Production Of Streptokinase Research Articles

Biotechnological Advancements in Streptokinase Production

This article offers a deep analysis of activating streptokinase which looks into the biotechnological challenges and future perspectives. One of the main problems associated with streptokinase is its short half-life, complex production process, and immunogenicity which hinders patients from getting a successful reperfusion. Yet, the development of biotechnology methods that would not face these drawbacks, such as the strategy of recombinant streptokinase production and enzyme engineering theories, is a promise. This paper aims to provide an overview of biotechnological breakthroughs in the production of streptokinase with a focus on discovering long-lasting formulations exhibiting less immunogenicity and in addition the economic viability and scalability of biotechnological production processes. This article also gives the comping image of known purification methods like chromatography, ultrafiltration, and I-A chromatography for the separation and purification of streptokinase. In addition, the author presents the advantage of such suitable production technologies as cell-free production systems and E. Coli system optimization, such as recombinant streptokinase synthesis in Yeast systems. Biotechnology is turning out to be a new area, having a future boom due to the rising innovation and demand that are associated with the prospects revealed by the market trends.

Detection of Streptokinase Production by Streptococcus pyogen using Real Time PCR

Tonsillitis which is commonly referred to a strep throat is caused by group A β-hemolytic streptococci (GABHS) or Streptococcus pyogenes. The bacteria is consider as one of the major human-specific bacterial pathogen and one of its virulence factors is the streptokinase; for this reason, 100 tonsillitis swabs were taken from Iraqi children suffering from symptoms of sore throat. Using conventional cultural methods, 60 bacterial isolates were characterized and subjected to biochemical tests. As a result, 40 isolates were identified as hemolytic bacteria and only 6 of them were identified as Streptococcus. pyogens using Bacitracin (0.04) and Vitek 2 system. The ability of the 6 isolates for streptokinase production was screened and the results showed that all of them were produce the enzyme with variable degrees. Molecular detection of streptokinase gene using real time PCR technique has been done with specific primers and the amplification results were present with all S.pyogens strains.

Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity

Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug. Optimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity . Synthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard. Seed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x10 6 IU/mg with 62.94 ± 2.3% clot lysis efficiency. A high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug.

Production and market comparison of urokinase and streptokinase as effective and cheap fibrinolytic agents for treatment of cardiovascular diseases

Failure of hemostasis and the formation of blood clots in the arteries are the main reasons that provoke the onset of cardiovascular diseases (CVDs) such as myocardial infarction and ischemic stroke. Cardiovascular diseases have become the primary cause of deaths and disabilities across the globe. Therefore, this problem needs to be addressed with urgency. The disintegration of blood clots requires fibrinolytic agents, which are involved in thrombolysis. Streptokinase and urokinase are fibrinolytic enzymes; the former is primarily produced from microbial sources and the latter is isolated from urine, respectively. Streptokinase and urokinase have been in use for a long time to treat cardiovascular diseases. This review explains in detail the comparison of employing streptokinase and urokinase for the said purpose in a cost-effective manner. The recombinant production of both the agents has been discussed in detail. Furthermore, the efficacy of both the agents has been compared based upon their side effects and retention time in the body. A thorough study has been made to compare the influence of using both the agents on the health of cardiovascular patients in the last decade.

Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

A Statistical Application for the Enhanced Production of Streptokinase from a Mutant Strain Streptococcus equinus VIT_VB2

The present study focuses on media optimization of streptokinase (SK) production from the mutant strain EMS1 of Streptococcus equinus VIT_VB2 which was originally isolated from bovine milk. A two-level Plackett–Burman design was applied to screen key ingredients of the media (fructose, tryptose, KH2PO4, MgCl2, NaHCO3, CH3COONa, temperature, pH and incubation time). The dependent variables influencing SK production was further carried out using central composite design using response surface methodology. Statistical analysis revealed that the second-order model was found to be significant with R2 value of 0.9895. The production of SK was found to be high with supplementation of fructose, tryptose and potassium dihydrogen phosphate. The mutant strain EMS1 gave 2.06-fold higher SK production in the presence of optimized medium (663.53 U mL−1) than that of parent strain (wild strain) in minimal basal medium (321.5 U mL−1). The optimization of process parameters using response surface methodology resulted in a twofold increase in the yield of fibrinolytic enzyme.

