Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media

Taneja, Kapila; Bajaj, Bijender Kumar; Kumar, Sandeep; Dilbaghi, Neeraj

doi:10.1007/s13205-017-0808-4

Cited by 36 publications

(23 citation statements)

References 39 publications

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“…Fibrinolytic protease by Bacillus subtilis K42 [76] and Serratia sp. [33] sustained 80-85% of its activity in the presence of organic solvents. Also, acetone, acetic acid, Tween 20, glycerol, isopropanol, DMSO, and SDS stimulated fibrinolytic activity [33].…”

Section: Effect Of Inhibitors/activators Metal Ions and Organic Solmentioning

confidence: 99%

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Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

et al. 2019

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The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett-Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20-80 • C and pH 5-10 with utmost activity at 50 • C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn 2+ , Zn 2+ , and Cu 2+ ). The enzyme kinetics revealed K m and V max values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI-TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.Biomolecules 2020, 10, 3 2 of 23 (active protease), converting soluble fibrinogen (glycoprotein) into fibrin polymer (insoluble blood clot) [5]. Fibrin clots are hydrolyzed by plasmin [12], which is stimulated from plasminogen by plasminogen activators (PAs) [13,14]. This natural dynamic equilibrium is disturbed when the process of natural fibrin clot hydrolysis undergoes pathophysiological shambles, leading to formation of fibrin clots. Such clots may cause hindrance in the circulation of blood, causing blockage in blood vessels, ultimately leading to cardiac disorders which are life-threatening [15,16].Approaches such as use of anti-coagulant agents, anti-platelet drugs, fibrinolytic enzymes, and surgical operations are employed for the treatment of thrombosis and to dissolve the blood clots [17]. Further, there are numerous side effects which may occur following the administration of the available anti-thrombotic strategies, as well as the high expense, which limit their scope. The effects of reperfusion, urticaria (allergic reaction), and hemorrhage are the major inimical consequences of such thrombolytic approaches on human health [18,19]. Other after effects include headache, dizziness, ulcers, increased clotting time, nausea and vomiting, etc. Management of thrombosis usin...

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Section: Effect Of Inhibitors/activators Metal Ions and Organic Solmentioning

confidence: 99%

“…Similar results were reported for fibrinolytic enzyme produced by Serratia sp. KG-2-1 [33], B. subtilis HQS-3 [58], and Pseudoalteromonas sp., IND11 [59]. Scientific reports indicate that most proteases are active in neutral to alkaline conditions, from pH 7.0 to 9.5 [60], and a few of extreme alkalophilic optima at pH 12-13 [61].…”

Section: Maldi-tof Ms Analysismentioning

confidence: 99%

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

et al. 2019

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“…Similarly to proteases that belong to the serralysin family in other bacteria, such as Photorhabdus, Erwinia, or Pseudomonas, S. marcescens PrtA has been implicated in cytotoxicity, immunomodulation, or virulence traits using different experimental models, including nematodes, insects, and mice (11)(12)(13)(14)(15)(16)(17). In addition, the potential application of the enzymatic properties of PrtA, for instance, as a detergent additive, has attracted industrial interest, and different purification strategies and the optimization of its catalytic activity have been reported (15,(18)(19)(20)(21)(22). In several countries, purified serralysins are also commercially available as potent analgesic and anti-inflammatory drugs (23).…”

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confidence: 99%

CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation

et al. 2018

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PrtA is the major secreted metalloprotease of Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of , there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in s, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a mutant background, is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of We demonstrate that metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in , the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is activated at 37°C and able to downregulate PrtA expression by direct interaction of CpxR with a binding motif located upstream of the gene. Moreover, we reveal that PrtA expression favors the ability of to develop biofilm, irrespective of the bacterial growth temperature. In this context, thermoregulation along with a highly conserved CpxR-dependent modulation mechanism gives clues about the relevance of PrtA as a factor implicated in the persistence of on abiotic surfaces and in bacterial host colonization capacity.

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“…The optimal pH of JP-I was similar to that of fibrinolytic enzyme from Bacillus pumilus BS15 (pH 8) (Yao et al, 2018), Bacillus subtilis JS2 (pH 8) (Yao et al, 2018), Bacillus velezensis BS2 (pH 8) (Yao et al, 2019), and Serratia sp. KG-2-1 (pH 8) (Kapila, Bijender, Sandeep, & Neeraj, 2017), while the optimal pH of JP-II was similar to that of fibrinolytic enzyme from Stenotrophomonas sp. (pH 10) (Kapila, Bijender, Sandeep, & Neeraj, 2019) (Table 2).…”

Section: Properties Of Purified Jp-i and Jp-iimentioning

confidence: 71%

“…The optimal temperature of JP-II was higher than that of fibrinolytic enzyme from Bacillus licheniformis KJ-31 (40°C) (Hwang et al, 2007), Bacillus pumilus BS15 (40°C) (Yao et al, 2018), Serratia sp. KG-2-1 (40°C) (Kapila et al, 2017) and Bacillus amyloliquefaciens Jxnuwx-1 (41°C) (Yang et al, 2019) (Table 2). To estimate the thermostability, after heat treatment at 40 to 60°C for a various time at pH 7.4, the residual activity (%) was determined and the enzyme activity of the nonheated enzyme was taken as 100%.…”

Section: Properties Of Purified Jp-i and Jp-iimentioning

confidence: 99%

A novel fibrinolytic enzymes from the Korean traditional fermented food—Jotgal: Purification and characterization

Kim¹,

Ri²,

Choe

2020

J Food Biochem

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The fibrinolytic activity in Korean traditional fermented food, Jotgal (pickled fish) was identified. Though the fibrinolytic activity could vary in different kinds of Jotgal, this activity seems to be produced by microorganisms during the natural fermentation stage. From Gonjaengijot (pickled opossum shrimp), two novel fibrinolytic enzymes named by JP‐I and JP‐II, have been purified by ethanol precipitation, Bio‐GEL P‐100 gel filtration, and DEAE‐cellulose ion‐exchange chromatography. Compared to the crude enzyme extract, the specific activity of the JP‐I and JP‐II increased 258, 85‐fold with the recovery of 22.1, 8.5%, respectively. The molecular weights of both enzymes were estimated as 36 kDa on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS–PAGE). The optimal condition for fibrinolytic activity of JP‐I was at 50°C and pH 8.1, while that of JP‐II was at 45°C and 9.9. Both enzymes were stable at a broad range of pH (5.0 to 10.5) and have metalloprotease nature. From these results, it concludes that these enzymes could be a novel potent thrombolytic agent. Practical applications The fibrinolytic enzyme is one of the clinical agents for cardiovascular diseases which is the leading cause of morbidity and mortality worldwide with 17 million deaths every year. A variety of fibrinolytic enzymes are found and characterized from various sources such as plants, animals, and microorganisms, and new sources for fibrinolytic enzymes continue to be explored. Jotgal, widely used in Korean people's diet, is a traditional Korean seafood prepared from many different types of fishes, fish eggs, fish intestines, and shellfishes. Through an amount of research, some of fibrinolytic enzymes were found and purified from Jotgal, however, no studies have been done on fibrinolytic enzyme from opossum shrimp. In this study, the purification, enzymatic characteristics, and fibrinolytic activity of the proteases, originated from Korean traditional fermented food, Jotgal were reported. These enzymes could be novel potent thrombolytic agent.

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Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media

Cited by 36 publications

References 39 publications

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation

A novel fibrinolytic enzymes from the Korean traditional fermented food—Jotgal: Purification and characterization

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