Fibrinolytic Enzyme Activity Research Articles

Fibrinolytic Protease Production: Impact of Initial pH and Temperature in Solid-State Fermentation by Rhizopus microsporus var. oligosporus FNCC 6010

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.

Isolation and Optimal Fermentation Conditions of Bacillus licheniformis SFD-Y5 for a New Douchi Fibrinolytic Enzyme Producer

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.

Screening of a Novel Fibrinolytic Enzyme-Producing Streptomyces from a Hyper-Arid Area and Optimization of Its Fibrinolytic Enzyme Production

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.

Therapeutic and Anti-Thrombotic Properties of Some Naturally Fermented Soybean Foods of the Eastern Himalayas

Naturally fermented soybean foods have anti-thrombotic properties due to the presence of microbial fibrinolytic enzymes. However, fibrinolytic enzyme-producing bacteria from naturally fermented soybean foods of the Eastern Himalayas viz. kinema, grep-chhurpi, peha, peron namsing, peruñyaan and bemerthu have not been screened yet. Therefore, this study aims to screen the fibrinolytic enzyme-producing bacteria from these naturally fermented soybean foods and also to assess their therapeutic properties such as antioxidant, anti-inflammatory, flavones, isoflavones, anti-obesity and anti-diabetic properties of samples. A total of 877 bacteria were isolated, out of which Bacillus subtilis (ten strains), B. velezensis (three strains), B. inaquosorum (two strains) and B. halotolerans (one strain) showed fibrinolytic enzyme activity ranging from 775.70 U/mL to 1230.61 U/mL, with a blood clot-degrading property of more than 50%. A remarkable DPPH radical scavenging property (>50%), flavone (278.98 ± 7.06 µg QE/g) and isoflavone (394.13 ± 11.68 µg GEN/g) contents were observed. The Himalayan fermented soybean foods have shown anti-thrombotic, antioxidant, anti-inflammatory, anti-diabetic and anti-obesity properties indicating their therapeutic values.

Патогенетичні особливості пептичної виразки шлунка та дванадцятипалої кишки в поєднанні з артеріальною гіпертензією та цукровим діабетом 2-го типу

Розвиток та перебіг пептичної виразки шлунка та дванадцятипалої кишки супроводжуються наявністю штамів H. pylori (СagA+ VacA+, СagA+ vacА–, CagA– VacA+, CagA– VacA–), що виявлено у хворих за наявності супутньої патології та коли її немає. У групі хворих на пептичну виразку шлунка та дванадцятипалої кишки в поєднані з артеріальною гіпертензією і цукровим діабетом 2-го типу виявлено штами СagA+ VacA+ у 14 осіб (45,16 %), СagA+ VacA– — у 4 (12,9 %), CagA– VacA+ — у 13 (41,43 %) та в групі хворих на пептичну виразку шлунка та дванадцятипалої кишки — відповідно в 4 (13,79 %), у 8 (27,59 %), у 16 (55,17 %) осіб. Вплив штамів H. pylori супроводжується змінами протеолітичної та фібринолітичної активності. З урахуванням супутньої патології та ролі штамів при розвитку основної патології виявлено вірогідне зниження рівня лізису альбуміну, лізису азоказеїну та підвищення лізису азоколу порівняно зі зниженням сумарної фібринолітичної активності, неферментативної фібринолітичної активності та підвищенням ферментативної фібринолітичної активності. Використання антихелікобактерної базисної терапії та пробіотиків (Lactobacterium, Bifidobacterium) дає позитивний результат.

Enhanced Production of Fibrinolytic Enzyme from Pseudomonas aeruginosa by Optimization Media Components

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.