Optimization and High Level Production of Recombinant Synthetic Streptokinase in E. coli Using Response Surface Methodology.

Streptokinase (SK) is an extracellular protein comprising 414 amino acids with considerable clinical importance as a commonly used thrombolytic agent. Due to its wide spread application and clinical importance designing more efficient SK production platforms worth investigation. In this regard, a synthetic SK gene was optimized and cloned in to pET21b plasmid for periplasmic expression. Response surface methodology was used to design a total of 20 experiments for optimization of IPTG concentration, post-induction period, and cell density of induction (OD600). The optimum levels of the selected parameters were successfully determined to be 0.28 mM for IPTG concentration, 9.889 H for post induction period, and 3.40768 for cell density (OD600). These settings result in 4.14fold increase in SK production rate of optimum expression conditions (7663 IU/mL) in comparison to the primary expression conditions (1853 IU/mL). Achieving higher yields of SK production in shake flask could lead to more cost effective industrial production of this drug which is the ultimate aim of SK production studies.

Enhanced Production Of Streptokinase By Chemical Mutagenesis Of Streptococcus agalactiae EBL-20

Abstract Streptokinase (SK) is an enzyme that is used for the treatment of cardiovascular diseases. The current study focused on the enhanced production of SK by inducing mutation in Streptococcus agalactiae EBL-20 and optimization of medium components and culture conditions for the maximum growth of mutant derived strain. S. agalactiae EBL-32 was selected as a potent mutant after exposure of S. agalactiae EBL-20 to EMS for 180 minutes. SK activity obtained from mutant derived strain was found to be 1.6 fold higher as compared to the activity achieved by wild strain. Nutritional requirements of the mutated strain were optimized by single factor analysis method suggesting glucose as the optimum carbon source; yeast extract and peptone as a suitable nitrogen sources and corn steep liquor (CSL) as an appropriate substrate for the maximum SK production. The culture conditions determined by response surface methodology (RSM) suggested that a temperature value of 37.5⁰C and pH 7 of the fermentation medium with 2.50 mL inoculum size for 36 hours of incubation was optimum for maximum yield of SK. Hence the optimization studies resulted into 1.92 fold increase in the yield of SK suggesting the new isolate suitable for commercial scale production of SK.

Enhanced Production of Streptokinase by UV- and Ethidium Bromide-Treated Streptococus equisimilis Mutant

Enhanced Production of Streptokinase by UV- and Ethidium Bromide-Treated Streptococus equisimilis Mutant

Optimization of culture media for extracellular expression of streptokinase in Escherichia coli using response surface methodology in combination with Plackett-Burman Design

Purpose: To investigate the enhancement of streptokinase extracellular expression in Escherichia coli by adjusting culture media.Methods: Screening of 10 chemical factors (EDTA, peptone, glycine, triton X-100, glycerol, K2HPO4, KH2PO4, Ca2+ (calcium chloride), yeast and NaCl) in order to increase the secretion of extracellular protein was carried out by response surface methodology (RSM). The method was also employed to optimize the concentrations of critical factors that had been determined in the screening step.Results: The results indicate that glycine, triton X-100 and Ca2+ were the most effective chemical factors in terms of increase in extracellular expression of streptokinase with optimum levels of 0.878, 0.479 and 0.222 %, respectively. Expression of streptokinase under optimum concentrations of critical permeabilizing factors led to a 7-fold increase in the quantity of secreted recombinant protein (5824 U/mL) compared to the initial level (802 U/mL).Conclusion: The results show that medium optimization using RSM is effective in improving extracellular streptokinase expression. The optimization medium is considered fundamental and useful for efficient production of streptokinase on a large scale.Keywords: Streptokinase, Response surface methodology, Membrane permeabilization, Extracellular secretion

