scholarly journals 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

2021 ◽  
pp. 246-255
Author(s):  
K. Gowthami ◽  
R. Jaya Madhuri
Keyword(s):  
Wound Healing ◽  
Enzyme Activity ◽  
Incubation Time ◽  
Specific Activity ◽  
Blood Clot ◽  
Inoculum Size ◽  
Fibrinolytic Enzyme ◽  
Physical Parameters ◽  
Bacterial Strains ◽  
Fibrinolytic Enzymes

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.

Biosynthesis and characterization of a novel fibrinolytic alkaline serine protease from newly isolated Bacillus flexusBF12 for biomedical applications

2020 ◽  
Vol 21 ◽  
Author(s):  
T. Sterlin Raj ◽  
S. Athimoolam ◽  
P. Vijayaraghavan
Keyword(s):  
Enzyme Activity ◽  
Enzyme Production ◽  
Blood Clot ◽  
Fibrinolytic Enzyme ◽  
Physical Factors ◽  
Alkaline Serine Protease ◽  
Fibrinolytic Enzymes ◽  
Nutritional Factors ◽  
Thrombolytic Agents ◽  
Bacillus Flexus

Background: Cardiovascular diseases (CVDs) such as stroke, high blood pressure, peripheral vascular disease, ischemic heart disease and acute myocardial infarction are some of the leading causes of death. To treat CVDs, commercially available thrombolytic agents are widely used. However, these thrombolytic agents have various side effects. Alternatively, fibrinolytic enzymes from bacterial sources are highly safe and have direct blood clot lytic activity. Methods: A fibrinolytic enzyme producing bacterial strain, Bacillus flexus BF12, was isolated from a solar saltpan in Kanyakumari District, Tamilnadu, India. Enzyme production was improved by optimizing physical factors and nutritional factors. Results: A novel fibrinolytic enzyme was isolated from a strain of the studied B. flexus BF12. Enzyme production was enhanced significantly by optimizing process parameters. The critical physical factors (pH and salinity) and influencing nutritional factors (carbon, nitrogen and ions) were optimized by one variable at a time approach, followed by statistical method. The strain BF12 was highly active at alkaline pH (>7.0) and between 4 and 6% NaCl concentration. The nutrients such as fructose (carbon source), beef extract (nitrogen source) and CaCl2 significantly influenced enzyme production. Central composite design and response surface methodology improved 3.2-fold enzyme yield than unoptimized culture medium. Fibrinolytic protease was purified by ammonium sulphate precipitation, dialysis and gel filtration chromatography. Discussion: The molecular weight of an enzyme was found to be 23 kDa. It was active at a broad temperature (40-60 °C) and pH (7.0-9.0) ranges. Enzyme activity was enhanced by Ca2+ and Co2+ ions. The purified protease retained 100% enzyme activity in the presence of ethanol and acetone. Acetonitrile, butanol, DMSO, methanol and chloroform showed enzyme activity of 63%, 92.5%, 94.7%, 92.3% and 90.4%, respectively. The purified enzyme degraded 100% of human blood clot. Conclusion: The Bacillus flexus BF12 fibrinolytic enzyme shows promising potentials in nutraceutical and food fortification applications. The application of fibrinolytic enzymes could prevent CVDs.

Screening and characterization of thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 / Bacillus amyloliquefaciens EGE-B-2d.1’den termostabil fibrinolitik enzimin taranması ve karakterizasyonu

2016 ◽  
Vol 41 (3) ◽  
Author(s):  
Birkan Slem ◽  
Yüksel Gezgin ◽  
Rengin Eltem
Keyword(s):  
Bacillus Amyloliquefaciens ◽  
Fibrinolytic Activity ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Fibrinolytic Therapy ◽  
Fibrinolytic Enzyme ◽  
Fibrinolytic Enzymes ◽  
Enzyme Thermostability ◽  
Natural Agent

