dietary consumption of natto, a traditional Bacillus-fermented food (BFF), can reduce the risk of CVDs. Nattokinase (NK), a natural, safe, efficient and cost-effective thrombolytic enzyme, is the most bioactive ingredient in natto. NK has progressively been considered to have potentially beneficial cardiovascular effects. Microbial synthesis is a cost-effective method of producing NK. Bacillus spp. are the main production strains. While microbial synthesis of NK has been thoroughly explored, NK yield, activity and stability are the critical restrictions. Multiple optimization strategies are an attempt to tackle the current problems to meet commercial demands. We focus on the recent advances in NK, including fermented soybean foods, production strains, optimization strategies, extraction and purification, activity maintenance, biological functions, and safety assessment of NK. In addition, this review systematically discussed the challenges and prospects of NK in actual application. Due to the continuous exploration and rapid progress of NK, NK is expected to be a natural future alternative to CVDs. [ABSTRACT FROM AUTHOR]

Petis is one of the fermentation product which is made with marine creatures such as fish or shrimp. A fibrinolytic in some fermentation food from marine creatures were discovered. Screening of bacteria that produce fibrinolytic enzyme using six petis from different home industry. The isolation of samples was obtained from Skim Milk Agar proteolytic test and fibrin plate test. Identification of the bacteria performed with gram staining test and sequencing 16S rRNA method. The sample was diluted with NaCl 0,9% up to 10-7 then inoculated on the media SMA and incubated in 37°C for 24 hours. All samples have the proteolytic activity that shown by a clear zone around the colony. There were seventeen proteolytic isolates bacteria tested fibrinolytic activity with fibrin plate then incubated in 37°C for 24 hours. The seventeen isolates showed positive result to fibrinolytic activity with highest fibrinolytic index was P3c. Isolate bacteria P3c was identified using sequencing gene 16s rRNA. The result of identifying, the bacteria that have strong fibrinolytic activity was Bacillus flexus. [ABSTRACT FROM AUTHOR]

Fibrinolytic enzymes are important components in the treatment of thrombosis-associated disorders. A new bi-functional fibrinolytic enzyme, versiase, was identified from a marine-derived fungus Aspergillus versicolor ZLH-1. The enzyme was isolated from the fungal culture through precipitation with ammonium sulfate at 90% saturation. Additionally, it was further purified by DEAE-based ion-exchange chromatography, with a recovery of 20.4%. The fibrinolytic enzyme presented as one band on both SDS-PAGE and fibrin-zymogram, with a molecular mass of 37.3 kDa. It was elucidated as a member of metalloprotease in M35 family by proteomic approaches. The homology-modeling analysis revealed that versiase shares significant structural homology wuth the zinc metalloendopeptidase. The enzyme displayed maximum activity at 40 °C and pH 5.0. The activity of versiase was strongly inhibited by the metalloprotease inhibitors EDTA and BGTA. Furthermore, versiase hydrolyzed fibrin directly and indirectly via the activation of plasminogen, and it was able to hydrolyze the three chains (α, β, γ) of fibrin(ogen). Additionally, versiase demonstrated promising thrombolytic and anticoagulant activities, without many side-effects noticed. In conclusion, versiase appears to be a potent fibrinolytic enzyme deserving further investigation. [ABSTRACT FROM AUTHOR]

Fibrinolytic enzyme is factor that lysis fibrin clots. This fibrinolytic factor has prospective use to treat cardiovascular diseases, such as stroke and heart attack. Cardiovascular diseases attracted worldwide attention for their elevation morbidity and mortality. Expensive cost and fatally undesired side effects associated with the available fibrinolytic agents to treat these diseases stimulated the researchers to investigate potentially better agents for curative applications. In the current investigation, fibrinolytic enzyme production from Pseudomonas aeruginosa isolated from injuries of wounds and burns patients. Parameters for the promoted production of the enzyme under minimal production media were optimized. It comprised carbon source (glucose), Nitrogen source (Yeast extract), Fibrinogen concentration (0.5 %), inoculum size (1 %), temperature (37°C), and PH (7). Enhanced fibrinolytic enzyme activity (136.2 IU/ml) was obtained after optimization Medium Components compared with that obtained with the minimal medium (60.2 IU/ml) which is 2.2 times higher than the same under non optimized production conditions. Media optimization researches for enhancement of fibrinolytic enzyme production from Pseudomonas aeruginosa in Iraq has not been performed so far. This may be the first study to optimization media for the production of fibrinolytic enzyme from Pseudomonas aeruginosa. The importance of this study lies in the enhancing the production of the fibrinolytic enzyme with high activity using these bacteria.

