Fibrinolytic Enzyme Research Articles

Gene cloning, expression and homology modeling of first fibrinolytic enzyme from mushroom (Cordyceps militaris)

Fibrinolytic enzymes are important thrombolytic agents for blood-clotting disorders like cardiovascular diseases. Availability of novel recombinant fibrinolytic enzymes can overcome the shortcomings of current thrombolytic drugs. With the objective of facilitating their cost-effective production for therapeutic applications and for gaining deeper insight into their structure-function, we have cloned and expressed the first fibrinolytic protease gene from Cordyceps militaris. Cordyceps militaris fibrinolytic enzyme (CmFE) has one open reading frame of 759 bp encoding “pre-pro-protein” of 252 amino acids. Recombinant CmFE was expressed as 28 kDa extracellular enzyme in Pichia pastoris which was capable of degrading fibrin clot. A structure homology model of CmFE was developed using urokinase-type plasminogen activator. The active site contains catalytic triad His41, Asp83, Ser177 and consensus sequence of GDSGG. The substrate binding residues are Asp (171), Gly (194) and Ser (192). Its trypsin-like specificity is determined by the critical Asp171 in S1 subsite. The “oxyanion hole” is formed by backbone amide hydrogen atoms of Gly-175 and Ser-177. CmFE contains six conserved cysteines forming three disulfide linkages. This is the first study describing cloning, expression and prediction of structure-function relationship of a mushroom fibrinolytic protease. Hence it has great relevance in application of fibrinolytic enzymes as thrombolytic agents.

Usage of vermiculite as promising carrier for solid-state fermentation for fibrinolytic enzymes production by Aspergillus species

Usage of vermiculite as promising carrier for solid-state fermentation for fibrinolytic enzymes production by Aspergillus species

Production, characterization and optimization of fibrinolytic protease from Bacillus pseudomycoides strain MA02 isolated from poultry slaughter house soils

Abstract Bacterial fibrinolytic enzyme possessed significant application in treatment and prevention of cardiovascular diseases, thus isolation of potential strains producing more fibrinolytic enzyme is of prime importance. In this present study, microorganisms were isolated from soil samples and sediments around poultry slaughter houses and were screened for protease activity. Positive protease producers were further screened for fibrinolytic activity and its efficacy were assessed by fibrinolytic enzyme assay. One strain showed maximum activity of 79.83 FU/mL and the strain was identified as Bacillus pseudomycoides strain MA02. The molecular weight of the protease was estimated to be 35 kDa. Response Surface Methodology (RSM) and Central Composite Rotary Design (CCRD) were used to optimize skimmed milk agar to study the effects of the components in the production of fibrinolytic enzyme by B. pseudomycoides. The highest enzyme activity of 284 FU/mL was obtained at 7.5 g/L of peptone, 45 g/L of sodium chloride and 5 g/L of skimmed milk, which was 3 fold higher than the un-optimized medium.

THE IDENTIFICATION, PRODUCTION AND CHARACTERIZATION OF NATTOKINASE, BACTERIOCIN FROM BACTERIAL SPECIES

The study was aimed at identification, production and characterization of nattokinase, bacteriocin from bacterial species. Nattokinase and bacteriocins finds a wide range of applications in Pharmaceutical industry, health care and medicine. Nattokinase is a highly active fibrinolytic enzyme secreted by Bacillus subtilis and bacteriocins are proteinaceous toxins produced by Lactobacillus to inhibit the growth of closely related bacterial strains. Bacillus subtilis and Lactobacillus isolates shown positive results to microscopic, biochemical analysis. The nattokinase and bacteriocins were produced by optimizing the media. The enzymes were purified by ammonium sulfate precipitation and HPLC. The enzyme activity for nattokinase was found at 7 mg/ml, pH 8.0 and temperature 48 ºC and the enzyme activity for bacteriocin was found at 3.9 mg/ml, pH 6.5 and temperature 30 °C. Bacteriocins from Lactobacillus showed good antagonistic activity against pathogenic bacteria. Nattokinase from Bacillus subtilis played a significant role in thrombolytic and anti-coagulation at in vitro. The results indicated that the pure enzyme has a potential in dissolving blood clot.