Optimization of Cultural Conditions for Maximum Production of Fibrinolytic Enzymes from the Local Marine Bacterial Isolates and Evaluation of their Wound Healing and Clot Dissolving Properties

Fibrinolytic enzymes find necessary applications to treat and prevent cardiovascular diseases. In this study, optimal conditions for enhancing the production of fibrinolytic enzyme from local marine bacterial strains were evaluated. The present study also focuses on screening of wound healing efficacy of the isolated fibrinolytic enzymes.Various physical parameters such as temperature, pH, incubation time and medium components viz. inoculum size, substrate (nitrogen and carbon) concentrations were optimized. A cultivation medium was designed using optimized conditions for mass production of fibrinolytic enzyme and specific activity of enzyme was analyzed. The maximum enzyme production was observed at 37 °C temperature, 8.0 pH,substrate concentration with 3 ml inoculum size and 32 h. of incubation time. Among the different carbon sources tested, Mannitol showed maximum enzyme activity i.e 538 U/ml. yeast extract was found to be the best nitrogen source with an enzyme activity of 498 U/ml. The best substrate for the production fibrinolytic enzyme was found to be kernelwith high activity of 1056U/ml. The crude enzyme displayed potent activity and digested blood clot completely in in vitro condition and exhibited potent activity on wound healing property in macrophages.

Optimization of Conditions for Using Rhizopus Chinesis 12 to Ferment and Produce Semen Sojae Praeparatum With High-activity Fibrinolytic Enzyme

To explore the optimal conditions for the production of high-activity thrombolytic enzymes by Rhizopus chinesis 12 fermented semen sojae praeparatum, the Rhizopus Chinesis 12 was used as fermentation strain, five kinds of beans (black beans, soybeans, red beans, mung beans, white kidney beans) were used as raw materials to screen the best raw materials and pretreatment methods for the processing of semen sojae praeparatum.Using the activity of fibrinolytic enzyme as the indicator, the fermentation process of semen sojae praeparatum was optimized by response surface test, and the product quality was analyzed.The results showed that, the black soybean as raw materials and indicated the optimal formulations as follows:Black soybean after germination treatment for 38 h, inoculation rate was 15% (m/m), fermentation temperature was 31℃, and fermentation time was 7 d. Under these optimal fermentation conditions, the fibrinolytic activity of Semen sojae praeparatum reached to (17122 ±392.7) U/g.Furthermore, the ammonia nitrogen of the prepared semen sojae praeparatum was significantly higher than that of a retail brand (p < 0.05), while, its hardness, elasticity and chewiness were significant lower than those of the retail brand (p < 0.05). In addition, the fibrinolytic enzyme activity of semen sojae praeparatum powder after drying was 16350 U/g, which was significantly higher than that of a certain brand of natto (22%) (p < 0.05).These results indicated that Rhizopus chinesis 12 could significantly increased he fibrinolytic enzyme activity of semen sojae praeparatum (p < 0.05), which would have a great significance for further development of the semen sojae praeparatum with high activity fibrinolytic enzyme.

Optimization of process parameters for the enhanced production of fibrinolytic enzyme by a newly isolated marine bacterium

Thrombotic disorders are the major cause of death worldwide. As the thrombolytic agents we use today were having lot of side effects, microbial enzymes, which have marked specificity to fibrin, gain attention. A marine bacterium with potent fibrinolytic activity was successfully isolated and identified as Serratia rubidaea KUAS001. Process parameters for the enhanced production of the enzyme under submerged fermentation were optimized. It includes, incubation time (48 h), inoculum size (0.8% v/v), pH (6.0), temperature (30 °C) and concentration of nutrient sources like maltose (1.5% w/v), yeast extract (1.5% w/v), NaCl (1.5% w/v) and skimmed milk powder (0.15% w/v). Enhanced fibrinolytic enzyme activity (394.9 IU/ml) was obtained after optimization, which is 2.57 times higher than the same under unoptimized conditions.

Tetramethylpyrazine from adlay (Coix lacryma-jobi) biotransformation by Bacillus subtilis and its quality characteristics.