The demand for microbial thrombolytic agents is alarmingly increasing, and the discovery of novel microbial species with significant level of fibrinolytic enzyme production are required to cope up with the current circumstances. Surprisingly, marine fungi remain largely unexplored in this regard, presenting an untapped potential for the production of fibrinolytic enzymes. A novel marine fungal strain (Penicillium steckii KU1) was isolated successfully from Kappil beach, Kerala. At the beginning Czapek-dox media was used for the production of fibrinolytic enzyme. Statistical optimization of the enzyme production was achieved initially by Plackett-Burman design (PB). Interactive effect of various process parameters was further scrutinized by Central Composite Design (CCD). Incubation time, inoculum size and concentration of casein were shown significant contribution towards production. Statistical optimization, resulted in a tenfold increment (454.49 ± 14.66 U/mL) [optimized conditions - 30 °C, pH 7, casein 2.7 % (w/v), inoculum 5.75 million spores/mL and 115 h incubation], against the predicted value 441.42 U/mL. [Display omitted] • Fibrinolytic enzyme producing marine fungi was isolated and identified as Penicillium steckii KU 1. • Process parameters were optimized for enzyme production using Response surface methodology. • Optimum conditions were incubation time 115 h, inoculum size 5.75 million spores/mL and casein concentration 2.7 % w/v. • The mean activity (454.49 ± 14.66 U/mL), was found to be near to the predicted activity from the model (441.42 U/mL). • Final optimization of process parameters resulted in a tenfold increment in enzyme yield. [ABSTRACT FROM AUTHOR]

Fibrinolytic enzymes are a kind of proteolytic enzymes that can hydrolyze fibrin and dissolve blood clots. They could be used as a therapeutic agent for treating thrombosis. It is important for the treatment of cardiovascular disease to find and develop new thrombolytic drugs. In order to explore new fibrinolytic enzymes, a strain named 214L-11 with protease and fibrinolytic enzyme activity, which was isolated from the Flaming Mountain of Xinjiang Province, was screened using the skimmed milk plate, the blood powder agarose plate and the fibrin plate methods. Phylogenetic analyses showed that strain 214L-11 shared the highest similarity with Streptomyces fumanus NBRC 13042T (98.88%), which indicated that it represented a potential novel species in the Streptomyces genus. The fibrinolytic enzyme produced by 214L-11 displayed thrombolytic and anticoagulant activities, and it could degrade a single specific protein in the thrombus, thereby destroying the thrombus structure. The fermentation medium optimized through response surface methodology was 15 g/L soluble starch, g/L KNO3 0.58, 0.43 g/L peptone, 0.01 g/L FeSO4·7H2O, 0.5 g/L MgSO4·7H2O, 0.2 g/L Mn2+, 0.5 g/L NaCl and 1 L distilled water, pH 8, and the maximum amount of fibrinolytic enzyme produced by strain 214L-11 in the optimal fermentation medium was 1255.3 FU/mL. Overall, the fibrinolytic enzyme-producing strain was screened from the Flaming Mountain of Xinjiang for the first time, which provided a basis for further research and the development of new efficient and safe hemolytic drugs.

Fibrinolytic enzymes are the most effective agents for the treatment of thrombotic diseases. In the present study, we purified and characterized an extracellular fibrinolytic serine metalloprotease (named Velefibrinase) that is produced by marine Bacillus velezensis Z01 and assessed its thrombolysis in vivo. SDS-PAGE and MALDI-TOF-MS analyses showed that the molecular mass of Velefibrinase was 32.3 KDa and belonged to the peptidase S8 family. The optimal fibrinolytic activity conditions of Velefibrinase were 40 °C and pH 7.0. Moreover, Velefibrinase exhibited high substrate specificity to fibrin, and a higher ratio of fibrinolytic/caseinolytic (1.48) values, which indicated that Velefibrinase had excellent fibrinolytic properties. Based on the degradation pattern of fibrin and fibrinogen, Velefibrinase could be classified as α/β-fibrinogenase. In vitro, Velefibrinase demonstrated efficient thrombolytic ability, anti-platelet aggregation, and amelioration of blood coagulation (APTT, PT, TT, and FIB), which were superior to those of commercial anticoagulant urokinase. Velefibrinase showed no hemolysis for erythrocyte in vitro and no hemorrhagic activity in vivo. Finally, Velefibrinase effectively prevented mouse tail thrombosis in a dose-dependent (0.22–0.88 mg/kg) manner. These findings suggested that Velefibrinase has the potential to becoming a new thrombolytic agent. [ABSTRACT FROM AUTHOR]