Selection of a Single Chain Variable Fragment Antibody (scFv) against Subtilisin BRC and its Interaction with Subtilisin BRC

Background: The focus of this study was the selection of a single chain variable fragment antibody (scFv) against subtilisin BRC, a fibrinolytic enzyme using phage display, and to characterize the interaction between the antibody and subtilisin BRC. Methods: The subtilisin BRC-specific phage clones were selected using Griffin.1 scFv phage library and sequenced. The gene of subtilisin BRC-specific scFv (scFv-BRC) from selected phage clone was expressed in E. coli and scFv-BRC was characterized. Molecular modeling of the three-dimensional (3D) structures of scFv-BRC was performed using MODELLER 9.19 modeling software and assessed by PROCHEK. Molecular docking of subtilisin BRC with scFv-BRC was carried out using PATCHDOCK. Results: The size of scFv-BRC gene is 635bp and it consists of 54bp of heavy chain region (VH), 336bp of light chain region (VL), 45bp of a linker. scFv-BRC was actively expressed by E. coli expression vector pET28a-scFv in E. coli BL21 (DE3), and the amount of expressed scFv-BRC was about 50 mg/L. Its molecular weight is ~26kDa. The CDR domain of scFv-BRC consists of 6 amino acids in CDR L1, 3 amino acids in CDR L2 and 9 amino acids in CDR L3. Docking results of subtilisin BRC and scFv-BRC showed global energy of - 56.29 kJ/mol. Furthermore, the results showed that amino acid residues in subtilisin BRC for binding with scFv-BRC are Tyr6, Ser182, Ser204, and Gln206. Conclusion: scFv against subtilisin BRC selected using phage display showed relatively strong binding energy with subtilisin BRC. The amino acid residues in subtilisin BRC for binding with scFv-BRC are not relevant to that in subtilisin BRC for binding with its substrates. These results suggested that scFv-BRC can be used as a ligand for detection and affinity purification of subtilisin BRC.

Fibrinolysin production by Alcaligenes faecalis strain 26 isolated from environment

Fibrinolytic drugs are commonly used for fibrin clot lysis but due to their inappropriate side effects, as well as their high costs, using fibrinolytic enzymes has been paid attention. Bacterial sources of this enzyme are a good alternative for this purpose. The aim was fibrinolysin production through screening of fibrinolysin producing bacteria from environmental samples. Bacterial isolation was performed from different environmental samples and was screened based on sheep blood clot digestion and culture on plasma plate. The most potent isolate was optimized for different growth parameters including temperature, pH and fibrinolysin production at optimum growth conditions. The stability of produced enzyme at various temperatures and pH and treatment with MgSO4, NiSO4, SDS and EDTA was then investigated. Finally this isolate was identified based on the 16S rRNA sequencing. As a result, from 79 different isolates, the most potent fibrinolysin producer was identified as Alcaligenes faecalis strain 26. This isolate produced 12 mm halo zone on plasma plate. Its optimum growth temperature and pH was 43°C and 7, respectively. The produced enzyme had the best stability at pH 7 and was also active up to 60°C. The fibrinolytic activity of this isolate was reduced following treatment with MgSO4, NiSO4 and also with protease inhibitors, such as SDS and EDTA. Based on the obtained results it can be suggested that Alcaligenes faecalis strain 26 has appropriate efficiency for fibrinolysin production that can be used in food industry and medicine.

PAI-1 Level Differences in Malignant Plural Effusion, Parapneumonic Pleuritis, and Cardiac Hydrothorax.

