Exploration of new effective fibrinolytic agent from Alcaligenes aquatilis PJS_1 from polluted soil

Aim: To isolate and identify Alcaligenes aquatilis PJS_1 from slaughter house soil samples for production of enzymatic fibrinolytic agent productionMethodology: Fibrinolytic enzyme producing bacterium was isolated from slaughter house soil samples and identified by biochemical tests and 16S rRNA sequencing. The fibrinolytic enzyme production media was optimized by various factors like energy sources, pH and temperature. Bioreactor used in the experiment was designed with suitable parameters for effective production and purification is by gel filtration chromatography. Blood clotting assay was performed to determine its anticoagulant property. Results: The isolated enzyme producing bacterium was identified as Alcaligenes aquatilis PJS_1. The medium with fructose and urea at pH 7.0 was found to have optimum production when incubated for 24 hr at 37ºC. The crude enzyme was purified by acetone precipitation followed by gel filtration chromatography. The enzyme showed a final specific activity of 629.32 Umg-1 with of 88.24% yield Interpretation: The present study provides information that the enzyme produced by Alcaligenes aquatilis PJS_1 acts as an effective fibrinolytic agent

Screening and identification of fibrinolytic enzyme-producing microbes in fermentation process of Sojae Semen Praeparatum / 中草药

Objective: To screen and identify the dominant strains which produce fibrinolytic enzyme during the processing of Sojae Semen Praeparatum (SSP, Dandouchi in Chinese). Methods: SSP was prepared according to the Chinese Pharmacopoeia (2020 edition), and samples were taken at different time points during the fermenting process of SSP.The casein plate method and fibrin plate method were used to screen the fibrinolytic enzyme-producing microorganisms in samples at different time points. The fibrinolytic enzyme-producing microorganisms were inoculated in the designated liquid medium to obtain single strain fermentation broth, and fibrin plate method was used to measure the fibrinolytic activity of the fermentation broth. The DNA sequences of fibrinolytic enzyme-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA universal primer by PCR respectively.The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was done by phylogenetic tree constructed by MEGA 4.1 software. Results: Three types of fibrinolytic enzyme-producing bacteria were screened out and identified in this study. They were Bacillus subtilis, Stenotrophomonas maltophilia and Micrococcus, respectively. The result of fibrin plate method showed that the fermentation broth of S. maltophilia had the highest fibrinolytic activity, reaching 527.49 IU/mL. Conclusion: There are fibrinolytic enzyme-producing dominant microorganisms existing in the fermenting process of SSP and the thrombolytic effect of SSP is worthy of further study. This study lays the foundation for revealing the formation mechanism of fibrinolytic enzyme in the fermentation process of SSP.

Improvement Effects of Fibrinolytic Enzyme from Sipunculus nudus on Hemorheology Disorder and Vascular Endothelium Injury of Naked Acute Blood Stasis Model Rats / 中国药房

OBJECTIVE： To study the improvement effects of fibrinolytic enzyme from Sipunculus nudus （SNFE） on hemorheology disorder and vascular endothelium injury in naked acute blood stasis model rats. METHODS： SD rats were randomly divided into control group， model group， aspirin group （100 mg/kg） and SNFE high-dose and low-dose groups （2 500， 5 000     U/kg）， with 10 rats in each group. They were given relevant medicine intragastrically once a day， for consecutive 7 d. One hour after the 6th day of administration， except for control group， other groups were given adrenaline hydrochloride 0.8 mg/kg subcutaneously， and then the acute blood stasis model was induced by ice-water bath. Blood was collected from abdominal aorta 2 h after the next day. Blood rheological parameters such as whole blood viscosity （high， medium and low shear rate）， plasma viscosity， hematocrit， erythrocyte aggregation index and erythrocyte deformability index were measured by automatic rheometer. The contents of NO and ET-1 in plasma and their ratio were determined by ELISA， and the damaged degree of vascular endothelium were observed by HE staining. RESULTS： Compared with control group， whole blood viscosity of high， medium and low-shear rate， plasma viscosity， erythrocyte aggregation index and ET-1 content were increased significantly in model group， while erythrocyte deformability index， NO content and NO/ET-1 ratio were decreased significantly， with statistical significance （P＜0.05 or P＜0.01）. Compared with model group， whole blood viscosity of high， medium and low-shear rate， plasma viscosity， hematocrit， erythrocyte aggregation index and ET-1 content were decreased significantly in SNFE high-dose groups. Erythrocyte deformability index， NO content and NO/ET-1 ratio were increased significantly， with statistical significance （P＜0.05 or P＜0.01）. In SNFE low-dose group， erythrocyte deformability index and NO/ET-1 ratio were increased significantly， while ET-1 content was decreased significantly， with statistical significance （P＜0.05 or P＜0.01）. Vascular endothelial staining showed that compared with control group， the structure of aorta layers in model group was loose and disordered， the endothelial defect was incomplete， the vacuoles increased， and the endothelial damage was obvious. The endothelium of rats in each administration group was damaged to varying degrees， but the degree of injury was lighter than in model group. CONCLUSIONS： SNFE can improve hemorheological abnormalities and vascular endothelial injury in rats with acute blood stasis.