Abstract Background As a main drug for diseased thrombus, some clinically used thrombolytic agents have various disadvantages, safer novel thrombolytic agents are of great demand. This study aimed to achieve high and efficient production of a fibrinolytic enzyme with superior enzymatic properties, by a combination strategy of codon optimization, gene dosage and process optimization in Komagataella phaffii (K. phaffii). Results After codon optimization, the fibase from a marine Bacillus subtilis was expressed and secreted in K. phaffii GS115. Recombinant strains harboring different copies of the fib gene (fib-nc) were successfully obtained via Geneticin (0.25–4 mg/ml) screening on minimal dextrose selection plates and assessment via real-time quantitative PCR. The respective levels of fibase produced by strains expressing fib-5.4c, fib-6c, fib-8c, fib-9c, and fib-12c were 4428, 5781, 7323, 7930, and 2472 U/ml. Levels increased as the copy number increased from 4 to 9, but decreased dramatically at copy number 12. After high cell density fermentation optimization, the highest fibase activity of the strain expressing fib-9c was 7930 U/ml in a shake flask and increased to 12,690 U/ml after 3 days of continuous culture in a 5-L fermenter, which is one of the highest levels of production reported. The recombinant fibase was maximally active at pH 9.0 and 45 °C, and was remarkably stable at pH levels ranging from 5 to 10 and temperatures up to 50 °C. As a metal-dependent serine protease, fibase did not cause hemolysis in vitro and preferentially degraded fibrin directly. Conclusions The combination of codon optimization, gene dosage, and process optimization described herein could be used for the expression of other therapeutic proteins difficult to express. The characteristics of the recombinant fibase suggest that it has potential applications for thrombosis prevention and therapy.

Purpose This study aimed to evaluate the effect of different additives on chemical composition, fermentation characteristics, and gas production parameters of tomato pomace and pumpkin waste silages. Method Treatments were: tomato pomace silage, pumpkin waste silage, tomato pomace and pumpkin waste silage mix (50:50), tomato pomace and pumpkin waste silage mix treated with the fibrinolytic enzyme (E), tomato pomace and pumpkin waste silage mix treated with LAB made inoculants (LMI), and tomato pomace and pumpkin waste silage mix treated with E+ LMI. Representatives of samples were packed manually into laboratory silos and allowed to ensile for 1, 3, 7, 21, 45, and 90 days. Results The results showed a significant difference between the experimental treatments in chemical composition (p<0.05). The treatment of pumpkin waste showed the lowest amount of dry matter (DM), insoluble fibers in neutral detergent (NDF), and insoluble fibers in acidic detergent (ADF). The value of crude protein (CP) showed a decreasing trend with increasing time after ensiling. The treatment with bacterial and enzymatic additives had a faster drop in pH and a lower final pH compared to other treatments. Conclusion Compared with the tomato pomace and pumpkin waste silage, treatments E and E + LMI had lower acetic and butyric acid contents. During aerobic exposure, tomato pomace and pumpkin waste had the lowest pH changes in silage. Generally, applying a combination of E and LAB inoculants improved both fermentation quality and aerobic stability of silage. [ABSTRACT FROM AUTHOR]

Objectives: The main aim of this study is to isolate and characterize fibrinolytic enzyme from Bacillus flexus. Methods: Fish meal of Sardinella longiceps and anchovy was optimized using a two-level full factorial design (25) and response surface methodology. The significant physical factors and nutrient sources (peptone, maltose, and magnesium chloride) were identified by statistical approach. The properties of a purified enzyme including their effect at different temperature, pH and the effect of metal ions were evaluated. Results: Enzyme yield was improved 3.5 fold than unoptimized medium. Central composite design optimized culture medium enhanced enzyme yield (4711 ± 29.3 U/g of substrate). The fibrinolytic enzyme was highly active at alkaline pH (8.0), 50 °C and the molecular weight was 32 kDa. Conclusions: From these findings, it concludes that this fibrinolytic enzyme could be a novel potent thrombolytic agent.