Background and Objectives: Plasminogen activator inhibitor-1 (PAI-1) is a fibrinolytic system enzyme whose role in various fibrinolytic processes is currently unknown. In clinical manifestations of pleural liquids of diverse etiology, various levels of fibrinolytic activity can be observed—parapneumonic processes tend to loculate in fibrin septa, while malignant pleural effusion (MPE) does not. The purpose of this study was to determine possible differences in PAI-1 levels in pleural effusions of varied etiology. Material and Methods: PAI-1 level in pleural effusion and serum was determined in 144 patients with pleural effusions of various etiology (cardiac hydrothorax—42 patients (29.2%), MPE—67 patients (46.5%), parapneumonic pleuritis—27 (18.8%), tuberculous pleuritis—6 patients (4.1%), pancreatogenic pleuritis—1 patient (0.7%) and pulmonary artery thromboembolism with pleuritis—1 patient (0.7%)). Results: The median PAI-1 level (ng/mL) was the highest in the parapneumonic pleuritis group both in the effusion and the serum, with values of 291 (213–499) ng/mL and 204 (151–412) ng/mL, respectively, resulting in a statistically significant difference (p < 0.001) from the cardiac hydrothorax and MPE groups. However, there was no statistically significant difference between PAI-1 levels in the pleural effusion and serum in the cardiac hydrothorax and MPE groups. Conclusion: The PAI-1 level in MPE and cardiac hydrothorax was statistically significantly lower than in parapneumonic pleuritis.

Computational modeling of culture media for enhanced production of fibrinolytic enzyme from marine bacterium Fictibacillus sp. strain SKA27 and in vitro evaluation of fibrinolytic activity.

The present study reports the optimized production and purification of an extremely active fibrinolytic enzyme from newly isolated marine bacterium Fictibacillus sp. strain SKA27, with a specific activity of 125,107.85U/mg and an apparent molecular weight of 28kDa on SDS-PAGE. Wheat bran extract used for submerged production proved to be highly beneficial and enhanced fibrinolytic enzyme production when combined with yeast extract and CaCl2. Optimization of culture media by response surface methodology (RSM) resulted in high root mean square error (RMSE), which led to the training of a back propagation multilayer artificial neural network (ANN) with 3-5-1 topology for better prediction quality. The prediction and optimization capabilities of regression and ANN were critically examined and ANN displayed higher proficiency with R 2 of 0.99 and RMSE of 2.0 compared to 0.98 R 2 and 48.9 RMSE of the regression model. An adept ANN linked genetic algorithm (GA) optimized the medium components to achieve 1.8-fold higher enzyme production (4175.41U/mL). Further, a new and improved in vitro qualitative analysis displayed high specificity of purified enzyme to fibrin.

Dichotomous Role of Plasmin in Regulation of Macrophage Function after Acetaminophen Overdose

Kupffer cells and monocyte-derived macrophages are critical for liver repair after acetaminophen (APAP) overdose. These cells produce promitogenic cytokines and growth factors, and they phagocytose dead cell debris, a process that is critical for resolution of inflammation. The factors that regulate these dynamic functions of macrophages after APAP overdose, however, are not fully understood. We tested the hypothesis that the fibrinolytic enzyme, plasmin, is a key regulator of macrophage function after APAP-induced liver injury. In these studies, inhibition of plasmin in mice with tranexamic acid delayed up-regulation of proinflammatory cytokines after APAP overdose. In culture, plasmin directly, and in synergy with high-mobility group B1, stimulated Kupffer cells and bone marrow-derived macrophages to produce cytokines by a mechanism that required NF-κB. Inhibition of plasmin invivo also prevented trafficking of monocyte-derived macrophages into necrotic lesions after APAP overdose. This prevented phagocytic removal of dead cells, prevented maturation of monocyte-derived macrophages into F4/80-expressing macrophages, and prevented termination of proinflammatory cytokine production. Our studies reveal further that phagocytosis is an important stimulus for cessation of proinflammatory cytokine production as treatment of proinflammatory, monocyte-derived macrophages, isolated from APAP-treated mice, with necrotic hepatocytes decreased expression of proinflammatory cytokines. Collectively, these studies demonstrate that plasmin is an important regulator of macrophage function after APAP overdose.

