Production of fibrinolytic protease from a halobacterium Bacillus licheniformis VITLMS isolated from marine sponges of Rameshwaram coast, India

2020 ◽  
Vol 16 ◽  
Author(s):  
Lalith Kumar .CK ◽  
Merlyn Keziah.S ◽  
Hemalatha. M ◽  
Mohanapriya.A ◽  
Mohanasrinivasan.V ◽  
...  
Keyword(s):  
Ammonium Sulfate ◽  
Bacillus Licheniformis ◽  
Fibrinolytic Activity ◽  
Gel Filtration ◽  
Marine Sponges ◽  
Clot Lysis ◽  
Malt Extract ◽  
Ammonium Sulfate Precipitation ◽  
Fibrinolytic Enzymes ◽  
Lysis Activity

Background: Marine bacteria serve as excellent sources of therapeutic enzymes, metabolites and natural products, which possess novel therapeutic properties. Increasing death rates due to cardiovascular diseases urges for cost effective production of fibrinolytic enzyme. Methods: Marine sponge samples were screened for potent fibrinolytic producing bacteria. Primary screening was done for protease production, clot lysis activity. Secondary screening was done for casein plasminogen activity and fibrinolytic activity. The strain which had potent fibrinolytic activity among them was further subjected to morphological, biochemical and molecular characterization. Media optimization was carried to enhance enzyme production. The enzyme produced was subjected to purification using ammonium sulfate precipitation, gel filtration and characterized using HPLC and FTIR analysis Results: Sponge was identified to be Desmapsamma anchorata. Thirteen bacterial isolates were isolated from the sponge sample. . The 16S rRNA sequencing revealed that the potential strain had 99% similarity with Bacillus licheniformis. Amongst the isolates most were found to be morphologically identical to Bacillus genus. Gram’s staining and SEM analysis of the potent isolate was performed to identify the spore formation and rod shaped morphology of the bacteria. The optimal temperature and pH for production of the enzyme was 37 °C and 8 respectively. The carbon source maltose and nitrogen sources were malt extract and yeast extract were found to be optimal. The optimum incubation time was found to be both 4 to 5 days. The crude supernatant was purified with ammonium sulfate precipitation and gel filtration chromatography. The retention time of 11.3 min and presence of functional groups show the purity of the enzyme. The partially purified enzyme showed 96.4 % clot lysis in artificial clot lysis activity. Conclusion: Although the secretion of fibrinolytic enzymes from Bacillus species is not new, based on our investigation there are no reports regarding Bacillus licheniformis being isolated from marine sponges. However, there are reports of Bacillus licheniformis secreting fibrinolytic enzymes isolated from fermented food samples This study identifies marine environment as a potential source of new exploration for drug discovery.

scholarly journals PRODUCTION AND PARTIAL CHARACTERIZATION OF FIBRINOLYTIC ENZYME FROM A SOIL ISOLATE ASPERGILLUS CARBONARIUS S-CSR-0007

2016 ◽  
Vol 8 (12) ◽  
pp. 142 ◽  
Author(s):  
Afini A.v. M. ◽  
Sooraj S. Nath ◽  
Smitha K. V. ◽  
Kunhi A.a. M.
Keyword(s):  
Ammonium Sulfate ◽  
Substrate Concentration ◽  
Fibrinolytic Activity ◽  
Blood Clot ◽  
Maximum Activity ◽  
Fibrinolytic Enzyme ◽  
Clot Lysis ◽  
Aspergillus Carbonarius ◽  
Lysis Activity ◽  
Wide Range