Adlay, as a traditional Chinese medicine, has been used in nourishing foods, which are rich in a variety of nutrients (special biological compounds). The study was designed to optimize the fermentation parameters of dehulled, polished and broken adlay fermented by Bacillus subtilis BJ3-2 with regard to tetramethylpyrazine (TMP) yield and fibrinolytic enzyme activity. Then the proximate and bioactive components of B. subtilis-fermented adlay were evaluated. Box-Behnken design results showed that the TMP yield was 6.93mg/g DW (dried weight) of B. subtilis-fermented polished adlay, which was about 136 times higher than that of B. subtilis-fermented soybean (BSB). The fibrinolytic enzyme activity was 2236.17 U/g in B. subtilis-fermented dehulled adlay, and slightlylessthan in BSB. B. subtilis-fermented adlay contained higher fat, free amino acids and fatty acids contents but lower protein and starch contents than raw adlay. Except for coixol and coixan, the levels of γ-aminobutyric acid, triterpenes, phenolics, flavonoids and coixenolide in B. subtilis-fermented adlay increased by 14.05, 2.02, 2.31 and 1.36 times, respectively. The contents of phenolic acids including caffeic, gallic, catechinic and chlonogenic acids in the free phenolic extracts significantly increased (p < 0.05). The results demonstrated thatthebiotransformation of high-yield TMP, fibrinolytic enzyme and other bioactive components of B. subtilis-fermented adlay products was realized. B. subtilis-fermented adlay could be a promising value-added food, and that is more suitable for human consumption.

Characterization of Fibrinolytic Enzymes Produced by the Halophilic Streptomyces Flaveolus and Streptomyces Galtieri Isolated from Wady El-Natron Area in North Egypt

AbstractBackground: Intravascular thrombosis is responsible for an increasing number of deaths every year. Lung blood clots in USA alone affect 1,000,000 patients annually. Clots formed from insoluble fibrin restrict the smooth flow of blood in blood vessels, leading to thrombosis and heart attacks. World-wide, over 29% of the total mortalities are due to thrombosis, thus antithrombotic therapy is of great interest. The three fibrinolytic (thrombolytic) agents that are currently being used for this purpose are urokinase, streptokinase and genet-ically engineered tissue-type Plasminogen Activator (t-PA). However, these enzymes are expensive, thermolabile, with low specificity and stability, large therapeutic doses and can produce undesirable side effects such as gastrointestinal bleeding, allergic reactions, and resistance to repercussion. This warrants the search for novel fibrinolytic enzymes from various sources with higher efficacy, safety, specificity and stability and preferably those direct acting activities. Though new fibrinolytic enzymes are being explored from microbes, microorganisms remain the preferred source due to their biochemical versatility, feasibility of mass culture and ease of genetic manipulation. Hence, many fibrinolytic enzymes have been isolated from a variety of microorganisms, including actinomycetes, bacteria, fungi, and algae.Aim of Study: To detect the characterization of fibrinolytic enzymes produced by the halophilic Streptomyces flaveolus and Streptomyces galtieri that were isolated from soil of Wady El-Natron region, in North Egypt, as a potential thrombolytic agent.Material and Methods: Fibrinolytic enzymes, extracted from Streptomyces flaveolus and Streptomyces galtieri, were purified by ammonium sulfate precipitation and gel filtration. By using SDS-Page electrophoresis, determining their molec-ular masses, classified by inhibitory acting materials, charac-terized by determining metal ions influences, anticoagulation clotting time delay with CaCl2, their proteolytic activity in units/mg, the active conc. in μg/ml and the least conc. in μl/ml of thrombolytic activity.Results: The fibrinolytic enzymes extracted from both Streptomyces flaveolus and Streptomyces galtieri are classified as metallo-protease enzymes, their molecular masses were 1 6kDa and 4 1kDa, their proteolytic activity are 1.4 and 2.3 units/mg of proteins, the anticoagulation clotting time assay showed 20 and 15min delay in clotting time with CaCl2, the active conc. of each enzyme was determined from the standard curve using streptokinase as standard fibrinolytic enzyme as 200 and 300μg/ml, and the least conc. of enzyme thrombolytic activity was measured by performing the modified Holmstrom method, as the crude thrombolytic activity were at concentra-tions of 80-100μl, whereas, precipitated enzymes, showed thrombolytic activity at concentrations 20μl and 30μl/ml respectively. Metal ions exhibited different influences on the fibrinolytic enzyme activity produced by Streptomyces flave-olus, such as Ca2+, Cu2+ and Fe2+ promoted the activity of the enzyme, whereas Mg2+ is strongly promoted it. But on the fibrinolytic enzyme activity produced by Streptomyces galtieri, Cu2+ promoted the activity of the enzyme, whereas Ca2+, partially inhibit the enzyme, but the Fe2+ and Mg2+ were strongly and completely inhibit it.Conclusion: These results suggest that the extracted enzymes from both isolated Streptomyces flaveolus and Streptomyces galtieri are with potent and potential applications as thrombolytic agent.