Effect of Fibrinolytic Microbes and Enzymes on Biochemical Blood Parameters in Crossbred Calves

Twenty crossbred (HF x Kankrej) calves were allotted randomly to five groups (each had one male and three female calves) almost with similar body weight (85.70±6.37 kg) and age (167.55±21.70 days). Experimental calves of the control group were offered hybrid napier untreated, and the other four groups were offered hybrid napier treated with (i) fungus- Aspergillus spp. (1×107 per g feed), (ii) fibrolytic bacteria- Escherichia spp. (106 CFU per g feed), (iii) xylanase (50 mL/kg having xylanase 1.2 IU/mL), and (iv) consortium of 1/3rd dose of fungus + bacteria + enzyme as treatments for 140 days. The blood samples were collected from each calf at a biweekly intervals in the EDTA vacuttee. Nutrients offered to crossbred calves were sufficient to satisfy the nutrients requirements. Bacteria fed calves had significantly higher (plessthan0.05) mean hemoglobin levels than control. Blood plasma glucose of calves in fibrolytic microbes and enzyme groups was statistically similar to control. Feeding of fungus, bacteria, enzyme, and consortium was without a significant effect on plasma total proteins.In comparison to control (7.53 mg/dL), the plasma phosphorus was significantly (plessthan0 0.05) higher in calves fed enzyme (7.87 ± 0.20 mg/dl) and lower in group fed fungus (7.13 mg/dL). In contrast, in bacteria (7.57 mg/dL) and consortium group (7.60 mg/dL) it was statistically similar to control. Plasma glutamic pyruvic transaminase concentration of crossbred calves was lower ( pless than 0.05) in enzyme and higher (p lessthan 0.05) in fungus, bacteria, and consortium groups than in control, while plasma glutamic oxaloacetate transaminase concentration in all groups was statistically similar. In conclusion, all blood parameters of different groups were within normal range and without adverse effect on the overall performance of calves.

Purification and characterization of a novel, highly potent fibrinolytic enzyme from Bacillus subtilis DC27 screened from Douchi, a traditional Chinese fermented soybean food

The highly fibrinolytic enzyme-producing bacterium was identified as Bacillus subtilis DC27 and isolated from Douchi, a traditional fermented soybean food. The DFE27 enzyme was purified from the fermentation broth of B. subtilis DC27 by using UNOsphere Q column chromatography, Sephadex G-75 gel filtration, and high-performance liquid chromatography. It was 29 kDa in molecular mass and showed the optimal reaction temperature and pH value of 45 °C and 7.0, respectively, with a stable fibrinolytic activity below 50 °C and within the pH range of 6.0 to 10.0. DFE27 was identified as a serine protease due to its complete inhibition by phenylmethysulfony fluoride. The first 24 amino acid residues of the N-terminal sequence of the enzyme were AQSVPYGVSQIKAPALHSQGFTGS. The enzyme displayed the highest specificity toward the substrate D-Val-Leu-Lys-pNA for plasmin and it could not only directly degrade but also hydrolyze fibrin by activating plasminogen into plasmin. Overall, the DFE27 enzyme was obviously different from other known fibrinolytic enzymes in the optimum substrate specificity or fibrinolytic action mode, suggesting that it is a novel fibrinolytic enzyme and may have potential applications in the treatment and prevention of thrombosis.

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.

Production of fibrinogenolytic and fibrinolytic enzymes by a strain of &lt;i&gt;Penicillium&lt;/i&gt; sp. isolated from contaminated soil with industrial effluent

Cardiovascular diseases associated with thrombosis are one of the main causes of death all around the world. Urokinase, streptokinase, and tissue plasminogen activator are the major thrombolytic agents used to treat thrombosis. However, the fact that these agents have several side effects and high prices has driven the search for safer and more economically viable compounds for the treatment of cardiovascular diseases. Thus, the aim of this study was to evaluate the potential of fungi isolated from industrial effluents to produce fibrino(geno)lytic enzymes. The selection of the protease-producing strains showed that only the BF20 strain was able to produce proteolytic halos in milk agar. This strain identified as belonging to the genus Penicillium was cultivated in submerged fermentation. Different media composition were tested to evaluate proteolytic activity, and the results showed that the medium containing 0.1% yeast extract and 1% skim milk, pH 5.0, present higher azocaseinolytic activity (0.24 U mL-1 min.-1). This sample also showed the ability to degrade fibrinogen and fibrin after 15 and 120 min. of incubation, respectively. These results indicate that the BF20 strain has a thrombolytic potential, effectively degrading fibrinogen and fibrin, having great application in the health area.

