Browsing by Author "Krishnamurthy, A."

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A novel fibrinolytic serine metalloprotease from the marine Serratia marcescens subsp. sakuensis: Purification and characterization
(Elsevier B.V., 2018) Krishnamurthy, A.; Belur, P.D.
This study demonstrates the purification and characterization of a fibrinolytic serine metalloprotease from the marine Serratia marcescens subsp. sakuensis (KU296189.1). The purified enzyme (1033 U/mg) had a molecular weight of 43 KDa, with optimum pH and temperature being 7 and 55 °C. The in vitro half-life of the fibrinolytic enzyme at 37 °C was found to be 19 h. The kinetic constants, Km and Vmax of the purified enzyme determined using fibrin as substrate was 0.66 mg/mL and 158.73 U/mL. The Kcat and catalytic efficiency of the enzyme was found to be 12.21 min?1 and 18.32 mL/(mg min) respectively. The fibrinolytic enzyme did not show any proteolytic activity towards blood plasma proteins like haemoglobin, ?-globulins and transferrin. In vitro studies revealed that the fibrinolytic enzyme displayed 38% clot lysis for a period of 3 h which was higher than that displayed by streptokinase and heparin. A total of seven peptide sequences were obtained after the LC-MS/MS-TOF analysis, out of which only four sequences showed 67% homology with the sequences of the other proteases. All these results suggest its novelty and potential application in thrombolytic therapy. © 2018 Elsevier B.V.
Improving the catalytic efficiency of Fibrinolytic enzyme from Serratia marcescens subsp. sakuensis by chemical modification
(2018) Krishnamurthy, A.; Mundra, S.; Belur, P.D.
Microbial fibrinolytic enzymes have gained increased attention due to their potential to prevent or cure cardiovascular diseases. Promising natural enzymes are often modified to improve/enhance the kinetic constants. Hence an attempt was made to chemically modify the fibrinolytic enzyme produced by marine Serratia marcescens subsp. sakuensis using amino acid specific modifiers. The aim was to enhance the kinetic constants and gather information on the vital amino acid residues involved in the catalysis. Modification of cysteine, histidine, tryptophan and serine residues resulted in drastic reduction in fibrinolytic activity indicating their presence in the active site. Modification of carboxylate residues resulted in a 19-fold increase in specific activity suggesting their presence in the catalytic site. Interestingly, ratio of fibrinolytic to fibrinogenolytic activity of the modified enzyme did not change significantly. There was a 507-fold reduction in Km value after chemical modification and due to that, 219-fold enhancement of catalytic efficiency was evidenced. Circular dichroism spectrum analysis of the modified and native enzyme revealed changes in ?- helix and -sheet conformation of the enzyme. Furthermore, the modified enzyme was more responsive to the presence of most of the metal ions tested. 2018 Elsevier Ltd
Improving the catalytic efficiency of Fibrinolytic enzyme from Serratia marcescens subsp. sakuensis by chemical modification
(Elsevier Ltd, 2018) Krishnamurthy, A.; Mundra, S.; Belur, P.D.
Microbial fibrinolytic enzymes have gained increased attention due to their potential to prevent or cure cardiovascular diseases. Promising natural enzymes are often modified to improve/enhance the kinetic constants. Hence an attempt was made to chemically modify the fibrinolytic enzyme produced by marine Serratia marcescens subsp. sakuensis using amino acid specific modifiers. The aim was to enhance the kinetic constants and gather information on the vital amino acid residues involved in the catalysis. Modification of cysteine, histidine, tryptophan and serine residues resulted in drastic reduction in fibrinolytic activity indicating their presence in the active site. Modification of carboxylate residues resulted in a 19-fold increase in specific activity suggesting their presence in the catalytic site. Interestingly, ratio of fibrinolytic to fibrinogenolytic activity of the modified enzyme did not change significantly. There was a 507-fold reduction in Km value after chemical modification and due to that, 219-fold enhancement of catalytic efficiency was evidenced. Circular dichroism spectrum analysis of the modified and native enzyme revealed changes in ?- helix and ß-sheet conformation of the enzyme. Furthermore, the modified enzyme was more responsive to the presence of most of the metal ions tested. © 2018 Elsevier Ltd
Methods available to assess therapeutic potential of fibrinolytic enzymes of microbial origin: a review
(Springer, 2018) Krishnamurthy, A.; Belur, P.D.; Subramanya, S.B.
Fibrinolytic enzymes are agents administered for the treatment of myocardial infarctions, strokes, cardiac and respiratory failure. Although several microorganisms are known to produce these fibrinolytic enzymes, only a few of such enzymes, along with the age-old oral anticoagulants, have been employed in the clinical and therapeutic applications in humans. The use of these agents is associated with drawbacks such as allergic reactions and bleeding complications; therefore, it necessitates frequent monitoring of drug levels in the blood. Due to this, there is an impetus on the current effort to identify newer potential candidates from the novel microbial sources which show longer half-life, higher fibrin specificity, higher therapeutic index and lesser allergic reactions. Various methods are available for the preliminary evaluation of a potential drug candidate for the therapeutic use. Choosing the right combination of in vitro and in vivo methods would give crucial insight on the therapeutic potential of the chosen test compound. This article discusses various assay techniques, in vitro trails and in vivo models available, to help researchers in choosing right biological methods and its combinations to evaluate efficacy of potential drug candidate. © 2018, The Author(s).
Methods available to assess therapeutic potential of fibrinolytic enzymes of microbial origin: a review
(Springer, 2018) Krishnamurthy, A.; Belur, P.D.; Subramanya, S.B.
