Bioprospecting of Clot Buster Enzyme Producing Staphylococcus sp from Bovine Milk Sample

 

V. Mohanasrinivasan, Gurav Rawat, Aishwarya Parashar, Merlin Keziah, Nivetha. A

School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu

 

ABSTRACT:

The current study was intended to mitigate the risks posed by Cardiovascular heart diseases(CVD) which is the major cause of mortality and morbidity in the recent years around the world. Staphylococcus produces an enzyme known as Staphylokinase(SAK). In this experiment the SAK producing Staphylococcus sp.,was isolated from the environmental sample (bovine milk) using the selective media (Mannitol salt agar) and further the isolate was allowed to grow on blood agar to check its hemolytic activity.Beta hemolytic colonies were observed on blood agar. The preliminaryscreening of the isolatewere done using Gram Staining, biochemical analysis (catalase and coagulase test) and casein hydrolysis agar assay (for proteolytic activity). The isolate was found to be positive in all preliminary screening tests. Following screening,16S rRNA sequencing was done for species characterization. The Staphylokinasewas produced using the production media(Satoh's media). The enzyme was further extracted and subsequently assayed by Modified Holmstrom method and heated plasma agar assay for thrombolytic and plasmolyticactivity respectively. In Modified Holmstromit was observed that the crude enzyme extracted from the isolate showed thrombolytic activity at concentrations of 100-125µl. Furthermore, the partial purification of the enzyme was done using Ammonium sulphate precipitation.The precipitated protein was then subjected to SDS-PAGE to determine its molecular weight. Also the precipitated protein was then purified using 14 kdaultra filtrationmembrane.As the enzyme was found to be potent in lysing the blood clot, it can be further utilized by the pharmaceutical industries for the production of a clot busting agent which will be useful in treating various cardiac diseases mainly myocardial infarction.

 

 

 

INTRODUCTION:

With the change in lifestyle among people cardiovascular diseases are on the rise due to various predisposing factors such as obesity, diabetes, stress, lack of exercise, high blood pressure and pollution ultimately leading to different forms of heart diseases. Once the heart vessels are damaged the mechanism is irreversible and thereby progressing to chronic stages.

 

The fibrinolytic activity of SAK produced by Staphylococcus was once ignored in the past but now have gained importance among researchers in the recent years due to its thrombolytic property¹. SAK is a 136 amino acid single chain extracellular protein which digest fibrin clot by activating plasminogen to form plasmin and can be a promising alternative candidate drug in treating Cardiovascular disease or Acute myocardial infarction²Conventional thrombolytic drugs include Streptokinase, Urokinase, Tissue plasminogen activators (t-Pa) . As Streptokinase is of bacterial origin it causes tolerance and allergic reactions when repeatedly used. Our approach in the research was to isolate Staphylococcus from environmental origin and develop a novel therapeutic agent for treating and managing CVD³. As different Staphylococcus spp. produce different amounts of SAK so screening for individual  isolate is necessary and laborious, it’s just like finding a needle in a haystack for the isolate which produces maximumSAK. So in the current study, the main objective is to isolate the SAK producing non pathogenic strain from the environmental sample so that the chances of further cross reaction will be less. Also, the use of the non pathogenic strain make the isolation and production procedures safe and  reduces the steps of purification and hence reduces the overall cost. The Staphylococci was isolated from bovine milk sample and the isolate was further subjected for enzyme (SAK) production in satoh's medium. The enzyme was screened for its various lytic activities and further the protein was precipitated using ammonium sulphate precipitation. The protein after precipitation was subjected to ultra filtration and finally the molecular weight was determined by SDS-PAGE. Finally, the protein was further purified using gel filtration chromatography.

 

MATERIALS AND METHODS:

Sample collection:

Early morning Cow's milk sample was collected aseptically from Vellore district, Tamil Nadu.

 

Isolation and morphological characterization of Staphylococcus:

The cocci in grape like clusters were isolated from the milk sample using pour plate method. Mannitol Salt Agar medium was prepared and sterilized. The milk was serially diluted up to 10 ̄⁶ dilution and 1 ml of dilution was dispensed in the sterile petriplate. The sterile medium was poured into the plate and incubated at 37°C for 24 hours. The yellowish cream colony with yellow zone in the media was taken and furtherly streaked on 5% Blood Agar medium(incubation -37°C for 24 hours).Further the Gram staining from blood agar plate was performed to characterize the morphological aspects of the isolate².

 

Biochemical Analysis:

The isolate was subjected to catalase and coagulase tests for its biochemical confirmation.

 

Casein Hydrolytic Assay and Heated Plasma Agar Assay was performed to analyze the proteolytic and plasmolytic activity of the isolate respectively³.

 

Production of staphylokinase:

The enzyme staphylokinase was produced by subjecting the isolate to the production media(Satoh's Medium)⁴. 2g nutrient broth,0.6g yeast extract,1g sodium chloride and 2ml of glycerol was suspended in 200 ml of water and the pH was adjusted to 6.8.This media was sterilized and isolated colony from the pure culture plate was inoculated. The flask was incubated in the rotatory shaker at 37°c on 100rpm for 24 hours.

Extraction of enzyme:

The enzyme was harvested by centrifuging the production media at 8,000 rpm for 10 minutes at 4°c.The supernatant was removed carefully and was kept in refrigerator for further analysis.

 

Enzyme Screening:

The enzyme was screened for its thrombolytic by modified holmstrom method⁴.Freshly drawn human blood  of 0.5 ml was dispensed in 11 eppendorf and was allowed to coagulate at room temperature for 1 hour. The serum was removed carefully from each eppendorf. The tubes were labelled from 1 to 10 and the 11^th one was kept as control. Further 25µl,50µl,75µl till 250µl crude enzyme was added to the 1^st, 2^nd, 3^rd till 10^th eppendorf respectively. The tubes were incubated at 37°C for 18 hours and after incubation were observed for haemolysis.

 

The enzyme was also screened for its proteolytic and plasmolytic activity again by boring the well on the agar plates and adding the enzyme instead of streaking the isolate.

 

Salt precipitation (Ammonium sulphate precipitation):

The protein was precipitated using different concentration of the salt i.e. 20%, 40%, 60%, 80%. Initially 20% of the salt was added in the crude enzyme and was continuously stir for 10 minutes. The suspension was centrifuged at 8,000rpm for 10 minutes at 4°C. After centrifugation, the suspension was dispensed back into the flask and the centrifuge tubes were washed with 1ml of 2mM Tric HCl. The same procedure was repeated with the rest of the concentrations^7-8

 

Ultra filtration of protein:

The 10Kda ultra filter membrane was used to further filter the precipitated protein.7 ml of the precipitated protein was poured into the 10kda membrane tube and was centrifuged at 6,000 rpm for 10 minutes.

 

SDS-PAGE:

The ultra filtered protein(80%) was further subjected to the SDS-PAGE for its molecular weight determination. In addition(80%) ammonium salt precipitated protein was also run along with 97 to 14 kda marker⁷.

 

Gel filtration chromatography:

The ultra filter enzyme was run in a column of sephadex gel(2g in 15 ml phosphate buffer) and 20 different fractions were collected for further analysis^8-9.

 

 

 

RESULTS AND DISCUSSION:

Isolation and morphological characterization:

Mannitol salt and blood agar plates:

After 24 hours of incubation, creamy yellowish colour colony developed on mannitol salt agar plate. The colour of the media turned orange to yellow which indicates complete fermentation of mannitol. The creamy yellowish colonies on the surface were identified asstaphylococcus species (Figure 1). On blood agar,colonies showed complete hemolysis indicates the  ß-hemolysis (Figure 2)

 

 

Figure 1: Mannitol fermentation on MSA

 

 

Figure 2: ß-hemolysis on blood agar plate

 

Biochemical tests:

Gram staining: Gram positive violet colour cocci in grape like bunches was seen under microscope (Figure 3).The isolate was positive with both catalase (Figure 4) and coagulase.

 

Figure 3: Gram Positive Cocci in grape like cluster

 

Fig.4: Catalase Negative

 

Modified Holmstrom Method

This method was performed to check the thrombolytic activity of the crude enzyme. Lysis of the clot was observed in all the eppendorf butthe maximum lysis was observed in the eppendorf with 125µl of enzyme. Fig 5 shows the lysis of blood clot in eppendorf with enzyme while there is no lysis in control.

 

 

Fig.5: Clot lysis by Modified Holmstrom Method

 

Gel Filtration Chromatography:

It depicts the elution profile of the fractions collected during gel filtration chromatography. The graph shows the O.D. in three ranges which indicate the presence of three different proteins in the sample. The maximum peak in the graph was observed in the first fraction which indicates that the protein with the highest molecular mass elutes first which may be corresponds to the one whose band comes around 14.5kda in SDS-PAGE

 

SDS-PAGE:

The SDS-PAGE was done to check the molecular weight of the enzyme. Different bands were observed after staining and destaining the membrane. In the ultra filtered sample three bands were observed and  in the salt precipitated sample four bands were observed. Fig 6 shows the presence of the 14.5 kda protein (staphylokinase)which was confirmed by cross matching the bands with the 97-14 kda marker.