Genetic Clearness Novel Strategy of Group I Bacillus Species Isolated from Fermented Food and Beverages by Using Fibrinolytic Enzyme Gene Encoding a Serine-Like Enzyme.

Fibrinolytic enzyme gene (fibE) is widely conserved among Bacillus spp. belonging to group I species. This is encoding a serine-like enzyme (FibE) secreted in extracellular medium. This present work aims to assess the molecular usefulness of this novel conserved housekeeping gene among group I Bacillus spp. to identify and discriminate some related strains in traditional fermented food and beverages in Republic of Congo. First of all 155 isolates have been screened for enzymatic activities using caseinolytic assays. PCR techniques and nested PCR method using specific primers and correlated with 16S RNA sequencing were used. Blotting techniques have been performed for deep comparison with molecular methods. As a result B. amyloliquefaciens (1), B. licheniformis (1), B. subtilis (1), B. pumilus (3), B. altitudinis (2), B. atrophaeus (1), and B. safensis (3) have been specifically identified among 155 isolates found in fermented food and beverages. Genetic analysis and overexpression of glutathione S-transferases (GSTs) fused to mature protein of FibE in Escherichia coli BL21 and TOP10 showed 2-fold higher enzymatic activities by comparison with FibE wild type one. Immunodetection should be associated but this does not clearly discriminate Bacillus belonging to group I.

Non-sterile Submerged Fermentation of Fibrinolytic Enzyme by Marine Bacillus subtilis Harboring Antibacterial Activity With Starvation Strategy.

Microbial fibrinolytic enzyme is a promising candidate for thrombolytic therapy. Non-sterile production of fibrinolytic enzyme by marine Bacillus subtilis D21-8 under submerged fermentation was realized at a mild temperature of 34°C, using a unique combination of starvation strategy and self-production of antibacterial agents. A medium composed of 18.5 g/L glucose, 6.3 g/L yeast extract, 7.9 g/L tryptone, and 5 g/L NaCl was achieved by conventional and statistical methods. Results showed efficient synthesis of fibrinolytic enzyme and antibacterial compounds required the presence of both yeast extract and tryptone in the medium. At shake-flask level, the non-sterile optimized medium resulted in higher productivity of fibrinolytic enzyme than the sterile one, with an enhanced yield of 3,129 U/mL and a production cost reduced by 24%. This is the first report dealing with non-sterile submerged fermentation of fibrinolytic enzyme, which may facilitate the development of feasible techniques for non-sterile production of raw materials for the preparation of potential drugs with low operation cost.

Research progress in tranexamic acid for reducing perioperative hidden blood loss in intertrochanteric fracture of femur

Intertrochanteric fracture of femur (IFF) is a common fracture type in traumatic orthopedics, which seriously affects patients' quality of life and health. Studies have shown that the fibrinolytic system can be activated in the invasive operation of IFF, which leads to massive blood loss during and after operation, especially hidden blood loss. Tranexamic acid, as an anti-fibrinolytic drug, can effectively reduce fibrinolytic activity by inhibiting fibrinolysis induced by fibrinolytic enzymes in order to stop bleeding. In recent years, with the diversified development of surgical hemostasis methods and the continuous exploration of hemostatic drugs, the application of anti-fibrinolytic drugs represented by tranexamic acid in perioperative period of IFF has gradually increased. Based on literatures at home and abroad, this paper aims to summarize the research progress of the application of tranexamic acid to reduce the perioperative hidden blood loss in intertrochanteric fracture, providing reference for clinical treatment decision. Key words: Tranexamic acid; Femoral fractures; Hidden blood loss

Community Composition and Metabolic Potential of Endophytic Actinobacteria From Coastal Salt Marsh Plants in Jiangsu, China.