Study on the Anti-coagulation Effect and Mechanism of Fibrinolytic Enzyme SNFE in Sipuculus Nudus / 中国药房

OBJECTIVE:To study the anti-coagulation effect and mechanism of fibrinolytic enzyme SNFE in sipuculus nudus, and provide reference for further development of SNFE. METHODS:40 mice were randomly divided into blank control group(nor-mal saline),Xueshuantong group(positive control,15 mg/kg)and SNFE low-dose,high-dose group(15,30 mg/kg),10 in each group. After intravenous injection in tail,tail bleeding time (BT) and clotting time (CT) were respectively determined to investi-gate the anti-coagulation effect of SNFE. After taking blood in abdominal aorta of rats,test was divided into blank control group, positive control group and SNFE low-mass concentration,medium-mass concentration,high-mass concentration groups (0.25, 0.50,1.00 mg/mL). Prothrombin time(PT),re-calcium time(PRT)(using orokinase as positive drug,100000 U/mL),and max-mum platelet aggregation rate (PAG) in 5 min under adenosine diphosphate (ADP) inducer (using asprin as positive drug,0.50 mg/mL) were respectively determined,and anti-coagulation effect mechanism of SNFE was analyzed. RESULTS:Compared with blank control group,BT,CT of mice in each group were prolonged,with statistical significance in Xueshuantong group and SNFE high-dose group (P<0.05 or P<0.01). Plasma PT of rats in positive control group,SNFE medium-dose,high-dose groups and PRT in each administration group were significantly prolonged(P<0.05 or P<0.01);and PAG in administration group was signifi-cantly reduced(P<0.01). CONCLUSIONS:The fibrinolytic enzyme SNFE in sipuculus nudus can play its anti-coagulant effect by inhibiting the activity of coagulation factors in internal and external sources and ADP-induced platelet aggregation.

Study on the Anti-coagulation Effect and Mechanism of Fibrinolytic Enzyme SNFE in Sipuculus Nudus / 中国药房

OBJECTIVE:To study the anti-coagulation effect and mechanism of fibrinolytic enzyme SNFE in sipuculus nudus, and provide reference for further development of SNFE. METHODS:40 mice were randomly divided into blank control group(nor-mal saline),Xueshuantong group(positive control,15 mg/kg)and SNFE low-dose,high-dose group(15,30 mg/kg),10 in each group. After intravenous injection in tail,tail bleeding time (BT) and clotting time (CT) were respectively determined to investi-gate the anti-coagulation effect of SNFE. After taking blood in abdominal aorta of rats,test was divided into blank control group, positive control group and SNFE low-mass concentration,medium-mass concentration,high-mass concentration groups (0.25, 0.50,1.00 mg/mL). Prothrombin time(PT),re-calcium time(PRT)(using orokinase as positive drug,100000 U/mL),and max-mum platelet aggregation rate (PAG) in 5 min under adenosine diphosphate (ADP) inducer (using asprin as positive drug,0.50 mg/mL) were respectively determined,and anti-coagulation effect mechanism of SNFE was analyzed. RESULTS:Compared with blank control group,BT,CT of mice in each group were prolonged,with statistical significance in Xueshuantong group and SNFE high-dose group (P<0.05 or P<0.01). Plasma PT of rats in positive control group,SNFE medium-dose,high-dose groups and PRT in each administration group were significantly prolonged(P<0.05 or P<0.01);and PAG in administration group was signifi-cantly reduced(P<0.01). CONCLUSIONS:The fibrinolytic enzyme SNFE in sipuculus nudus can play its anti-coagulant effect by inhibiting the activity of coagulation factors in internal and external sources and ADP-induced platelet aggregation.

Screening and Identification of Fibrinolytic Bacteria from Malaysian Fermented Seafood Products.