 

 

CONCLUSION:

The isolation of bacteria was done on mannitol salt agar and further cultivated on blood agar which showed characteristic ß-haemolytic colonies, the isolate was found to be gram positive with morphological characteristic of grape like bunches or clusters. The isolate was also coagulase and catalase positive when biochemical analysis was done. The production media(Satoh’s) was inoculated with pure culture under aseptic conditions and incubated in rotatory shaker at 100 rpm overnight. The crude enzyme was harvested by centrifuging production media at 8000 rpm for 10 minutes at 4°C .The extracted crude enzyme was screened by modified Holmstrom method which showed maximum thrombolytic activity at 125µl. The crude was further partially purified by ammonium precipitation method with different salt concentration of 20% , 40%, 60%, 80% and finally purified by ultracentrifugation with  10kDa membrane.Partially purified enzyme and purified enzyme of 80% ammonium sulphate were then subjected to SDS-PAGE which showed four and three bands respectively. Among these bands Staphylokinase was present in each of these whose molecular weight was 14.5kDa when compared with the marker. As more than one bands were observed so further purification was done by gel filtration technique and different fractions were collected thus among the collected fractionswhichever of these fraction will show positive result for Modified Holmstrom method will be the purified staphylokinase.

 

As staphylokinase produced in the experiment is from environmental origin thus chances of allergic reactions are less. The genes encoding the enzyme can be tracked down by various tools of genetic engineering and molecular biology, the SAK genes can then be inserted into a desired host and the enzyme can then be made to produce by that particular host resulting in cheap, reliable and novel therapeutic drug in pharmaceutical industry to manage and cure cardiovascular diseases which we all know is one of the leading cause of deaths in the modern world.

 

REFERENCE:

1.        Maria Bokarewa I, Tao Jin, Andrej Tarkowski,  Staphylococcus aureus : Staphylokinase. The International journal of biochemistry and cell biology. 2006; volume 38,504-509.

2.        Subathra Devi C, Dipti Sinha, Vaibhav Sharma and  Mohanasrinivasan V. Screening for staphylokinase producing Staphylococcus Spp. from different environmental samples. Asian Journal of Pharmaceutical and Clinical Research. 2012; volume 5,suppl 4.

3.        Pulicherla KK, Gadupudi GS, Rekha VPB, Seetharam K, Anmol Kumar, Sambasiva Rao KRS. Isolation, Cloning and Expression of Mature Staphylokinase from Lysogenic Staphylococcus aureus Collected from a Local Wound Sample in a Salt Inducible E.coli Expression Host. International Journal of Advanced Science and Technology. 2011; 30:35-42.

4.        Mohanasrinivasan V, Subathra Devi C, Akata Saha, Madhumanti Mondal, Sudeshna Dhar,Sughnthi V,  Selvarajan E. Screening for Staphylokinase producing Staphylococcus Spp. From Bovine Milk Sample. International Journal Of  Pharmacy and Pharmaceutical Sciences. 2013; volume 5, Issue 2.

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6.        Sweta Prasad, Rajpal S Kashyap, Jayant Y Deopujari, Hemant J Purohit, Girdhar M Taori, Hatim F Daginawala.Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thrombosis Journal. 2006;4:14 - 18

7.        Maria Goretti M, Purwanto. The role and efficiency of ammonium sulphate precipitation in purification process of papin crude extract. Science direct, Procedia chemistry. 2015; volume 18, 127-131.

8.        Bokarewa MI, Jin T, Tarkowski A. Staphylococcus aureus: Staphylokinase. International Journal of Biochemical Cell Biology. 2006; 38: 504-509.

9.        Mohanasrinivasan V, Subathra Devi C, Monidipa Banerjee, Jannatul Firdous Siddiqui, Pragya Lakshmi, Jemimah Naine S, Vaishnavi B. Production of fibrinolytic staphylokinase from UV Mutated staphylococcus aureus VITSDVM7. International Journal of ChemTech Research, 2014; volume 6, no. 8.

 

 

 

Accepted on 20.08.2017           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(5):1800-1803.