The diversity and functional roles of the plant associated endophytic actinobacteria in unique habitats remain poorly understood. In this paper, we examined the phylogenetic diversity and community composition of endophytic actinobacteria associated with native coastal salt marsh plants in Jiangsu, China using a combination of cultivation and 16S rRNA gene-based high-throughput sequencing (HTS) methods. Further, we evaluated the antifungal, fibrinolytic activities and the secondary metabolite biosynthesis potential of isolates via gene screening. A total of 278 actinobacterial isolates were isolated from 19 plant samples. 16S rRNA gene sequencing revealed that the isolates were highly diverse and belonged to 23 genera within the Actinomycetales order, with Streptomyces, Saccharopolyspora, and Pseudonocardia comprising the most abundant genera. In addition, more than 10 of the isolates were novel actinobacterial taxa distributed across eight genera. HTS analyses of seven representative plant root samples revealed that Actinobacteria phylum constituted 0.04–28.66% of root endophytic bacterial communities. A total of four actinobacterial classes, 14 orders, 35 families, and 63 known genera were detected via HTS, and these communities were found to be dominated by the members of the order Actinomycetales including the genera Streptomyces, Mycobacterium, Arthrobacter, Nocardioides, and Micromonospora. In addition, 30.4% of the representative isolates exhibited antifungal activities, 40.5% of them showed fibrinolytic activities, while 43.0% of the strains harbored secondary metabolite biosynthesis genes. These results demonstrated that coastal salt marsh plants in the Jiangsu Province represented an underexplored new reservoir of diverse and novel endophytic actinobacteria that may be of potential interest in the discovery of bioactive compounds with potential as biocontrol agents and for fibrinolytic enzyme production.

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters

BackgroundNattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800.ResultsSix recombinant strains harboring different plasmids with a single promoter (PP43, PHpaII, PBcaprE, PgsiB, PyxiE or PluxS) were constructed, and the analysis of the fibrinolytic activity showed that PP43 and PHpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by PP43 and PHpaII reached 140.5 ± 3.9 FU/ml and 110.8 ± 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive PP43 or PHpaII increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of PP43 or PHpaII could effectively enhance NK production. Eight triple-promoters with PP43 and PHpaII in different orders were constructed, and the highest yield of NK finally reached 264.2 ± 7.0 FU/ml, which was mediated by the promoter PHpaII-PHpaII-PP43. The scale-up production of NK that was promoted by PHpaII-PHpaII-PP43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL.ConclusionsOur studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by PHpaII-PHpaII-PP43, which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes.

Cost-effective fibrinolytic enzyme production by Bacillus subtilis WR350 using medium supplemented with corn steep powder and sucrose

The goal of this study was to develop a cheap and simple medium and to optimize fermentation parameters for fibrinolytic enzyme production by Bacillus subtilis WR350. A low-cost medium containing 35 g/L sucrose, 20 g/L corn steep powder and 2 g/L MgSO4·7H2O was developed via single-factor and orthogonal experiments. A cheap nitrogen source, corn steep powder, was used to replace the soy peptone present in the initial medium. The highest fibrinolytic activity of 5865 U/mL was achieved using the optimized medium in a 100-L fermenter with an aeration rate of 1.0 vvm and an agitation speed of 200 rpm. The resulting enzyme yield was among the highest described in the literature with respect to fibrinolytic activity, as determined by the fibrin plate method. Techno-economic evaluation indicated that the cost of the optimized medium was only 8.5% of the cost of the initial medium, and the total fermentation cost of fibrinolytic enzyme production using the optimized medium was 23.35% of the cost of using the initial medium.

Intrapleural Administration of Fibrinolytic Enzymes as an Adjunct Therapy of Parapneumonic Purulent Pleural Exudation: Does it Affect Outcome?

In this comparable study, sixty patients were studied between March 2016 to November 2018. All of them were diagnosed having parapneumonic purulent pleural exudation (defined by the presence of purulent pleural effusion or pleural fluid with a pH below 7.2 associated with signs of chest infection and/or proven bacterial invasion of the pleural space).