Screening for Streptokinase producing Streptococcus sp. from Food and Soil samples

The objective of the study is to identify a potent streptokinase producing streptococcus sp. from different samples. Various food samples and soil samples were collected and screened for potent streptokinase producing Streptococcus strains. Isolates were confirmed as Streptococcus sp. based on its morphological and biochemical characterization. Further they were screened for the streptokinase activity. Isolates from curd sample and bore soil samples showed good activity and they were taken for further analysis and production process. They were compared based on their activity of clot lysis and casein digestion. Isolate from curd sample showed partial hemolysis and the isolate from bore soil sample showed complete hemolysis on blood agar medium. Production of streptokinase from these Streptococcal strains were done and partially purified.

Cell‐free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care.

Strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1

Screening of isolates for their potency to produce streptokinase was an important criterion of this research. The current study emphasizes the strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1 isolated from bovine milk. The study was carried out on samples collected from milk sample. Primary screening and characterization is used as an excellent source for the isolation of β-hemolytic organisms. Strain improvement was done by both physical & chemical mutagenesis. The enzyme activity was checked by clot lysis assay and confirmed by fibrin plate method. The partially purified and crude enzyme were analysed by high-performance liquid chromatography. Molecular weight & enzyme purity was checked by SDS–PAGE, further confirmed by fibrin zymography. Out of the 3 isolated strains, only one isolate expressed β-haemolysis with streptokinase (SK) activity. Based on the results of radial caseinolytic assay and blood clot dissolving assay, isolate TNA-M1 demonstrated the highest streptokinase activity. Based on morphological, biochemical and molecular characterization, it was identified as Streptococcus uberis and the strain was named as Streptococcus uberis TNA-M1. The results indicated that ultra-violet (UV) and ethyl methane sulfonate (EMS) were effective mutagenic agents for strain improvement of Streptococcus uberis TNA-M1 and enhanced SK productivity. HPLC analysis was performed in order to confirm the presence of streptokinase with the similar retention time (0.875 min) with its standard (0.854) min. SDS-PAGE of the enzyme showed protein band of approximately 47 kDa and confirmed by fibrin zymography. It exhibited fibrinolytic activity, which was more potent than other fibrinolytic enzymes. Glucose and peptone were recorded to be the optimum carbon and nitrogen sources respectively. Thus this study presents its novelty by highlighting the potential of Streptococcus uberis TNA-M1 as a significant source for the production of fibrinolytic enzymes.

High level production of active streptokinase in Pichia pastoris fed-batch culture

Streptokinase is a biological macromolecule involved in dissolution of fibrin blood clot and favourably used in various clinical applications. This protein is poorly expressed in soluble form due to its toxic effects on host physiology. The extracellular expression of recombinant streptokinase (SK) with and without 6xHis tag was obtained by cloning its gene under the α-mating factor signal sequence and alcohol inducible AOX1 promoter. Host-vector combinations were optimized to select a hyper producer. From shake flask optimization studies, a maximum expression of 582mg/L of rSK (non-tagged) and 538mg/L of rSK-His (His-tagged) protein was obtained when cells were induced at OD600 of 20. The high cell density fermentation increased the volumetric product concentration of rSK-His to a level of 4.25g/L with a 7.9 folds increase from shake flask results. The specific product yield (YP/X) was 49.75mg/g DCW along with a high volumetric productivity of 57.43mg/L/h. The protein was predicted to have 15.43% α-helix and 26.43% β-sheet with tryptophan emission maxima of around 347nm. The highest specific activity of rSK-His was 64,903IU/mg with 1.48 folds purification whereas specific activity of rSK was 55,240IU/mg with 1.22 folds purification.

Delivery of tissue plasminogen activator and streptokinase magnetic nanoparticles to target vascular diseases

Thrombolytic therapy for acute myocardial infarction standardly makes use of the medications streptokinase (SK) and tissue plasminogen activator (tPA). In this study, the potential of silica-coated magnetic nanoparticles (SiO2-MNPs) as nanocarriers clinical thrombolytic therapy was investigated. SiO2-MNPs for use in targeted therapeutic delivery of tPA and SK were prepared using a combined technique incorporating controlled precipitation and hydrothermal methods. Response surface methodology (RSM) was employed to evaluate the efficiency of the SiO2-MNPs. The production of SK secreted from Streptococcus equi was enhanced using random mutagenesis. The tPA and SK A were encapsulated by means of a silanizing agent with a surface rich in 3-aminopropyltrimethoxysilane layered around the SiO2-MNPs. Blood clot lysis assays and fibrin-containing agarose plates were used to carry out in vitro thrombolysis testing. The optimum conditions for producing MNPs were found to be at pH=13 and at a temperature of 75°C for 45min. Culture conditions of 2.75% NaCl concentration at initial pH=7.5 for 90s under UV resulted in maximum SK activity. The tPA/SK-conjugated SiO2-MNPs (SiO2-MNP-tPA-SK) increased operating stability in whole blood and storage stability in a buffer by 92%. More effective thrombolysis using magnetic targeting was indicated by a 38% reduction in blood clot lysis time achieved with SiO2-MNP-tPA-SK compared to administering the SiO2-MNPs without guidance. The silica-coated magnetic nanocarriers developed in this study show potential for improved clinical thrombolytic therapy.

IMPROVED STREPTOKINASE PRODUCTION

Several strains of beta-hemolytic Streptococci produce streptokinase enzymethat can bind and activate human plasminogen to plasmin. Streptokinase degrades the fibrinlump by its explicit lysine joining site and so it is applied as a remedy in thrombolytic therapy.The purpose of the study was to subject wild strain of Streptococcus equisimilis to straindevelopment technique, using random mutagenesis by UV irradiation for enhanced productionof streptokinase. Objective: To evaluate the hyper production of streptokinase after mutagenesisof wild Streptococcus equisimilis by means of UV irradiation. Study Design: Randomized study.Period: 2012-2014. Setting: Enzyme Biotechnology Laboratory, Department of Biochemistry,University of Agriculture, Faisalabad-Pakistan. Materials and Methods: UV lamp (TUP 40wlamp which has about 90% of its radiation at 2540-2550 A0) was used for the mutation ofStreptococcus equisimilis cells (1x 107 cells mL-1) for enhanced production of streptokinase. 10mL fresh inoculum was transferred to sterile petri plates, which were exposed to UV light for 30,60, 90, 120, 150, 180, 210, 240 and 270 minutes. The exposure was carried out at distance of20cm from the centre of lamp. A dose producing 87% killing was selected as optimum dose,after preparing kill curve. The kill/ survival curve was prepared and time of exposure giving(210 minutes) 3 log kill was selected for mutation of the Streptococcus equisimilis for hyperproduction of streptokinase enzyme. Results: Enzyme assay was performed for both wildand mutant strains. Dose of 210 minutes was selected as best dose which was followed bythe selection using triton X-100. Finally the selected strain S. equisimilis EBL-UV-210 showed480 U mL-1 of streptokinase activity in quantitative blood clot liquefaction test, which is quitehigher than wild strain (370 U mL-1). This maximum yield of streptokinase was obtained after24h, at CSL 4%, pH 7.5, 37oC, KH2PO4 0.04%, K2HPO4 0.05%, MgSO4. 7H2O 0.04%, NaHCO30.15%, CaCO3 0.004%, CH3COONa. 3H2O 0.10%, FeSO4. 7H2O 0.04%, MnCl2. 4H2O 0.02%,glucose 2%, yeast extract 3% and 5% inoculum size in liquid state fermentation. Conclusions:Results showed that mutated strain gave enhanced streptokinase activity in comparison tothe wild strain. Our current study focused on streptokinase production from this UV mutatedstreptococcus equisimilis species and purification of this enzyme by ammonium sulfateprecipitation, Ion exchange and gel filtration chromatography. The activity of streptokinase wasdetermined by using quantitative blood clot liquefaction method.

Exploring the in vitro thrombolytic potential of streptokinase-producing β-hemolytic Streptococci isolated from bovine milk.

The aim of this study was to isolate and characterize streptokinase-producing β-hemolytic Streptococcus sp. from bovine milk. A total of 50 milk samples were collected randomly from different breeds of cow and goat (Vellore, Tamil Nadu, India). The samples were characterized and screened for streptokinase-producing isolates using microbial and biochemical analysis. About 97 colonies were isolated from milk samples showing hemolytic patterns of α (19.6%), β (24.7%) and γ (55.6 %). Out of 20β-hemolytic isolates, only 6 colonies (VB2, VB3, VB8, VB14, VB16, and VB17) were identified as β-hemolytic Streptococci as potent producers of streptokinase. VB2 and VB14 showed the greatest streptokinase activities of 265 U mL(-1) and 225 U mL(-1), respectively. Based on biochemical and molecular characterization, the potent isolates VB2 and VB14 were identified and confirmed as S. equinus and S. agalactiae, respectively. The identified strains were named Streptococcus equinus VIT_VB2 (GenBank accession no. JX406835) and Streptococcus agalactiae VITVS5 (GenBank accession No. KF186620) The strains isolated from bovine milk provide a variance in the fibrinolytic activity on blood clots. The current study has demonstrated that the isolation of streptokinase producers from bovine milk, and the production of streptokinase from novel strain, enhanced the fibrinolytic activity. This study is the first to report that Streptococcus equinus produces streptokinase.

Production and Characterization of Streptokinase Enzyme by Using Streptococcus mutans Strain in Liquid State Fermentation through Corn Steep Liquor (CSL) Substrate

Background: Enzymes play a very important and central role in the chemical reactions, occurring in biological systems. The study of enzymes has an immense practical importance. They have high catalytic power, many times greater than the synthetic catalysts. Vascular blockage can cause serious consequences leading to death by a thrombus (blood clot) developed in the circulatory system. Streptokinase is an extracellular protein Trans-located by many strains of beta-hemolytic Streptococci. By converting the plasminogen, to fibrin lytic enzyme, plasmin, it favor’s the blood clot lysis. Results: The main objective of this research was the production of streptokinase in liquid state fermentation using CSL substances as substrate from Streptococcus species which was grown on blood agar media. Streptococcus mutans was selected for streptokinase production using corn steep liquor as substrate. Corn steep liquor (CSL) was applied in different concentrations ranging from 0.1% to 0.8% in liquid state fermentation culture medium and fibrin clot lysis method was used for enzyme assay. Conclusion: Among different concentrations of CSL 0.3% was selected as optimum concentration, 44% blood lysis was observed by CSL in liquid state fermentation culture medium for streptokinase production.

Process strategies for enhancing recombinant streptokinase production in Lactococcus lactis cultures using P170 expression system

The production of recombinant proteins in Lactococcus lactis is often limited by several process and biological constraints. Recombinant gene expression in the P170 system of L. lactis is triggered at a pH below 6.5 by lactic acid accumulation. This work has used static flask, batch bioreactor and chemostat studies to independently investigate various factors affecting production of recombinant streptokinase. These factors are induction strength, complex-nutrient supplementation, specific growth rate and the cellular response to acid-stress termed as acid tolerance response (ATR). In the P170 system, induction strength is mainly a function of lactate concentration. Recombinant protein production is enhanced by increasing induction strength and complex-nutrient supplementation. However, acid-induced cellular stress-response has a deleterious effect on recombinant protein productivity. It is seen that suppression of ATR has the most predominant effect on enhancing recombinant protein productivity. The insights obtained from batch studies were used to investigate fed-batch processes, both during complete development of ATR and during suppression of ATR. During complete ATR-development, a 25-fold enhancement of volumetric productivity was achieved in fed-batch culture, due to a synergistic combination of increased glucose-feed rates and addition of complex-nutrients to the feed. A combination of these factors and ATR-suppression gave rise to nearly 60-fold increase in volumetric productivity in fed-batch culture over batch processes.