Extraction of Serralysin: A Fibrinolytic enzyme from Serratia sp. isolated from soil

 

Archana. L, Madhura Shevkar, Merlyn keziah. S., Subathra Devi C^*

Department of Biotechnology, School of Biosciences and Technology, VIT University,

Vellore -632014, Tamil Nadu, India.

 

ABSTRACT:

Serralysin is a type of metalloprotease originally isolated from digestive system of silkworm.This research shows the purification and characterization of metalloprotease (serralysin) extracted from Serratiaspisolated from soil. Out of 5 soilsamples collected, only one potent strain was observed to be an effective producer of fibrinolytic enzyme which was screened by heated plasma and skim milk agar plate assay. This species was found to have better growth in tryptosesoybroth agar and the color production of Serratia was also improved in tryptose soy agar at pH of 7.The purified enzyme demonstrated a zone of hydrolysis on heated plasma plate. This protein macro molecule was more stable over a wide range of pH (6–10) and the temperatures up to 60 ^◦C. It showed optimum enzyme activity at pH 9.0 and at a temperature of 37 ^◦C. The purified enzyme showed an apparent molecular mass of approximately 50 kDa in SDS-PAGE. The study also suggested that this serralysin is having potential application towards thrombolytic therapy.

 

 

 

INTRODUCTION:

Metalloproteases are those enzymes whose catalytic mechanism involve a metal, which plays an important role in pathogenesis, hence have advantage in health care industries. Most of pharmarelatedmetalloproteases are dependent on zinc and a few uses with cobalt, iron and manganese. Serralysin is a type of metalloprotease originally isolated from digestive system of silkworm.Subsequently, this enzyme also reported from different bacterial strains including Pseudomonas aeruginosa, Serratiamarcescens, Proteus mirabilis and Escherichia freundii.^[1] Serralysin differs from other metallo-endopeptidases in the catalytic reaction, where the enzyme preferentially cleaves the peptide bonds associated with hydrophobic residues.

 

Cardiovascular diseases (CVD), including acute myocardial infraction, ischemic heart disease, valvular heart disease, peripheral vascular disease, arrhythmias, high blood pressure and stroke, are the leading causes of death throughout the world. The World Health Organization (WHO) World Health Report 2006 reported that 18 million people die from CVD every year. Fibrin is the primary protein component of blood clots, which are formed from fibrinogen by thrombin.² The insoluble fibrin fiber is hydrolyzed into fibrin degradation items by plasmin, which is produced from plasminogen by plasminogen activators, for example, tissue plasminogen activator, vascular plasminogen activator, blood plasminogen activator, urokinase, Hageman variable, and streptokinase plasminogen complex.

 

The fibrinolytic operators accessible today for clinical utilize are for the most part plasminogen activators, for example, tissue-sort plasminogen activator, urokinase-type plasminogen activator and the bacterial plasminogen activator, streptokinase.³ Regardless of their across the board utilize, these specialists show low specificity to fibrin and cause undesired reactions, for example, the hazard for inner discharge inside the intestinal tract when orally administrated, have provoked analysts to hunt down less expensive and more secure assets. In this manner, microbial fibrinolytic proteins have additionally pulled in a great deal more medicinal enthusiasm amid late decades. Therefore, the hunt proceeds down other fibrinolytic proteins from different hotspots to explore thrombolytics.

 

MATERIALS AND METHODS:

Isolation of the organism:

The strain was isolated from the soil sample from VIT University. The soil sample isolated was serially diluted and spread plated on nutrient agar medium, incubated at 37°C for 24h. The orange pigmented colonies were selected on tryptosesoybroth agar and enhanced pigmentation was observed. This strain was further grown ontryptosesoybroth agar slants at 30 ^◦C and sub-cultured regularly in the same media. For growth and inoculum development, nutrient broth was used.