AbstractObjective: To screen fibrinolytic enzyme-producing Bacillus isolates (n=210) and to characterize of thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 that had the highest level of fibrinolytic activity together with the highest thermostability.Methods: Firstly, a total of 210 isolates were screened for their fibrinolytic enzyme production. The potent fibrinolytic enzyme producing isolates were evaluated for the thermostability of their fibrinolytic enzymes and one isolate showing prominent fibrinolytic activity was identified as molecular. Fermentation process was carried out on the isolate that had both the highest level of fibrinolytic activity and enzyme thermostability. The thermostable fibrinolytic enzyme from this isolate was then purified and characterized.Results: The fibrinolytic enzyme activities of 21 Bacillus sp. isolates in Nutrient Yeast Salt Medium were found to be in the range of 0.176-1.734 U/ml. The fibrinolytic activity of the enzyme purified from the culture supernatant of Bacillus amyloliquefaciens EGE-B-2d.1 was relatively stable at pH 7.0-11.0 for 24 h and also showed stability at a temperature of 60°C for 60 min. The enzyme degraded the fibrin clots by direct fibrinolysis. The specific activity and the molecular weight of the purified enzyme were estimated to be 44.46 units/mg protein and 30 kD respectively.Conclusion: The thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 was purified and characterized. This enzyme might also be used as a natural agent for oral fibrinolytic therapy or thrombosis prevention.

scholarly journals Cloning and Expression of Levansucrase Gene of Bacillus velezensis BM-2 and Enzymatic Synthesis of Levan

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 317
Author(s):  
Min Xu ◽  
Lixia Zhang ◽  
Fangkun Zhao ◽  
Jingyue Wang ◽  
Bo Zhao ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Enzymatic Reaction ◽  
Production Capacity ◽  
Cloning And Expression ◽  
Buffer System ◽  
Bacterial Strains ◽  
Bacillus Velezensis ◽  
Fusion Enzymes ◽  
Optimal Reaction Temperature

Levan is a versatile and valuable fructose homopolymer, and a few bacterial strains have been found to produce levan. Although levan products have numerous specific functions, their application and promotion were limited by the production capacity and production cost. Bacillus velezensis BM-2 is a levan-synthesizing strain, but its levan production is too low to apply. In this study, the levansucrase gene of B. velezensis BM-2 was cloned to plasmid pET-32a-Acma-zz, and the recombinant plasmids were transferred to Escherichia coli BL21. A transformed clone was selected to express and secrete the fusion enzymes with an Acma-tag efficiently. The expressed products were further purified by a self-developed separating material called bacterial enhancer matrix (BEM) particles. The purification efficiency was 93.4%, with a specific activity of 16.589 U/mL protein. The enzymatic reaction results indicated that the optimal reaction temperature is 50 °C, the optimal pH of the acetate buffer is 5.6, and the buffer system greatly influenced the enzyme activity. The enzyme activity was enhanced to 130% in the presence of 5 mM Ca2+, K+, Zn2+, and Mn2+, whereas it was almost abolished in the case of Cu2+ and Fe3+. The values of Km, kcat, and kcat/Km were 17.41 mM, 376.83 s−1, and 21.64 mM−1s−1, respectively. The enzyme amount of 20 U/g sucrose was added to the system containing 400 g/L sucrose, and the levan products with a concentration of 120 g/L reached after an incubation of 18 h, which was 8 times that of the yield before optimization. The results of molecular docking analysis indicated that the Asp86 might act as a nucleophilic catalytic residue for sucrose, Arg246 and Asp247 act as transition state stabilizer of transfructosylation, and Glu340 and Arg306 were recognized as general acid donors. They formed the catalytic-groups triad. The unique properties and catalytic activity of the levansucrase suggest that it deserves further research and might have good industrial application prospects.