The purpose of this investigation was to produce fibrinolytic enzyme by utilizing sugarcane bagasse as low cost substrate through solid state fermentation in an effective way. The potential producer of microorganism, Alcaligenes aquatilis PJS_1 was isolated and identified from slaughter house soil samples. In small scale level, higher production of microbial fibrinolytic enzyme was observed when the conditions provided like Glucose for carbon, Casein for nitrogen and (NH4)2SO4 for mineral sources when maintained at 60 h with 100% moisture. The sugarcane bagasse medium with glucose and casein was found to have optimum production when incubated for 60 h at 35°C. The culture conditions were optimized to improve the production of fibrinolytic enzyme (3832 U ml−1) from Alcaligenes aquatilis PJS_1 through statistical approach. Blood clotting assay was performed to determine its anticoagulant property. The fibrinolytic property of the synthesised enzyme was confirmed when it dissolved blood clot sample effectively. Thus, the enzyme it could be used in medicine to produce as a thrombolytic agent.

Background: The fibrinolytic enzymes from Bacillus sp. are proposed as therapeutics in preventing thrombosis. Computational-based analyses of these enzymes' amino acid composition, basic physiological properties, presence of functional domain and motifs, and secondary and tertiary structure analyses can lead to developing a specific enzyme with improved catalytic activity and other properties that may increase their therapeutic potential., Methods: The nucleotide sequences of fibrinolytic enzymes produced by the genus Bacillus and its corresponding protein sequences were retrieved from the NCBI database and aligned using the PRALINE programme. The varied physiochemical parameters and structural and functional analysis of the enzyme sequences were carried out with the ExPASy-ProtParam tool, MEME server, SOPMA, PDBsum tool, CYS-REC tool, SWISS-MODEL, SAVES servers, TMHMM program, GlobPlot, and peptide cutter software. The assessed in-silico data were compared with the published experimental results for validation., Results: The alignment of sixty fibrinolytic serine protease enzymes (molecular mass 12-86 kDa) sequences showed 49 enzymes possess a conserved domain with a catalytic triad of Asp196, His242, and Ser569. The predicted instability and aliphatic indexes were 1.94-37.77, and 68.9-93.41, respectively, indicating high thermostability. The random coil means value suggested the predominance of this secondary structure in these proteases. A set of 50 amino acid residues representing motif 3 signifies the Peptidase S8/S53 domain that was invariably observed in 56 sequences. Additionally, 28 sequences have transmembrane helices, with two having the most disordered areas, and they pose 25 enzyme cleavage sites. A comparative analysis of the experimental work with the results of in-silico study put forward the characteristics of the enzyme sequences JF739176.1 and MF677779.1 to be considered when creating a potential mutant enzyme as these sequences are stable at high pH with thermostability and to exhibit αβ-fibrinogenase activity in both experimental and in-silico studies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

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Pseudomonas aeruginosa was isolated from injuries of patients' wounds and burns, and to ensure that the isolate was belonging to P. aeruginosa, several tests were performed, such as staining techniques, a biochemical test, morphological test, Vitek 2 system, and sensitivity test. The results of the gram stain test showed rod pink gramnegative bacteria, demonstrating that the isolate belonged to P. aeruginosa. Growth optimization of bacterial was performed by assessing different combinations of pH and temperatures. It is revealed that the best conditions for increasing the number of bacteria were achieved at 37°C with the bacterial number of 5.53×108 and pH 6 with the bacterial number of 5.87×108. Fibrinolytic enzyme is an agent that lysis fibrin clots. This fibrinolytic factor has prospective use to treat cardiovascular diseases, such as stroke and heart attack. Cardiovascular diseases have attracted worldwide attention for their elevation morbidity and mortality. Fibrinolytic enzyme was extracted by centrifugation at 10000 × g at 4°C for 10 min, the supernatant was kept and the pellet having bacterial cells was discarded. Purification of the fibrinolytic enzyme was achieved using salt precipitation, ion exchange, and gel filtration chromatographic techniques. The results showed that the gel filtration chromatography had optimal specific activity and purification fold at 562.6 U/ml, and the final specific activity of the purified enzyme increased 4.1 times. The molecular weight of the fibrinolytic enzyme was determined at26 kDa by gel filtration chromatography. The purified fibrinolytic enzyme had optimum activity atpH 7 and40°C.The pH stability for the enzyme activity was found in pH 6-7 and the range of 10-40°C. [ABSTRACT FROM AUTHOR]