Natural fibrinolytic enzymes have been focused recently when compared to chemically synthesize thrombolytic drugs as they are more economical and believe to have less adverse reaction. Belacan (shrimp paste), Budu (fish sauce) and Cencaluk (shrimp sauce) were traditional fermented Malay seafood products used as sources for this study. The isolation and screening resulted in the discovery of 43 potential isolates with the ability as an extracellular fibrin degrader. Each isolate were unique due to differences in morphology characteristics and the amounts of fibrinolytic enzyme secretes under standard growth condition. Furthermore, it has been demonstrated that some strains posses the ability to act as tissue plasminogen activator (t-PA) which in deed has beneficial to many medicinal and therapeutic purposes. Of these 43 strains investigated for the fibrinolytic enzyme production, three species, mainly the best producer from each food sample were chosen for further molecular identification. This lead to the discovery of B. cereus 13BN, B. subtilis 2CN and B. subtilis 9BD strains, which posses both high fibrinolytic and t-PA activities, making it extremely valuable and promising producer.

Response Surface Methodology Based Optimization of a New Isolate Bacillus pumilus ZR LS S2 with Fibrinolytic Activity.

Aims: The work aims to isolate potential fibrinolytic enzyme producing isolates from various samples with the aim of developing a suitable optimization strategy using response surface methodology. Study Design: Plackett- Burman and Face centered central composite design was used. Place and Duration of Study: Department of Life Sciences, University of Calicut. Methodology: Various samples were screened using fibrin agar plates for the isolation of fibrinolytic enzyme producing bacteria and was identified by 16 s rRNA sequencing. Further the physical parameters and media components were optimized using Plackett- Burman design and face centered central composite design. Results: A novel isolate with fibrinolytic potential was isolated and was identified as Bacillus pumilus ZR LS S2. A novel media was formulated using this data, for the production of fibrinolytic enzyme, which contains peptone, casein, MgSO4 and NaCl. The isolate produced 6738.384 U/ml of enzyme in optimized conditions. Conclusion: Initial studies were performed for the production of the fibrinolytic enzyme by the novel isolate, further studies are required to effectively validate the potential of the fibrinolytic enzyme and to develop effective application for the same.

Separation and screening of components with fibrinolytic enzyme activity from twelve Chinese herbal medicines / 国际中医中药杂志

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.

Evaluation of cytotoxic and anti-tumor activity of partially purified serine protease isolate from the Indian earthworm Pheretima posthuma

Objective: To isolate, partially purify and evaluate the cytotoxic and antitumor activity of a serine protease from the chosen Indian earthworm Pheretima posthuma.Methods:Whole animal extract was prepared and purified its protein constituents by size and charge based chromatographic separation techniques using Sephadex G-50 and DEAE-Cellulose resin respectively. Average molecular weight of the protein isolate was determined and analyzed for its cytotoxic property against Vero cells in different dilutions (1: 20 and 1: 40) and anti-tumor activity by MTT assay (a colorimetric assay) using breast cancer cell line MCF-7, with tamoxifen as standard.Results:One of the protein constituents after purification was characterized as serine protease by Caseinolytic plate diffusion assay. Average molecular weight of this purified isolate was determined, by SDS-PAGE analysis with standard protein ladder, as of 15 kDa. The performed tests suggested that the 15kDa fraction has potent cytotoxic activity and satisfactory antitumor activity as well in vitro.Conclusions:Exact molecular mechanism of the cytotoxic and antitumor activities is yet to be explored and currently we are working on ultra-purification and biophysical characterization of this fraction. Further investigation into the mechanism(s) of cytotoxic and antitumor activities at molecular level would be useful in treatment of various classes of cancer and viral infections in future.

Gene Expression of the Micrococcus luteus Fibrinolytic Enzyme (MLFE) in Bacillus subtilis WB600 / 微生物学通报

MLFE (Micrococcus luteus fibrinolytic enzyme) is a fibrinolytic enzyme produced by Micrococcus luteus ML909 strain. The promoter and signal peptide-coding sequence of ?-amylase gene from Bacillus amyloliquefaciens DC-4 was cloned and fused to the sequence coding for mature peptide of MLFE (Gen-Bank: EU232121), forming the fusion gene called amymlfe. This hybrid gene was inserted into the Escherichia coli-Bacillus subtilis shuttle plasmid vector pSUGV4 and expression plasmid pSU-AmyMLFE was constructed. After transformation with B. subtilis WB600, transformant WB600/pSU-AmyMLFE was obtained and produced clear hydrolyzed zones on fibrin plates. The fibrinolytic activity in supernatants of WB600/pSU-AmyMLFE fermented for 24 hours was tested and found to be 238 UKU/mL. The results of SDS-PAGE analysis showed that there was indeed recombinant protein in supernatants. The Western blotting showed that the molecular weight of the expressed protein was the same as expected. These results indicate that the gene, amymlfe, is successfully expressed in B. subtilis WB600.