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

2019 ◽  
Vol 2 (4) ◽  
pp. 91
Author(s):  
Lal Krishna
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Pathogenic Bacteria ◽  
Blood Clot ◽  
Bacterial Species ◽  
Antagonistic Activity ◽  
Bacterial Strains ◽  
Wide Range

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.

scholarly journals PEMANFAATAN PERLIT YANG TELAH DIMODIFIKASI UNTUK AMOBILISASI ENZIM BROMELAIN

2015 ◽  
Vol 3 (1) ◽  
pp. 40
Author(s):  
Elida Mardiah
Keyword(s):  
Enzyme Activity ◽  
Protein Content ◽  
Incubation Time ◽  
Specific Activity ◽  
Native Enzyme ◽  
Enzymes Activity ◽  
Lowry Method ◽  
Enzyme Specific Activity

 ABSTRACT Amobilization of bromelaine enzyme extracted from ananas fruit (Ananas comusus) acetone has been done. Modified perlite in the amino silica phosphat (ASP) is used as matrix amobilization.  The protein content has determined by Lowry method, while enzyme activity were determined by Anson method caseine as substrate. It native enzyme specific activity was 0.1281 unit/mg with konsentration 20000 ppm, pH 7.0, incubation time 35 minutes, and  temperature 40°C. Amobil bromelaine specific activity was 0.7438 units/mg with substrate on 20000 ppm pH 7.0, incubation time 30 minutes and temperature 37°C. This bromelaine enzymes activity was increased six times than native enzim and showed the activity for several repeatation. Keywords: perlit, enzim bromelain, amobilization

scholarly journals OPTIMASI WAKTU INKUBASI PRODUKSI PROTEASE DAN AMILASE ISOLAT BAKTERI ASAL TERASI IKAN TERI Stolephorus sp.

2018 ◽  
Vol 10 (3) ◽  
pp. 719-725
Author(s):  
Yulia Oktavia ◽  
Shanti Dwita Lestari ◽  
Susi Lestari ◽  
. Herpandi ◽  
Miftahul Jannah
Keyword(s):  
Enzyme Activity ◽  
Incubation Time ◽  
Specific Activity ◽  
Amylase Activity ◽  
Test Results ◽  
Bacterial Isolates ◽  
Optimum Time ◽  
Time Production ◽  
Protein Levels ◽  
Two Stages

ABSTRAKPenelitian ini bertujuan untuk menentukan waktu optimum produksi enzim protease dan amilase isolat bakteri asal terasi ikan teri Stolephorus sp. Penelitian ini dilakukan dalam dua tahap yaitu pengukuran pertumbuhan isolat bakteri dan penentuan waktu optimum produksi enzim protease dan amilase. Pengujian yang dilakukan meliputi uji aktivitas enzim protease dan amilase dan  kadar protein dari tiap-tiap enzim tersebut. Data hasil pengujian dianalisis secara deskriptif. Ada 4 isolat bakteri, 2 isolat merupakan bakteri penghasil protease yaitu isolat P2 dan P4, dan 2 isolat yang merupakan bakteri penghasil amilase yaitu A2 dan A4. Aktivitas protease optimum terjadi pada jam ke-36 untuk isolat P2 sebesar 0,073 U/mL dengan aktivitas spesifik sebesar 1,632 U/mg dan isolat P4 yaitu 0,057 U/mL dengan aktivitas spesifik 4,91U/mg. Aktivitas amilase optimum terjadi pada jam ke-36 untuk isolat A2 sebesar 0,360 U/mL dengan aktivitas spesifik sebesar 7,73 U/mg dan aktivitas amilase optimum pada isolat A4 sebesar 0,239 U/mL dengan aktivitas spesifik 5,24 U/mg. ABSTRACTThe purpose of this research was to know the optimization of incubation time in the production of protease and amylase by bacterial isolates of anchovy Stolephorus sp. paste origin. This research was conducted in two stages, namely the growth of bacterial isolates measurement and determination the optimum time production of the enzyme protease and amylase. Testing conducted include the test proteases and amylase enzyme activity and protein levels of each of these enzymes. The data will be analyzed test results are descriptive. There are 4 bacterial isolates, where 2 isolates, which is a protease-producing bacteria isolates that is P2 and P4, and 2 isolates, which is the amylase-producing bacteria that is A2 and A4. The activity of the protease optimum occurs at to-36 hours to isolate P2 of 0.073 U/mL with a specific activity of 1.632 U/mg and isolates P4 that is 0.057 U/mL with a specific activity of 4.91 U/mg. Whereas amylase activity, optimum occurs at to-36ohours for A2 isolates of 0.360 U/mL with a specific activity of 7.73 U/mg and activity of amylase optimum on A4 isolates of 0.239 U/mL with a specific activity of  5.224 U/mg.