Optimization of Fibrinolytic Enzyme from Streptomyces althioticus BN22

Fibrinolytic enzyme plays an important place in the medical field for cardiovascular disease. Only few studies have reported the fibrinolytic enzyme production from marine actinomycetes, and Streptomyces sp. In this study 114 actinomycetes isolates from different marine soil samples were isolated by using starch casein agar media. These 114 actinomycetes strains were tested for fibrinolytic activity by fibrin plate method. 14 isolates showed fibrinolytic activity amongst the 114 actinomycetes strains, and this was identified based on the clear zone of formation around the colonies. The potent strain of Streptomyces althioticus BN22 was isolated and this was confirmed by 16SrRNA sequencing. The optimization of nutritional and physical parameters for fibrinolytic enzyme production from marine soil was studied for the isolate Streptomyces althioticus. The effects of nutritional and physical parameters were studied for the optimum production of a fibrinolytic enzyme from Streptomyces althioticus BN22 by submerged fermentation. The effects of initial pH, temperature and incubation time were studied. The maximum enzymatic activity was obtained with an initial pH7.5 (45,765 Eu/ml), incubation temperature 50°C (50,827 Eu/ml) and incubation time for (41,175 Eu/ml). The effects of various carbon and nitrogen sources were studied. The results indicated that glucose (51,570 Eu/ml) as carbon source and yeast extract (54,625 Eu/ml) as nitrogen source showed the highest fibrinolytic enzyme activity among them. Thus this study showed its originality by focus, that marine Streptomyces althioticus is an important source for fibrinolytic enzyme production.

Microbial diversity and chemical analysis of Shuidouchi, traditional Chinese fermented soybean

Shuidouchi is a traditional Chinese fermented soybean product and its quality is largely affected by the microbes involved in the fermentation. In this study, eleven Shuidouchi samples were collected from southwest China and the microbial diversity and its correlations with chemical characteristics were investigated. Bacterial community was detected using 16S rRNA sequencing, along with bacterial and fungal viable plate counts. Biogenic amines and other chemical characteristics were determined by HPLC and corresponding chemical reaction methods. Among eleven Shuidouchi samples, 21 phyla and 356 genera were identified. Firmicutes, Bacteroidetes and Proteobacteria were the predominant phyla while Bacillus, Bacteroides and Lactobacillus were the main genera. The average cell number of bacteria, lactic acid bacteria and fungi were 1.6 × 106, 5.9 × 104 and 7.6 × 103 CFU/g, respectively. HPLC results showed that the mean concentration of tryptamine, β-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine were 23.11, 3.66, 12.21, 7.12, 8.13, 22.98, 24.72, and 39.00 mg/kg, respectively. The average content of other characteristics including amino acid nitrogen, titratable acidity, and reducing sugar were 2.08, 3.44, and 25.78 g/kg, respectively. Shuidouchi samples were slightly acidic or neutral. Fibrinolytic enzyme activity was detected only in one sample. Among top 52 identified genera, 9 genera showed positive correlations with the chemical characteristics of Shuidouchi while 15 genera were negatively associated. Our results indicated that Shuidouchi contained rich microbial resources and were edible safety based on the tested indexes. The associations identified between microbes and chemical characteristics could be further utilized in the food fermentation industry.

Enhanced production of fibrinolytic enzyme by a new Xanthomonas oryzae IND3 using low-cost culture medium by response surface methodology.