Identification of a Bacillus sp. Strain with Fibrinolytic Activity and Primary Study on Fermentation Process / 微生物学通报

An extracellar fibrinolytic strain was isolated from fermented shrimp paste. In addition to general physiological and biochemical properties, the strain was identified by 16S rDNA sequence and systematic analysis. The results showed that 16S rDNA sequence of the strain had high similarity with AY601723 and AB195282, suggesting that the strain is a subspecies of Bacillus sp. It was named as Bacillus sp. nov. SK006 by CCTCC. The medium composition and fermentation conditions for fibrinolytic enzyme production were also optimized in the research.

Optimization on Fermentation and Purification of the Fibrinolytic Enzyme from Stenotrophomonas maltophilia / 中国生物工程杂志

The optimization on liquid fermentation and purification of the fibrinolytic enzyme from Stenotrophomonas maltophilia(DR-929) was investigated.The results showed the best fermentation are amidulin 2.0%,soya flour 1.0%,yeast extract 0.5%,NaCl 1.0%,CaCl2 0.02%,MgSO4 0.05%,inoculum of 36 hours,fermental time 4d,initial pH 8.0 or 9.0,temperature 25℃,volume of media 30ml,volume of inoculum 5% or 6%.The purification process includes the following steps: removing cells by the centrifugation,25%~70% saturation ammonium sulfate precipitation,HIC with Phenyl FF(high sub),IEC with Q-Sepharose FF,gel filtration chromatography with Superdex 75.SDS-PAGE electrophoresis was used to examine the purification effect,and the results indicated that homogeneous strap in SDS-PAGE and has a molecular weight around 28.3kDa.The purification factor and activity recovery of the fibrinolytic enzyme are 271.5 and 24.5%,respectively.

Reviews on Foodborne Fibrinolytic Enzyme / 中国生物工程杂志

Thrombolytic therapy is a safe and effective way to cure thrombosis. Developing the safe,effective and cheap therapeutic thromolytic agents are important to prevent and cure thrombosis. In recent years,several fibrinolytic enzymes have been found in the resources of Asian traditional fermented foods,such as Japanese natto,Korea Chungkook-Jang,Chinese Douchi and fermented shrimp paste. These fibrinolytic enzymes secreted by food-grade microorganisms are safe and effective,and are potent thrombolytic agents. The physical and biochemical properties,thrombolytic characteristics,and the potential applications of these fibrinolytic enzymes from Asian fermented food,are reviewed.

Thrombolytic effect of non-hemorrhagic fibrinolytic enzyme purified from Agkistrodon acutus on animal thrombosis / 中国药理学通报

AIM To study the thrombolytic effect of non hemorrhagic fibrinolytic enzyme from Agkistrodon acutus venom on animal thrombosis. METHODS By means of pulmonary embolism,arterial thrombosis model in rabbits and venous thrombosis model in rats. The non hemorrhagic fibrinolytic enzyme from Agkistrodon acutus was given by intravenous injection at 0 14, 0 7, 1.4 mg?kg -1 three different doses respectively. Normal saline was used as negative control and thrombolytic effects was observed. RESULTS It was showed that moist weights of arterial thrombosis, venous thrombosis and pulmonary embolism were markedly lightened and has statistical significance compared with normal saline ( P

Study on the antithrombotic effect of fibrinolytic proteins from Scolopendra subspinipes mutilans / 中国药理学通报

Aim The aim is to purify fibrinolytic enzyme from Scolopendra subspinipes mutilans L.Koch and to study the thrombolytic and anticoagulant effect.Methods Scolopendra subspinipes mutilans L.Koch fibrinolytic enzyme(SSFE) was purified by ammonium sulphate precipitation,DEAE-cellulose and SephadexG-75 column chromatography from Scolopendra subspinipes mutilans L.Kochby.And fibrinolytic activity was determined by fibrin plate.The anticoagulant effect was measured on mice with haemolytic test and hemorrhagic test.The thrombolytic effect was measured with rats In vitro and in vivo,and the activated partial thromboplastin time(APTT),plasma prothrombin time(PT),thrombin time(TT) were measured.Results SSFE was single component with fibrinolytic activity and without any hemolyzation and hemorrhagic activity.All doses of SSFE(2,5,10 mg?kg-1) could obviously prolong activated partial thromboplastin time(APTT) and thrombin time(TT) ;Middle dose of SSFE(5 mg?kg-1) could prolong plasma prothrombin time(PT) while high dose of SSFE(10 mg?kg-1) didn't prolong obviously.Conclusion SSFE has obvious thrombolytic effect and anticoagulant effect.