 

Screening for serralysin:

The isolated colonies were screened for serralysin production by heated plasma agar and skim milk agar assay. Depending upon the zone of clearance, the strains were isolated. The isolated strain was characterized by cultural, morphological characters according to Bergey’s Manual of Systematic Bacteriology.⁴

 

Skim milk agar assay

Skimmed milk agar supplemented with extra 2 % caesin was prepared. The supernatant of the production medium was added in the wells and the plates were kept for overnight incubation at 37°C to observe the zone of hydrolysis.⁵

 

Plasminolytic assay:

Hydrolysis was performed by incorporating fresh human serum and casein in 5% and 2% respectively in 1% nutrient agar. Well bore diffusion plate technique was used to check the plasminolyticactivity of the enzyme present in the supernatant of the production medium and incubated overnight at 37°C for 24 h.⁶

 

Extraction of crude Enzyme:

The production of serralysin was carried out in a nutrient agar medium containing 1% Peptic digest of animal tissue, 0.3% yeast extract, 0.5% sodium chloride with pH 7.2 adjusted with 1N NaOH or 1N HCl. 1% of the seed culture was inoculated in the production medium and maintained at 37°C for 24h in a shaker incubator (200 rpm). After complete fermentation, the whole fermentation broth was centrifuged at 8000 rpm for 15min at 4°C, and the clear supernatant was obtained. The crude enzyme supernatant was subjected to further purification.⁷

 

Partial Purification of Enzyme:

The crude enzyme supernatant was fractionated by precipitation with ammonium sulfate between 30%, 50%, 80% of saturation carried out at 4°C. All subsequent steps were carried out at 4 °C. The protein was suspended in 10mM TrisHCl buffer, pH 7.0. Dialysis was carried out for 4 h in 4°C and the purified enzyme extract was obtained and analyzed for the fibrinolysis activity by adding the enzyme to the blood at various concentration and the lysis of the clot was observed at various concentration of enzyme added.

 

SDS PAGE:

The purity of protease was analyzed by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass (using a standard molecular weight marker) was determined by SDS-PAGE according to the method of Laemmli. () After electrophoresis, the gel was soaked and washed twice for 20 min in 2.5% (v/v) Triton X-100 at room temperature to remove the SDS. Enzyme activity of protease was analyzed by incubating the gel for 12 h in 50 mM Glycine–NaOH buffer pH 9.0 at 37◦C. After incubation, gel was stained with 0.1% Coomassie brilliant blue R- 250 in methanol–glacial acetic acid–water (40:10:60) followed by destaining with methanol–glacial acetic acid–water (40:10:60) for visualization of protease activity bands.[^8]

 

RESULTS AND DISCUSSION:

Soil samples were collected from 5 different locations of VIT university. From 5 soil samples, 3 isolates were selected and screened for fibrinolytic activity. Characterization of the isolates was done by microscopic and morphological examination, which showed a gram negative rods and mucoid colonies which have entire margins and umbonate elevation with orange pigmentation.(Fig.1 &2)

 

                            [a]                                                          [b]

Fig.1: Serratiasp. on [a] Nutrient agar plate [b] Tryptose soy broth

                            [a]                                                         [b]

Fig 2 : [a] Orange color colonies of Serratia sp. [b] Microscopic view (100X)of Serratia sp.

 

The conformation of the serralysin production by Serratia sp. was done by skim milk agar assay and plasminolytic activity, which showed a zone of clearance. The zone was measured as 1.8 cm for 50µl, 2.4 cm for 75 µl and 2.5 cm for 100 µl of the supernatant added to the well.(Fig.3)

 

                          [a]                                                         [b]

Fig.3:  Zone of hydrolysis [a] Skim milk agar plate  [b] Plasminogen assay

 

The efficiency of the enzyme extracted was tested by clot lysis assay, in which the clot formed by blood was added with a 125 µl of enzyme, which showed a partial lysis and when clotted blood was subjected to 175 µl , it showed a complete lysis of clot. Further this enzyme will be used in medical field for clot lysis.

 

              [a]               [b]                     [c]  

Fig.4: Clot lysisassay[ [a]Negative control ;[b] positive control  ;[c]serralysin]

 

 

The purifed enzyme was then verified by SDS-PAGE suggested that the purified enzyme is homogeneous with molecular mass of 50 kDa.  The observed molecular weight of the enzyme is in accordance with literature reported extracellular metallo- protease of different S. marcescens where the reported molecular weight ranging from 44 to 60 kDa.

 

Fig 5 SDS-PAGE

 

CONCLUSION:

A protease extracted from the Serratiasp. was shown to be effective in fibrinolysis. Purification and characterization of the enzyme reveals they have some industrial important activity. Also the fibrinolytic activity showed that it can be effectively used as thrombolytic drugs.

 

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Accepted on 23.08.2017                             © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(7): 2911-2913.