Cardiovascular diseases (CVDs) cause high mortality throughout the world. Existing fibrinolytic agents are highly expensive and have many side effects. Microbial fibrinolytic enzymes are very much considered as novel therapeutic candidate for the treatment of CVDs. Reports on fibrinolytic enzyme from Xanthomonas sp. is lacking. This study reports fibrinolytic enzymes from Xanthomonas oryzae IND3 as it shows hyperactivity on fibrin-agarose plates. This organism utilized various agro-industrial wastes for enzymes production. Among all, cow dung enhanced more enzyme production, hence it was used as the low-cost substrate for statistical optimization of fibrinolytic protease in Solid state fermentation. Response surface methodology was employed to optimize the factors and enhanced yield by 4-fold. The interactions among the variables, viz, sucrose, yeast extract, and pH of the medium were investigated using Central Composite Design (CCD). The predicted fibrinolytic enzyme activity was 2340 U/g, and the observed fibrinolytic enzyme activity was 2294 ± 12.8 U/g. The fibrinolytic enzyme degraded blood clot in vitro completely. This study is the first report on statistical optimization of fibrinolytic enzyme production in SSF from Xanthomonas sp. The crude extract has immense activity on proteinaceous wastes. The production of fibrinolytic protease using the low-cost substrate could reduce the production cost of enzyme.

Purification and partial characterization of TFase, a fibrinolytic enzyme from the fruiting bodies of the medicinal and edible mushroom, Tremella fuciformis

A fibrinolytic metalloprotease-like enzyme named TFase, was purified from the fruiting bodies of the medicinal fungus Tremella fuciformis. Purification of TFase was performed by a chromatography on DEAE Sepharose CL-6B and Superdex-75. TFase showed a molecular weight of 38 kDa by SDS-PAGE and displayed a strong fibrin zymogram lysis band corresponding to a similar molecular mass. The enzyme demonstrated wide temperature optimum from 10 to 40°C and pH optimum at pH 8.0. The fibrinolytic activity of TFase was strongly inhibited by EDTA and EGTA, which are metalloprotease inhibitors. The K m and V max values for enzyme using the artificial substrate pyroGlu-Gly-Arg-pNA were 3.81 mM and 4.88 µmoles mg–1 min–1, respectively. TFase of T. fuciformis partially dissolved a fibrin clot directly and specifically cleaved the Aα and Bβ chains of fibrinogen. In addition, the enzyme prolonged the coagulation time, and increased the activated partial thromboplastin time and prothrombin time. These results suggest that TFase of T. fuciformis exhibits properties of a metalloprotease-like enzyme, shows novel fibrin(ogen)olytic enzyme activity, and has the potential to be a therapeutic agent for thrombosis.

Isolation and Optimization of Growth Condition of Bacillus sp. from Fermented Shrimp Paste for High Fibrinolytic Enzyme Production

The fibrinolytic enzyme has been found in the traditional fermented foods including the traditional shrimp paste product. In this study, six Vietnamese traditional shrimp paste products collected in three regions (north, south and middle of Vietnam) were screened for fibrinolytic enzyme and the related bacteria were isolated and identified. The fibrinolytic enzyme was found in all Vietnamese shrimp paste products, in which the products in the middle of Vietnam had higher fibrinolytic enzyme activity (2.43–2.95 FU/ml) than those in other regions. The isolated bacterium which produced the highest fibrinolytic enzyme was identified as a strain of Bacillus sp., closely related to the species of Bacillus weihenstephanensis with 99 % identities. The optimal fermentation conditions were also investigated using response surface methodology based on Box–Behnken design for high fibrinolytic enzyme production by the isolated strain. As a result, the fibrinolytic enzyme activity reached to 6.85 FU/ml at the optimal fermentation condition of 1.50 % of shrimp shell powder, 1.44 % of NaCl, at 33 °C and 32-h fermentation. Thus, the fibrinolytic enzyme produced by the strain of Bacillus sp. in this study might be used as a thrombolytic agent in pharmaceutical industries.