Separation, Purification and Analysis of the Components of Earthworm Fibrinoly tic Enzymes / 生物化学与生物物理进展

The earthworm fibrinolytic enzymes (EFE) were separated by affinity ch romatography using soybean trypsin inhibitor as a matrix. The enzymes were furth er separated and purified into 12 components after DEAE-32 chromatography and p reparative electrophoresis. The pI of these components gradually decreased f rom pH 4.0 according to electrophoresis mobility from higher to lower on PAGE. The molecular weights were in the range of 22～34 ku. 6.5 and 7 w ere glycoproteins proved by staining with the shiff reagent and thymol/sulfuric acid. The fibrinolytic activity of 7 was highest as determined usi ng chromzym UK and chromzym PL as specific substrates.

Anti-metastasis Activity of Earthworm Fibrinolytic Enzyme on Hepatoma Cell in Vivo / 中药新药与临床药理

Objective To investigate the inhibitory effect of earthworm fibrinolytic enzyme (EFE) on the metastasis of human hepatoma cells.Methods A metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation was established.After the modeling for 7 days,the mice were divided randomly into the control group,low-dose EFE group (800 uku/kg?d-1),and high-dose EFE group (1600 uku/kg?d-1).After administration for 30 days,the implanted tumors were weighted,and the intrahepatic transmission rate and abdominal cavity seeding rate were calculated after examination by naked eyes.Then the pulmonary metastasis were examined under microscope,and the expression of focus adhesion kinase (FAK) and ?1-intigrin were detected through RT-PCR and western blotting method.Results Compared with the model control group,the mean weight of the orthotopic tumor was reduced,and the intrahepatic transmission rate and abdominal cavity seeding rate were decreased in FEF groups (P

Purification of fibrinolytic enzyme from Perinereis aibuhitensis / 中草药

Object To extract and purify a novel fibrinolytic enzyme from Perinereis aibuhitensis Grube Methods The enzyme was precipitated from the extract by ammonium sulfate, dialyzed, chromatographed on Sephadex G 100 column, and then purified by polyacrylamide gel electrophoresis Its fibrinolytic activity was assessed with fibrin plate method Results The purified enzyme showed an isoelectric point (PI) around 4 5 as tested by gel isoelectric focusing It consisted of two polypeptide chains with molecular weights around 33 000 u and 14 400 u, respectively Conclusion This was a novel fibrinolytic enzyme discovered from P aibuhitensis for the first time

Study on anticoagulant and thrombolytic effects of a novel marine fibrinolytic enzyme in vitro / 中国海洋药物

Objective To estimate the anticoagulant and thrombolytic effects of a novel fibrinolytic enzyme with a low molecular weight for further carrying on animal test and clinic research.Methods Using pH7.4,0.9 % NaCl as negative control and lumbrukinase as positive control,the anticoagulant and thrombolytic effects of the novel marine fibrinolytic enzyme were investigated in vitro with fresh blood from wistar rat and rabbit,respectively.Results The results showed that there were apparent anticoagulant effect as well as thrombolytic effects in the self-made marine fibrinolytic enzyme,and the status of the released blood cells from thrombus using this enzyme was also better than that of using lumbrukinase.Conclusion The novel self-made fibrinolytic enzyme is a potent thrombolytic agent.

Screening of a high yield protease producing strain and studies on its fermentation conditions / 中国海洋药物

A1 strain with fibrinolytic activity was isolated from sea water in Qingdao. After combination treatment with ultraviolet light and MNNG, a mutant C22 producing 2947.60 u?mg -1 protein of fibrinolytic enzyme in culture broth was obtained.Its enzyme activity was 4.23 fold of wild stain A1.The optimum medium for fermentation consisted of 2.5% soy bean cake meal, 0.1% yeast extract,0.2%NaCl,0.05%MgSO 4?7H 2O,0.001%FeSO 4?7H 2O.They were dissolved with 0.05mol?L -1 ,pH7.4 phosphoric buffer.The strain producing maximum fibrnolytic activity after growth at 25℃ for 40h on a rotary shaker.The initial optimum pH was 7.0~7.5.