<p><strong>Objective: </strong>This work was undertaken with the aim of isolating and screening fungal soil isolates with fibrinolytic activity.</p><p><strong>Methods: </strong>Soil sample near slaughter house was collected and screened for fibrinolytic activity by using fibrin-agar. Enzyme production was optimized under various parameters like pH, temperature, substrate concentration and purified partially by ammonium sulphate precipitation. The stability of the partially purified enzyme was analyzed under the influence of a wide range of pH, temperature, and substrate concentrations.<strong></strong></p><p><strong>Results: </strong>Among the seven isolates screened, <em>Aspergillus carbonarius</em> S-CSR-0007 exhibited largest clear zone and was selected for further studies. Among the various substrates tested casein was found to support the highest caseinolytic activity of 816 U/ml and fibrinolytic activity of 510 U/ml. The culture supernatant of <em>A. carbonarius</em> S-CSR-0007 was fractionated by ammonium sulfate precipitation followed by dialysis, and maximum activity was obtained in the fraction with 80% ammonium sulfate, with an enzyme activity of 1200 U/ml using tyrosine as standard. The partially purified fibrinolytic enzyme showed optimal activity at 45 °C and pH 7.0. The enzyme was stable up to a temperature of 50 °C and pH 8.0, and the optimum substrate concentration was 4%.</p><p><strong>Conclusion: </strong>The crude enzyme showed high blood clot lysis activity, which may be a good candidate in the pharmaceutical industry. However, more studies need to be carried out to establish its clinical use.</p>

scholarly journals Novel Fibrinolytic Protease Producing Streptomyces radiopugnans VITSD8 from Marine Sponges

Marine Drugs ◽  
2019 ◽  
Vol 17 (3) ◽  
pp. 164 ◽  
Author(s):  
Dhamodharan D ◽  
Jemimah S ◽  
Merlyn S ◽  
Subathra C
Keyword(s):  
Tamil Nadu ◽  
Specific Activity ◽  
Blood Clot ◽  
Exchange Chromatography ◽  
Marine Sponges ◽  
Protease Production ◽  
Clot Lysis ◽  
Carbon And Nitrogen ◽  
Lysis Activity

Fibrinolytic enzymes have received more attention due to their medicinal potential for thrombolytic diseases. The aim of this study is to characterize the in vitro fibrinolytic nature of purified protease producing Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifera. Three varieties of sponge were collected from the Rameshwaram Sea coast, Tamil Nadu, India. The fibrinolytic activity of Streptomyces sp. was screened and determined by casein plasminogen plate and fibrin plate methods respectively. The crude caseinolytic protease was purified using ammonium sulfate fractionation, affinity and ion-exchange chromatography. Based on the morphological, biochemical, and molecular characterization, the isolate VITSD8 was confirmed as Streptomyces radiopugnans. Maltose and peptone were found to be the best carbon and nitrogen sources for the production of fibrinolytic protease. The carbon and nitrogen source peptone showed (781 U/mL) enzyme activity. The optimum pH and temperature for fibrinolytic protease production was found to be 7.0 and 33 °C respectively. The purified enzyme showed a maximum specific activity of 3891 U. The blood clot lysis activity was compared with the standard, and it was concluded that a minimum of 0.18 U (10 µL) of purified protease was required to dissolve the blood clot. This is the first report which exploits the fibrinolytic protease activity of Streptomyces radiopugnans VITSD8 extracted from a marine sponge. Hence the investigation suggests a potential benefit of purified fibrinolytic protease which will serve as an excellent clot buster alternative.

Adenosine Aminohydrolase from Halobacterium cutirubrum

1973 ◽  
Vol 51 (5) ◽  
pp. 621-626 ◽  
Author(s):  
Randy J. Bauer ◽  
David M. Carlberg
Keyword(s):  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Halophilic Bacterium ◽  
Ammonium Sulfate Precipitation ◽  
Two Phase ◽  
Liquid Polymer ◽  
Optimum Ph ◽  
Pyrimidine Bases

The extreme halophilic bacterium Halobacterium cutirubrum was examined for base, nucleoside, and nucleotide aminohydrolase activity on pyrimidine bases and their nucleosides and nucleotides. Only adenosine aminohydrolase activity was demonstrated. The enzyme was extracted from the cells and purified about 70-fold by liquid polymer two-phase fractionation, ammonium sulfate precipitation, and gel filtration. The enzyme has an apparent Km for adenosine of 4 × 10−5 M. The optimum pH is 7.5. The enzyme requires 4.0 M NaCl for maximal activity, but is irreversibly inactivated regardless of the concentration of NaCl when stored for extended periods of time.

