Screening of Marine Actinomycetes for Fibrinolytic Enzymes
K. Viswanathan*, L. Jeyanthi Rebecca
Department of Industrial Biotechnology, Bharath Institute of Higher Education and Research, Chennai, Tamilnadu, India
*Corresponding Author E-mail: viswanathan.bio@gmail.com
ABSTRACT:
Objective: This study aims to isolate the potential fibrinolytic enzyme from actinomycetes strains. Methods: Screening of actinomycetes from marine soil samples by using starch casein agar plate method, Protease activity by skim milk agar plate method and fibrinolytic enzyme activity by Fibrin plate method. Results: We have isolated 114 actinomycetes strains by starch casein agar plate method and the morphology was identified by coverslip method. Out of the 114 actinomycetes strains, 40 actinomycetes strains showed protease activity by skim milk agar plate method. Protease producing actinomycetes strains were tested for fibrinolytic activity by Fibrin plate method which was indicated by growth and clear zone of fibrinolysis. Out of 40 isolates, the fibrinolytic activity was highest in 12 actinomycetes strains. Conclusion: Based on the maximum fibrinolytic activity, BN22 was found to be a potential strain for further investigation.
KEYWORDS: Actinomycetes, Protease activity, Fibrinolytic activity.
INTRODUCTION:
Cardiovascular diseases (CVD) require more effective medications for treatment. The fibrinolytic enzymes such as staphylokinase, urokinase, streptokinase and nattokinase are used to treat blood clot inside a blood vessel, but these enzymes have many disadvantages such as immunogenicity, haemorrhagic side effect, and high cost. Hence the need for new fibrinolytic agent to overcome these drawbacks, the cost of fibrinolytic enzyme production and downstream processing are the major concerns of pharmaceuticals industry.
There are some fibrinolytic enzymes which are still in the development phase prior to commercial use. Nattokinase enzyme, which was purified from natto, which is a traditional Japanese fermented food, acts directly to lyse the thrombi in vivo[1,2], but can also enhance fibrinolytic activity in plasma and increase the amount of t-PA by oral administration[3].
According to the report by World Health Organization (WHO), 17.5 million people died from cardiovascular diseases in 2015[4] including heart disease, cerebrovascular disease, peripheral artery disease, hypertension, Rheumatic heart disease, congenital heart diseases and heart failure every year.
The major protein component of the blood clot is fibrin, which is formed from the fibrinogen via proteolysis by thrombin. Thrombolytic agents were administered orally or injected to lyse the thrombi in the blood vessels[5]. Antithrombotic, thrombolytic and anticoagulant reagents from various sources have been identified and used in the treatment of such cardiovascular diseases.The thrombolytic agents or fibrinolytic enzyme converts plasminogen to plasmin and lyse the clot by breaking down the fibrin contained in the clot; thrombosis is serious because it induces the cerebral and myocardial infarction
Plasmin plays a vital role in fibrinolysis and breakdown of fibrin polymers formed during the blood clotting process. Tissue plasminogen activators (tPA) are used to treat medical conditions such as myocardial infarction, embolic or thrombotic stroke and pulmonary embolism [6]. Urokinase plasminogen activator is the most common thrombolytic agent and it is used for peripheral vascular thrombosis and lysis of coronary thrombi[7].
The increase in the prevalence of cardiovascular diseases leads to increased need for fibrinolytic enzymes like Urokinase[8], staphylokinase[9], streptokinase[10] produced from microorganisms.
Actinomycetes are a special group of microorganisms, which have been demonstrated to be excellent producers of bioactive and structurally novel metabolites[11]. The actinomycetes strains were isolated from different sources, which produce several activities like antibacterial[12-15], antimicrobial[16], antiviral[17], nanoparticles[18] and novel therapeutic Drugs[19]. The present study aims to identify fibrinolytic enzyme producing actinomycetes strains from marine soil samples.
MATREIALS AND METHODS:
Reagents and Chemicals:
All the chemical and culture media used in the present study were of analytical reagents grade. Starch, Casein, agar, thrombin, fibrinogen, human fibrin and human plasma were purchased from Hi-Media ltd, Mumbai, India. Other reagents and chemicals used for the study were sourced purchased from Armats Biotek research center at Chennai.
Sample collection:
The different soil samples were collected from Chennai Beach Region, Tamilnadu.