scholarly journals PRODUKSI DAN PENENTUAN KONDISI OPTIMUM ENZIM XILANASE Bacillus amyloliquefaciens FUKUMOTO PADA SUBSTRAT XILAN JERAMI

2015 ◽  
Vol 2 (1) ◽  
pp. 74
Author(s):  
Widiyanti Sekatresna ◽  
Abdi Dharma ◽  
Periadnadi
Keyword(s):  
Enzyme Activity ◽  
Rice Straw ◽  
Incubation Time ◽  
Substrate Concentration ◽  
Bacillus Amyloliquefaciens ◽  
Specific Activity ◽  
Enzymatic Reaction ◽  
Optimal Conditions ◽  
Enzyme Specific Activity

 ABSTRACT The production and determination of  optimal condition of xylanase produced by Bacillus amyloliquefaciens on rice straw xylan were investigated in this study. The parameters to be observed were optimal conditions of pH, temperature, substrate concentration and incubation time. Xilanase activity was determined by measuring the amount of reducing sugar formed in the enzymatic reaction based on Somogyi Nelson method. Optimal conditions needed for the production of xylanase were at pH 7, temperature 27°C and six days of incubation time. While optimal conditions of xylanase action were reached at pH 8.2, temperature 45°C, substrate concentration 3.5%(w/w) and 15 minutes of incubation time with enzyme activity and enzyme specific activity of 1.285 U/mL and 0.738 U/mg respectively. As a comparison, xylanase was also produced on pure xylan  (birchwood), enzyme activity and enzyme specific activity obtained were 2.701 U/mL and 1.658 U/mg respectively. Cellulase content in enzyme produced on rice straw xilan showed the enzyme activity of 0.094 U/mL.  Keywords : xylanase, Bacillus amyloliquefaciens, rice straw xilan

Isolation, production and application of fibrinolytic enzyme from fermented food sources

2021 ◽  
Author(s):  
Nitika Singh ◽  
Shailendra Singh Shera
Keyword(s):  
Cardiovascular Diseases ◽  
Blood Clot ◽  
Fermented Food ◽  
Fibrinolytic Enzyme ◽  
Food Sources ◽  
Bacillus Species ◽  
Cow Dung ◽  
Fermented Foods ◽  
Fibrinolytic Enzymes ◽  
Wide Range

The accumulation of the fibrin in bacterial fibrinolytic enzymes finds applications to treat and prevent cardiovascular diseases which fail in hemostasis that leads to the formation of undesirable blood clots in the blood vessels leading to condition called thrombosis. The fibrinolytic enzymes from food grade organisms are useful for thrombolytic therapy. Conventional thrombolytic agents such as streptokinase, nattokinase etc.  Nattokinase is one such fibrinolytic enzyme with a wide range of applications in Pharmaceutical industry, health care and medicine etc. Hence, potent blood-clot dissolving protein used for the treatment of cardiovascular diseases is produced by the bacterium Bacillus subtilis during the fermentation of soybeans to produce Natto. The health benefits of some fermented foods are synthesis of nutrients, prevention of cancer, diabetes due to presence of functional microorganisms, which possess probiotics properties, antimicrobial, antioxidant, etc. The first report of fibrinolytic enzyme production of cow dung used as a cheap substrate from Bacillus species in SSF has been given earlier. This review describes different isolation methods which enable the screening and selection of promising organisms for industrial production. The purification and properties of these fibrinolytic proteases is discussed, and the use of fibrinolytic enzyme. In order to obtain Bacillus species producing fibrinolytic enzymes, the fermented food sample such as sprouted grain and processed grain etc were used. The heat tolerant isolates initially were selected for catalase test. Fibrinolytic activity of the selected isolates was determined by using Fibrin plate assay.  From the above work, it can be concluded that the fibrinolytic enzyme produced by Bacillus from fermented food samples had the ability to degrade the fibrin and hence can be used for functional food formulation.