Two types of xylanases of alkalophilic Bacillus sp. No. C-125

1985 ◽  
Vol 31 (6) ◽  
pp. 538-542 ◽  
Author(s):  
H. Honda ◽  
T. Kudo ◽  
Y. Ikura ◽  
K. Horikoshi
Keyword(s):  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bacillus Sp ◽  
Ammonium Sulfate Precipitation ◽  
Molecular Weights ◽  
Protein Molecules ◽  
Activity Curve ◽  
Alkalophilic Bacillus ◽  
Hydrolysis Of

One alkalophilic Bacillus sp. strain C-125 (FERM No. 7344) was isolated from soil. From this organism, two types of xylanases, designated xylanase A and xylanase N, were purified by an ammonium sulfate precipitation followed by Biogel P-30 gel filtration, DEAE-cellulose chromatography, and Sephadex G-75 gel filtration. The molecular weights of xylanase A and N were estimated as 43 000 and 16 000, respectively. Immunological experiments indicated that xylanase A and xylanase N were entirely different protein molecules. Xylanase N was most active at pH 6.0–7.0, but xylanase A had a very broad pH activity curve (pH 6–10) and was still active even at pH 12.0. The maximum hydrolysis of xylan by the enzymes was about 25%. Both enzymes split xylan and yielded xylobiose and higher oligosaccharides but could hydrolyze neither xylobiose nor xylotriose. Trans xylosidation activities were detected in both enzymes.

A Bacteriocin Produced by Lactobacillus brevis KLDS1.0373 Isolated from “Jiaoke”, a Traditional Fermented Cream from China

2011 ◽  
Vol 183-185 ◽  
pp. 1132-1136
Author(s):  
Hui Wang ◽  
Han Sheng Gong ◽  
Xiang Chen Meng ◽  
Li Li Man
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulfate ◽  
Inner Mongolia ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Lactobacillus Brevis ◽  
Ammonium Sulfate Precipitation ◽  
Gel Filtration Chromatography

A bacteriocin produced by Lactobacillus brevis KLDS1.0373 which was isolated from “Jiaoke”, a traditional, naturally fermented cream from Inner Mongolia in China was reported in this article. The bacteriocin was partially purified by ammonium sulfate precipitation followed by sequential gel filtration chromatography, and the apparent molecular weight of the partially purified bacteriocin was estimated at approximately 3.8 kDa.

Fibrinolytic Properties Of Microbial Proteases

1981 ◽  
Author(s):  
N S Egorov ◽  
N S Landau ◽  
G V Andreenko ◽  
V G Kreyer ◽  
S S Pokrovskaya ◽  
...  
Keyword(s):  
Blood Plasma ◽  
Intravenous Injection ◽  
Fibrinolytic Activity ◽  
Clot Lysis ◽  
Albino Rats ◽  
Ecological Groups ◽  
Fibrinolytic Enzymes ◽  
Thrombolytic Effect

Our study of 820 microorganisms from various systematic and ecological groups revealed that 72% of the cultures contained enzymes caoable to dissolve human fibrin clots in vitro. Fibrinolytic enzymes are formed during growth of producers on strictly specific media. Changes in the composition of media and conditions of enzymatic synthesis were found to increase the specificity of microbial proteases to fibrin and to decrease their sensitivity to fibrinolytic enzymes inhibitors. A thrombolytic enzyme (TE) has been isolated and purified from the cultural fluid of actinomycetes. An intravenous injection of TE to albino rats results in an increase of fibrinolytic activity of the blood plasma euglobulin fraction. Time of euglobulin clot lysis is thereby decreased by 32%. The fibrinolytic activity measured from euglobulin precipitate on un-heated fibrin plates is increased 2.3-fold. However, the fibrinolytic activity of non-diluted blood plasma remains unchanged probably due to a sharp rise in antiplasmin content following TE injection. TE exerts marked thrombolytic effect in vivo. After intravenous injection of TE to animals with artificial clots in a v.jugularis segment a rapid clot lysis and reconstitution of blood flow are observed.

scholarly journals Extraction and Partial Purification of Mouse Uterine Alkaline Phosphatase

1979 ◽  
Vol 32 (2) ◽  
pp. 153 ◽  
Author(s):  
RN Murdoch ◽  
DJ Kay ◽  
WJ Capper
Keyword(s):  
Alkaline Phosphatase ◽  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation ◽  
Pregnant Mice ◽  
Triton X

Alkaline phosphatase in uterine homogenates from day 7 pregnant mice was solubilized using 0�2 % (v/v) Triton X-100 and extracted with 20% (v/v) n-butanol. The procedure, which resulted in 182- fold purification, included ammonium sulfate precipitation, DEAE-cellulose anion exchange chromatography and Sephadex 0200 gel filtration.