Isolation of actinomycetes:
Add 1g of calcium carbonate to 10g of soil sample and incubate for 3 days. After this, the soil sample was sterilized by distilled water and these suspensions are to be considered as10-1. From10-1 Suspension, 1 ml is to be transferred into 9 ml of sterile distilled water and subsequently it was serially diluted to 10-2 - 10 -10. From the dilution (10-2 - 10 -3) 0.1ml suspension has to be drawn and poured into starch casein agar medium (containing (g/l): Starch 10g, Vitamin free casein 0.3g, CaCO3 0.02g, K2HPO4 2.0g, FeSO4 0.01g, KNO3 2.0g, NaCl2.0g, MgSO4 0.05g, Agar 20.0g and pH 7.0) for isolation of actinomycetes, and the plates were incubated at 28±2˚C for six to seven days [20].
Identification of marine actinomycetes by coverslip method:
Each actinomycetes strain was identified based on morphology and straining [21].
SCREENING OF PROTEASE AND FIBRINOLYTIC ACTIVITY:
Screening of Protease:
The isolated 114 actinomycetes strains were tested for protease activity by skim milk agar plate method containing: casein 5 g/l, skim milk 28 g/l, dextrose 1 g/l, yeast extract 2.5 g/l, agar 20 g/l and pH 7.0. The media was sterilized and plated on sterile petriplates. The test actinomycetes strains are then streaked on the respective substrate skim milk agar medium and the plates incubated at room temperature for five to six days. After incubation the plates was observed for clear zone of formation occurred around the colonies. The subculture was stored at 4˚C for further investigation [22].
Screening of Fibrinolytic:
The protease producing actinomycetes strains were tested for fibrinolytic enzyme activity by fibrin plate method containing: sodium chloride 5 g/l, fibrin 20 g/l, agar 15 g/, peptone 10 g/l and pH 7.0. The media was sterilized and plated on sterile petriplates. The test actinomycetes (protease producing) strains are then streaked on the respective substrate fibrin agar medium and the plates incubated at room temperature for five to six days. After incubation the plates was observed for clear zone of formation occurred around the colonies. The Subculture was stored at 4˚C for further investigation [23].
RESULTS:
Sample collection and Screening of Actinomycetes:
In present study, the marine soil samples were collected from three different places, namely Marina beach, Besant nagar and Neelangarai beach. The collected soil samples were screened for actinomycetes strains, therefore 114 actinomycetes strains isolated from the different soil samples by starch casein agar plate method. In the 114 actinomycetes strains, 57 isolates from marina beach, 40 isolates from Besant nagar and 17 isolates from Neelangarai beach. This isolates were incubated at room temperature for 15 to 21 days and dry amyloidal colonies with aerial and substrate mycelia were observed. After isolation of the pure actinomycetes colonies it was transferred from the SCA plate to SCA slants and incubated room temperature for pure colonies growth, and the SCA slants were maintained at 4˚C for further investigation.
Macroscopic Examination:
The isolated actinomycetes strains showed different colony colors from the collected soil samples. These colors were categorized in to grayish, light Brownish, dark brownish and light whitish.
Protease activity:
Totally, 114 actinomycetes isolates were tested for protease activity by using skim milk agar plate method in that, 40 isolates showed protease activity as measured by clear zone in skim milk agar plate; rest of them did not showed protease activity. In that 40 isolates, 10 isolates from marina beach showed good and very good inhibition, from Besant nagar beach 6 isolates showed very good inhibition and 9 isolate showed good inhibitions and from neelankarai beach 3 isolates showed good and very good inhibition. This 40 protease producing actinomycetes strains were further tested for fibrinolytic activity (Table 1).
Table 1: Protease Enzyme Activity
|
S.No |
Isolates |
Source (Soil Sample) |
Protease Activity |
|
1 |
MB22, MB41, MB16, MB47, MB36, MB15, MB55, MB08, MB11, MB19 |
Marina Beach |
+++ |
|
BN22, BN07, BN03, BN09, BN16, |
Besant Nagar Beach |
+++ |
|
|
NK17, NK15, NK13 |
Neelankarai Beach |
+++ |
|
|
2 |
MB03, MB10, MB27, MB30, MB33, MB37, MB39, MB45, MB43, MB20 |
Marina Beach |
++ |
|
BN10, BN17, BN15, BN19, BN31, BN27, BN34, BN05, BN36 |
Besant Nagar Beach |
++ |
|
|
NK05, NK07, NK11 |
Neelankarai Beach |
++ |
|
|
3 |
MB01, MB02, MB04, MB05, MB06, MB07, BN08, MB09, MB12, MB13, MB14, MB17, MB18, MB21, MB23, MB24, MB25, MB28, MB29, MB31, MB32, MB34, MB35, MB38, MB40, MB42, MB44, MB46, MB48, MB50, MB51, MB52, MB54, MB56, MB57, MB26, MB49, MB53 |
Marina Beach |
|
|
BN01, BN02, BN04, BN06, BN11, BN12, BN14, BN18, BN20, BN21, BN23, BN24, BN25, BN26, BN28, BN30, BN32, BN33, BN35, BN37, BN38, BN40, BN13, BN39, BN29 |
Besant Nagar Beach |
- |
|
|
NK01, NK02, NK04, NK06, NK08, NK09, NK10, NK12, NK14, NK16, NK03 |
Neelankarai Beach |
- |
+++ = Very good Inhibition, ++ = Good Inhibition, + = Moderate inhibition, - = No inhibition
Fibrinolytic activity:
Totally, 40 protease producing actinomycetes strains were tested for fibrinolytic activity by using fibrin plate method in that, 12 isolates showed that maximum fibrinolytic activity as measured by clear zone in fibrin plate; rest of them did not showed fibrinolytic activity. In that 12 isolates, 4 isolates from marina beach showed very good inhibition and 2 isolates good inhibition, from Besant nagar beach 2 isolates showed very good and good inhibitions and from neelankarai beach 2 isolates showed very good inhibition (Table 2). Based on the maximum fibrinolytic activity, BN22 was found to be a potential strain for further investigation (Figure 1).