Bacterial fibrinolytic proteases achieved more attention in the prevention and treatment of cardiovascular diseases, so purification, characterization, and activity enhancement are of prime importance. In this study, a fibrinolytic serine metalloprotease was purified from the culture supernatant from Bacillus sp. BC1. It was purified to homogeneity by a two-step procedure with a 24-fold increase in specific activity and a 33.1% yield. It showed 28 kDa molecular weight, while its optimal pH and temperature were obtained 8 and 50–60 °C. The cross-link enzyme aggregates of this fibrinolytic BC1 successfully immobilized on magnetic chitosan nanoparticles. A 52% activity enhancement was obtained by immobilized enzyme at pH 6.0, compared to free protease. Km values of the free and immobilized proteases were obtained about 0.638 and 0.61 mg/ml, respectively. The free and immobilized enzymes did not show any activity concerning transferrin, γ-globulins, and hemoglobin, as blood plasma proteins. The in vitro blood clot lysis test of the free and immobilized proteases showed a maximum of 42 and 50% clot lysis, which was comparatively higher than that revealed by streptokinase and heparin at the same condition. These results indicated that the free and immobilized proteases have the potential to be effective fibrinolytic agents. [ABSTRACT FROM AUTHOR]

Nattokinase is a fibrinolytic enzyme identified and isolated in 1987 from a specific Japanese food called natto (a kind of vegetable cheese). Since its discovery and until now, nattokinase has been studied experimentally and clinically almost exclusively in Japan, China, Korea and other countries in the respective geographical area, resulting in a complex picture of potentially useful properties in clinical practice. Nattokinase was approved for use in Europe in 2016 by EFSA (European Food Safety Authority) as a food supplement.

An aqueous protocol was developed for nattokinase purification under scalable conditions using a combination of salting out, anion exchange chromatography and ultra-filtration. The enzyme was firstly washed off from the fermented soybeans. The whole proteins were precipitated by ammonium sulfate and purified by anion exchange chromatography. The purified enzyme was finally refined by size exclusion chromatography and ultra-filtration at laboratory and industry-compatible level, respectively. There was a 476.18-fold increase in enzymatic activity with a 48.3% yield at the laboratory level and 429.75-fold increase in enzymatic activity with a 42.7% yield at the industrial-compatible scale. The protocol was universally adaptive for purifying the fibrinolytic enzymes produced by bacillus tequilensis, bacillus amyloliquefaciens, and bacillus cereus, showing a purification efficiency of 329.76-fold with 42.7% yield, 221.78-fold with 32.5% yield, and 288.56-fold with 38.7% yield, respectively. All procedures are scalable, aqueous, simple, cost-effective, and suitable for fibrinolytic enzymes industrial production.

With the dramatic increase in mortality of cardiovascular diseases (CVDs) caused by thrombus, this has sparked an interest in seeking more effective thrombolytic drugs or dietary nutriments. The dietary consumption of natto, a traditional Bacillus-fermented food (BFF), can reduce the risk of CVDs. Nattokinase (NK), a natural, safe, efficient and cost-effective thrombolytic enzyme, is the most bioactive ingredient in natto. NK has progressively been considered to have potentially beneficial cardiovascular effects. Microbial synthesis is a cost-effective method of producing NK. Bacillus spp. are the main production strains. While microbial synthesis of NK has been thoroughly explored, NK yield, activity and stability are the critical restrictions. Multiple optimization strategies are an attempt to tackle the current problems to meet commercial demands. We focus on the recent advances in NK, including fermented soybean foods, production strains, optimization strategies, extraction and purification, activity maintenance, biological functions, and safety assessment of NK. In addition, this review systematically discussed the challenges and prospects of NK in actual application. Due to the continuous exploration and rapid progress of NK, NK is expected to be a natural future alternative to CVDs.