Influence of Moderate and Strenuous Daily Physical Activity on Fibrinolytic Activity of Blood: Possibility of Plasminogen Activator Stores Depletion

1979 ◽  
Vol 41 (04) ◽  
pp. 745-755 ◽  
Author(s):  
Dušan Keber ◽  
Mojca Stegnar ◽  
Irena Keber ◽  
Bojan Accetto
Keyword(s):  
Physical Activity ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Regular Physical Activity ◽  
Clot Lysis ◽  
Moderate Activity ◽  
Lysis Time ◽  
Strenuous Activity ◽  
Activity Measurements ◽  
Clot Lysis Time

SummaryFibrinolysis was studied in 10 alpinists during regular physical activity of different intensity. Blood was sampled at rest and after exposure to submaximal workload on the treadmill on three occasions: before and after 6 months physical conditioning (moderate physical activity), and after 6 weeks of an alpinistic expedition (strenuous physical activity). Measurements included submaximal working capacity, fibrinogen, euglobulin clot lysis time (ELT), whole plasma clot lysis time, and estimations derived from ELT - percent increase in fibrinolytic activity after exercise (RFS), and absolute increase in fibrinolytic activity after exercise (PAR).Regular moderate activity increased the resting level of ELT, but strenuous activity decreased is. After each treadmill testing, a marked increase in fibrinolytic activity was observed. RFS was unaltered at all three testings. PAR increased after moderate activity, but decreased after strenuous activity.The results indicate that regular physical activity can lead from enhanced to decreased resting activity of plasminogen activator in blood. It is presumed that increased release of activator during prolonged stress causes partial depletion of endothelial stores with the consequence of decreased activator activity in the blood.

Effects of Ellagic Acid upon the Rabbit Fibrinolytic System

1972 ◽  
Vol 27 (01) ◽  
pp. 063-071
Author(s):  
S. G Iatridis ◽  
P. G Iatridis
Keyword(s):  
Continuous Infusion ◽  
Ellagic Acid ◽  
Animal Experimentation ◽  
Inhibitory Effect ◽  
In Vivo Studies ◽  
Clot Lysis ◽  
Hageman Factor ◽  
Lysis Activity ◽  
Site Of Action

SummaryThe present investigation deals with in vivo studies of possible relations of active Hageman factor (HFa) to the problems of thrombolysis. The study is based upon animal experimentation in which 40 normal, 5 dicumarolized and 5 heparinized rabbits each received ellagic acid (Elac 10-2 M) by intravenous continuous infusion at a rate of 1 ml/min for a period of 25 min. The data suggest that the Elac infusion induced in vivo activation of HF. Streptokinase (SK) injection 25 min from the start of Elac i. v. infusion failed to induce clot lysis in blood drawn one min after its injection. The phenomenon was more prominent with low (SK 250 U or 500 U) concentrations of SK. With higher concentrations, SK-induced clot lysis activity was not affected by Elac infusion.In dicumarolized and heparinized rabbits Elac infusion still counteracted the fibrinolysis activating effect of low concentration of SK. The possibility that the above described phenomenon was due to either hypercoagulability or to a non-specific inhibitory effect of Elac upon SK was explored and excluded.It is concluded that HFa and SK have the same site of action. Thus it seems that HFa may block the precursor upon which SK acts by forming a complex with it. It is stressed that activation of this precursor by HFa requires a suitable surface.

Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

2013 ◽  
Vol 10 (2) ◽  
pp. 29
Author(s):  
Normah Ismail ◽  
Nur' Ain Mohamad Kharoe
Keyword(s):  
Molecular Weight ◽  
Protein Content ◽  
Ammonium Sulfate ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Protease Activity ◽  
Protein Concentration ◽  
Temperature Stability ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage. 

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