Fibrinolytic enzymes are agents administered for the treatment of myocardial infarctions, strokes, cardiac and respiratory failure. Although several microorganisms are known to produce these fibrinolytic enzymes, only a few of such enzymes, along with the age-old oral anticoagulants, have been employed in the clinical and therapeutic applications in humans. The use of these agents is associated with drawbacks such as allergic reactions and bleeding complications; therefore, it necessitates frequent monitoring of drug levels in the blood. Due to this, there is an impetus on the current effort to identify newer potential candidates from the novel microbial sources which show longer half-life, higher fibrin specificity, higher therapeutic index and lesser allergic reactions. Various methods are available for the preliminary evaluation of a potential drug candidate for the therapeutic use. Choosing the right combination of in vitro and in vivo methods would give crucial insight on the therapeutic potential of the chosen test compound. This article discusses various assay techniques, in vitro trails and in vivo models available, to help researchers in choosing right biological methods and its combinations to evaluate efficacy of potential drug candidate. Â© 2018, The Author(s).
A novel fibrinolytic serine metalloprotease from the marine Serratia marcescens subsp. sakuensis: Purification and characterization
(2018) Krishnamurthy, A.; Belur, P.D.
This study demonstrates the purification and characterization of a fibrinolytic serine metalloprotease from the marine Serratia marcescens subsp. sakuensis (KU296189.1). The purified enzyme (1033 U/mg) had a molecular weight of 43 KDa, with optimum pH and temperature being 7 and 55 C. The in vitro half-life of the fibrinolytic enzyme at 37 C was found to be 19 h. The kinetic constants, Km and Vmax of the purified enzyme determined using fibrin as substrate was 0.66 mg/mL and 158.73 U/mL. The Kcat and catalytic efficiency of the enzyme was found to be 12.21 min?1 and 18.32 mL/(mg min) respectively. The fibrinolytic enzyme did not show any proteolytic activity towards blood plasma proteins like haemoglobin, ?-globulins and transferrin. In vitro studies revealed that the fibrinolytic enzyme displayed 38% clot lysis for a period of 3 h which was higher than that displayed by streptokinase and heparin. A total of seven peptide sequences were obtained after the LC-MS/MS-TOF analysis, out of which only four sequences showed 67% homology with the sequences of the other proteases. All these results suggest its novelty and potential application in thrombolytic therapy. 2018 Elsevier B.V.
Production of fibrinolytic enzyme by the marine isolate serratia marcescens subsp. sakuensis and its in-vitro anticoagulant and thrombolytic potential
(2017) Krishnamurthy, A.; Belur, P.D.; Rai, P.; Rekha, P.D.
Marine ecosystems house a plethora of microorganisms capable of producing therapeutically important metabolites, but remains largely unexplored. The aim of this work was to find a new marine bacterium capable of producing a fibrinolytic enzyme, which is fibrin specific, stable at the physiological pH, exhibiting both anticoagulant and thrombolytic effect in vitro. Once a potential bacterium was found, an effort was made to optimize medium. Based on the screening studies, the isolate C7 was chosen for further studies and was identified as Serratia marcescens subsp. sakuensis. Medium optimisation using Plackett-Burman design and one-factor experiments suggested that nitrogen sources have a profound effect on enzyme production. Fibrinolytic enzyme activity increased to 2.5 fold (487.29 U/mL) in the optimised medium as compared to unoptimised medium (191.65 U/mL). Current study demonstrates that seawater could be an excellent source for novel marine organisms capable of producing fibrinolytic enzymes. Fibrinolytic enzyme produced by Serratia marcescens subsp. sakuensis appears to be a potential candidate for thrombolytic therapy due to its high specific fibrinolytic activity, high ratio of fibrinolytic to fibrinogenolytic activity, activity at the physiological pH, anticoagulant and thrombolytic effects.
Production of fibrinolytic enzyme by the marine isolate serratia marcescens subsp. sakuensis and its in-vitro anticoagulant and thrombolytic potential
(Journal of Pure and Applied Microbiology micro_drkhan@yahoo.com 54, Near Post Office, Thana Street, Shahjahanabad Bhopal 462 001, 2017) Krishnamurthy, A.; Belur, P.D.; Rai, P.; Rekha, P.D.
Marine ecosystems house a plethora of microorganisms capable of producing therapeutically important metabolites, but remains largely unexplored. The aim of this work was to find a new marine bacterium capable of producing a fibrinolytic enzyme, which is fibrin specific, stable at the physiological pH, exhibiting both anticoagulant and thrombolytic effect in vitro. Once a potential bacterium was found, an effort was made to optimize medium. Based on the screening studies, the isolate C7 was chosen for further studies and was identified as Serratia marcescens subsp. sakuensis. Medium optimisation using Plackett-Burman design and one-factor experiments suggested that nitrogen sources have a profound effect on enzyme production. Fibrinolytic enzyme activity increased to 2.5 fold (487.29 U/mL) in the optimised medium as compared to unoptimised medium (191.65 U/mL). Current study demonstrates that seawater could be an excellent source for novel marine organisms capable of producing fibrinolytic enzymes. Fibrinolytic enzyme produced by Serratia marcescens subsp. sakuensis appears to be a potential candidate for thrombolytic therapy due to its high specific fibrinolytic activity, high ratio of fibrinolytic to fibrinogenolytic activity, activity at the physiological pH, anticoagulant and thrombolytic effects.