Studies on a Fibrinolytic Enzyme from Bacillus species

Two species of Bacillus, namely, Bacillus amyloliquefaciens and Bacillus licheniformis , isolated from spoilt milk and soy flour, respectively, exhibited fibrinolytic enzyme (Nattokinase) activity. In the laboratory scale studies, of these two cultures, B. amyloliquefaciens produced the Fibrinolytic enzyme in higher quantities, 28.98 FU/mL, compared to 26.63 FU/mL in B. licheniformis . The maximal activities were obtained after 72 h. The optimum conditions at laboratory for maximal production of the fibrinolytic (Nattokinase) enzyme were: pH 7.2, temperature 37 C and agitation 200 rpm. In scale up trials in a 7 L fermentor, the fibrinolytic activity of B. amyloliquefaciens was 55.6 at 72 h. The molecular weight of the enzyme was estimated to be about 38 kDa. The enzyme had exhibited excellent blood clot dissolving property and therefore may be considered for further scale up and commercial exploitation.

Separation and screening of components with fibrinolytic enzyme activity from twelve Chinese herbal medicines

Objective To separate and screen components with fibrinolytic enzyme activity from twelve Chinese herbal medicines. Methods The components were extracted with water and precipitated with salt, and they were tested by fibrinolytic protein plates method. The active components with fibrinolytic activity were separated and screened which were compared with urokinase. Results Eleven of the twelve extracts showed fibrinolytic activity, while Trichosanthes kirilowii got the biggest fibrinolytic zone after 36 hours, followed by Alisma plantago-aquatica and Leonurus japonicus, and the Radix Astagali got the smallest one. According to the concentration of the protein, the area of the fibrinolytic zone and the specific activity of the components, the extract from Angelica sinensis exhibited the best specific activity at level of 48.46U/mg. Conclusion The extracts from Chinese herbal medicines except Semen Persicae exhibit fibrinolytic enzyme activity which can dissolve the fibrin in different degrees. Key words: Chinese herbal medicine; Fibrinolytic enzyme; Separation; Specific activity

Improvement of the Nutritional Quality and Fibrinolytic Enzyme Activity of Soybean Meal by Fermentation of B acillus subtilis

Solid-state fermentation condition of soybean meal (SBM) by Bacillus subtilis XZI125 for producing fibrinolytic enzyme and soybean peptides was optimized, and the effect of microbe fermentation on decreasing anti-nutritional factors was studied. The optimum fermentation conditions were obtained by one-factor-at-a-time and orthogonal array design as follows: fermentation temperature 30C, fermentation time 48 h, grinding particle size of soybean meal 40 mesh, pH 7.0, soybean meal loading 10 g in a 250-mL flask, 1.5% glucose, initial moisture content and inoculum sizes were 1.0 mL/g and 3%, respectively. Under the optimum fermentation condition, fibrinolytic activity and soybean peptides reached 1347.35 IU/g and 171.32 mg/g, respectively. Furthermore, the trypsin inhibitor, phytic acid and urease content were reduced by 33.57, 42.65 and 67.31%, respectively. In our knowledge, it was first reported that the nutritional quality of SBMs was improved and fibrinolytic enzyme in fermented soybean meal (FSBM) was produced by fermentation. Practical Applications Soybean meal (SBM) has traditionally been used as an important dietary protein in the feed industry. However, SBM may be used as food for human consumption if anti-nutritional factors are reduced by fermentation with food-grade microbes. Solid-state fermentation condition of SBM by Bacillus subtilis XZI125 to produce fibrinolytic enzyme and soybean peptides was optimized. The FSBM complemented the fibrinolytic enzyme and peptides, and simultaneously reduced anti-nutritional factors, such as trypsin inhibitor, phytic acid and urease, indicating that the nutritional and health function of FSBM was upgraded through fermentation, and it could become a potential health food.

Statistical optimization of fibrinolytic enzyme production using agroresidues by Bacillus cereus IND1 and its thrombolytic activity in vitro.

A potent fibrinolytic enzyme-producing Bacillus cereus IND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (P < 0.05). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h. In vitro assays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent.