Abstract:www.nature.com/scientificreports/ (M1245)) were obtained from Himedia, India with a purity standard greater than 95%. All the chemicals used and their purification method is detailed in Table 3.
“…The purity fold obtained in this report was greater than the purity fold of pectinase (15.2) investigated by Amid et al using Triton X and sorbitol for phase separation 21 . A similar study on menthol based natural deep eutectic solvents (NADES) reported purity fold 21.2 for the recovery of fibrinolytic protease 2 . Figure 5 Response surface methodology based optimization of SDES concentration, Salt concentration and pH of medium for effective recovery of fibrinolytic protease.…”
Section: Resultsmentioning
confidence: 80%
“…Intravascular thrombosis is described as a pathophysiological disorder in which excessive accumulation of blood clots (thrombus) occurs throughout the blood vessels. This chronic condition obstructs blood flow to numerous internal organs and has been reported to be the major cause of cardiovascular abnormalities worldwide 1 , 2 . Conventionally, recombinant therapeutic enzymes such as streptokinase and urokinase are being used widely for treating these thrombotic abnormalities.…”
Fibrinolytic protease (FLP) is a therapeutic enzyme used in the treatment of thrombolytic diseases. The present study proposed the concept of pH-driven swappable micellar two-phase extraction for the concurrent production and purification of FLP from Bacillus subtilis at cloud point extraction. Extractive fermentation was carried out with a pH swap mechanism and FLP was extracted to the top phase by surfactant deep eutectic solvents (SDES). Shrimp waste was chosen as a sustainable low-cost substrate that yielded a maximum protease of 185 U/mg. Six SDESs were synthesized with nonionic surfactants as hydrogen bond donors and quaternary ammonium salts as hydrogen bond acceptors and their association was confirmed by H1 NMR. Thermophysical investigation of the synthetic SDES was accomplished as a function of temperature. Response surface methodology for extractive fermentation was performed with the concentration of SADES (35% w/v), Na2SO4 (15% w/v) and pH (6.3) as variables and the enzyme activity (248 IU/mg) as a response. Furthermore, purification using gel filtration chromatography was used to quantify the amount of enzyme obtained in the extraction phase (849 IU/ml). After final purification with an anion exchange column, the maximum purity fold (22.32) with enzyme activity (1172 IU/ml) was achieved. The in-vitro fibrinolytic activity has been confirmed using a fibrin plate assay.
“…The purity fold obtained in this report was greater than the purity fold of pectinase (15.2) investigated by Amid et al using Triton X and sorbitol for phase separation 21 . A similar study on menthol based natural deep eutectic solvents (NADES) reported purity fold 21.2 for the recovery of fibrinolytic protease 2 . Figure 5 Response surface methodology based optimization of SDES concentration, Salt concentration and pH of medium for effective recovery of fibrinolytic protease.…”
Section: Resultsmentioning
confidence: 80%
“…Intravascular thrombosis is described as a pathophysiological disorder in which excessive accumulation of blood clots (thrombus) occurs throughout the blood vessels. This chronic condition obstructs blood flow to numerous internal organs and has been reported to be the major cause of cardiovascular abnormalities worldwide 1 , 2 . Conventionally, recombinant therapeutic enzymes such as streptokinase and urokinase are being used widely for treating these thrombotic abnormalities.…”
Fibrinolytic protease (FLP) is a therapeutic enzyme used in the treatment of thrombolytic diseases. The present study proposed the concept of pH-driven swappable micellar two-phase extraction for the concurrent production and purification of FLP from Bacillus subtilis at cloud point extraction. Extractive fermentation was carried out with a pH swap mechanism and FLP was extracted to the top phase by surfactant deep eutectic solvents (SDES). Shrimp waste was chosen as a sustainable low-cost substrate that yielded a maximum protease of 185 U/mg. Six SDESs were synthesized with nonionic surfactants as hydrogen bond donors and quaternary ammonium salts as hydrogen bond acceptors and their association was confirmed by H1 NMR. Thermophysical investigation of the synthetic SDES was accomplished as a function of temperature. Response surface methodology for extractive fermentation was performed with the concentration of SADES (35% w/v), Na2SO4 (15% w/v) and pH (6.3) as variables and the enzyme activity (248 IU/mg) as a response. Furthermore, purification using gel filtration chromatography was used to quantify the amount of enzyme obtained in the extraction phase (849 IU/ml). After final purification with an anion exchange column, the maximum purity fold (22.32) with enzyme activity (1172 IU/ml) was achieved. The in-vitro fibrinolytic activity has been confirmed using a fibrin plate assay.
“…The in uence of these salts on the phase ratio depends on its corresponding salting out capacity. It could be noted from the results that individual salt in combination with various NADES shows a diverse phase volume ratio in extractive fermentation 42 .…”
Section: Effect Of Choice Of Salt and Nades On Extraction Of Narringinasementioning
Sustainable one-pot production and purification of naringinase using natural deep eutectic solvent based extractive fermentation is being executed in the current investigation. Tofu wastewater was used as alternative low-cost media for sustainable production of naringinase. Five natural deep eutectic solvents were prepared and their physicochemical properties were determined as a function of temperature. Optimization of essential variables in extractive fermentation for determining effective concentration of NADES at (74.5% w/w), Na2SO4 (15% w/v) and tofu wastewater (1.5% w/w) to achieve optimal naringinase yield of (249.6 U/ml). Scale up of naringinase production with a 3 L custom made desktop bioreactor was accomplished and effective regeneration of NADES was established. Quantification of the resulting active enzyme was done by size exclusion chromatography (736.85 U/mg). Ultrapure enzyme fraction was obtained with anion exchange chromatography yielding maximum purity of (63.2) and specific enzyme activity of (3516 U/mg). The in-vitro debittering activity of resulting ultrapure naringinase was determined with grape juice.
“…An 18.3-fold purification with specific activity of 52.4 U/mg was observed [28]. Maximum 21.2-fold purification with specific activity of 2607.8 U/mL was obtained on the subsequent purification of fibrinolytic protease from Bacillus cereus with an anion exchange column (DEAE-sepharose) [169].…”
Section: Recovery and Purification Of Fibrinolytic Enzymesmentioning
Cardiac disorders such as acute myocardial infarction, embolism and stroke are primarily attributed to excessive fibrin accumulation in the blood vessels, usually consequential in thrombosis. Numerous methodologies including the use of anti-coagulants, anti-platelet drugs, surgical operations and fibrinolytic enzymes are employed for the dissolution of fibrin clots and hence ameliorate thrombosis. Microbial fibrinolytic enzymes have attracted much more attention in the management of cardiovascular disorders than typical anti-thrombotic strategies because of the undesirable after-effects and high expense of the latter. Fibrinolytic enzymes such as plasminogen activators and plasmin-like proteins hydrolyse thrombi with high efficacy with no significant after-effects and can be cost effectively produced on a large scale with a short generation time. However, the hunt for novel fibrinolytic enzymes necessitates complex purification stages, physiochemical and structural-functional attributes, which provide an insight into their mechanism of action. Besides, strain improvement and molecular technologies such as cloning, overexpression and the construction of genetically modified strains for the enhanced production of fibrinolytic enzymes significantly improve their thrombolytic potential. In addition, the unconventional applicability of some fibrinolytic enzymes paves their way for protein hydrolysis in addition to fibrin/thrombi, blood pressure regulation, anti-microbials, detergent additives for blood stain removal, preventing dental caries, anti-inflammatory and mucolytic expectorant agents. Therefore, this review article encompasses the production, biochemical/structure-function properties, thrombolytic potential and other surplus applications of microbial fibrinolytic enzymes.
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