scholarly journals Bacterial Strain for Bast Fiber Crops Degumming and Its Bio-Degumming Technique

2021 ◽  
Author(s):  
Shengwen Duan ◽  
Bingrong Xu ◽  
Lifeng Cheng ◽  
Xiangyuan Feng ◽  
Qi Yang ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Bacterial Strain ◽  
Inoculum Size ◽  
Activity Levels ◽  
Bacterial Strains ◽  
Bast Fiber ◽  
Extracellular Secretion ◽  
Apocynum Venetum ◽  
Slow Progress ◽  
Kenaf Bast

Abstract The R&D of bio-degumming technology is under a slow progress due to the shortage of proper efficient bacterial strains and processes. A degumming bacterial strain—Pectobacterium wasabiae (PW)—with broad-spectrum degumming abilities was screened out in this study. After the fermentation for 12 h, the residual gum contents of kenaf bast, ramie bast, hemp bast, flax bast, and Apocynum venetum bast were all lower than 15%. This bacterial strain could realize the simultaneous extracellular secretion of pectinase, mannase, and xylanase with the maximum enzyme activity levels of 130.25, 157.58, and 115.24 IU/mL, respectively. The optimal degumming conditions of this bacterial strain were as follows: degumming time of 12 h, bath ratio of 1:10, temperature of 33 ℃, and inoculum size of 2%. After the bio-degumming through this bacterial strain, the COD in wastewater was below 4,000 mg/L, which was over 60% lower than that in boiling-off wastewater generated by chemical degumming. This technology achieves higher efficiency, higher quality, and lower pollution.

Simultaneous optimization of activity and stability of xylose reductase from D. nepalensis NCYC 3413 using statistical experimental design

2020 ◽  
Vol 27 ◽  
Author(s):  
Shwethashree Malla ◽  
Sathyanarayana N. Gummadi
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Reductase Activity ◽  
Xylose Reductase ◽  
Life Time ◽  
Economic Feasibility ◽  
Simultaneous Optimization ◽  
Physical Parameters ◽  
Statistical Experimental Design

Background: Physical parameters like pH and temperature play a major role in the design of an industrial enzymatic process. Enzyme stability and activity are greatly influenced by these parameters; hence optimization and control of these parameters becomes a key point in determining the economic feasibility of the process. Objective: This study was taken up with the objective to optimize physical parameters for maximum stability and activity of xylose reductase from D. nepalensis NCYC 3413 through separate and simultaneous optimization studies and comparison thereof. Method: Effects of pH and temperature on the activity and stability of xylose reductase from Debaryomyces nepalensis NCYC 3413 were investigated by enzyme assays and independent variables were optimised using surface response methodology. Enzyme activity and stability were optimised separately and concurrently to decipher the appropriate conditions. Results: Optimized conditions of pH and temperature for xylose reductase activity were determined to be 7.1 and 27 ℃ respectively, with predicted responses of specific activity (72.3 U/mg) and half-life time (566 min). The experimental values (specific activity 50.2 U/mg, half-life time 818 min) were on par with predicted values indicating the significance of the model. Conclusion: Simultaneous optimization of xylose reductase activity and stability using statistical methods is effective as compared to optimisation of the parameters separately.

Sign in / Sign up

Export Citation Format

Share Document