Table 2: Fibrinolytic Enzyme Activity
|
S. No |
Isolates |
Source (Soil Sample) |
Fibrinolytic Activity |
|
1 |
MB22, MB41, MB16, MB47 |
Marina Beach |
+++ |
|
BN16, BN22 |
Besant Nagar Beach |
+++ |
|
|
NK17, NK15 |
Neelankarai Beach |
+++ |
|
|
2 |
MB15, MB19 |
Marina Beach |
++ |
|
BN03, BN09 |
Besant Nagar Beach |
++ |
|
|
3 |
MB36, MB55, MB08, MB11,MB03, MB10, MB27, MB30, MB33, MB37, MB39, MB45, MB43, MB20 |
Marina Beach |
- |
|
BN07, BN10, BN17, BN15, BN19, BN31, BN27, BN34, BN05, BN36 |
Besant Nagar Beach |
- |
|
|
NK05, NK07, NK11, NK13 |
Neelankarai Beach |
- |
+++ = Very good Inhibition, ++ = Good Inhibition, + = Moderate inhibition, - = No inhibition
Figure 1: Fibrinolytic activity
Strain Names BN22, BN22 & MB41
Morphology:
Actinomycetes isolates were identified by coverslip method under microscopy [24, 25]. Isolate no BN22 was observed to be spore chains morphology and strain was found to be a potential strain for further genus identification process [Figure 2].
Figure 2: Photograph showing spore chain morphology of Actinomycetes
Strain BN22
DISCUSSION:
The fibrinolytic enzymes have an important role in pharmaceutical industries. Many types of fibrinolytic enzymes are available today for the treatment of cardiovascular diseases. Streptokinase, urokinase and natokinase from bacterial sources namely Natokinase from B.subtilis, Streptokinase from β-haemolytic streptococci and urokinase from Pseudomonasesp in this reported in literature[26].
Fibrinolytic enzymes were produced from different sources of microorganism, fungi, plants, animals, mushroom and fermented foods.
Now a day’s a tissue type plasminogen activator (t-PA), an urokinase type plasminogen activator and bacterial plasminogen activator are used for cardiovascular diseases.
Urokinase enzyme was treated for pulmonary embolism, deep vein thrombosis and thrombosis of the eye[27]. The urokinase enzyme preparation and purification process was difficult in the pharmaceutical industry, the yield of production is low and the cost of product is high.
Therefore the search for fibrinolytic enzymes from various sources is essential and is being pursued by few researchers. Hence the present study is very relevant in this context.
In this study we have isolated 114 actinomycetes isolates from different soil samples, 40 isolates showed protease activity and 12 isolates showed fibrinlytic activity by fibrin plate method. Fibrinolytic enzymes mostly used for hydrolysis of polypeptides to small molecular entities[28]. Microorganisms are the important source for fibrinolytic enzymes.
The fibrinolytic enzymes producing microbial source have been reported from different species viz.,Bacillus [29], Staphylococcus[30], mushrooms[31], Penicillium[32], Streptomyces[33,34], Penicillium chrysogenum[35], algae [36], Actinomyces thermovulgaris[37] and Actinomyces Spheroides[38].
CONCLUSION:
From the results obtained in the present study, the actinomycetes strain BN22 was found to be having maximum fibrinolytic enzyme activity. The activity and stability profile of the fibrinolytic enzyme from marine actinomycetes strain BN22 is to be further investigated for the application in medical management of cardiovascular diseases.
CONFLICT OF INTEREST:
Nil.
ACKNOWLEDGMENT:
We wish to extend our sincere gratitude to Bharath Institute of Higher Education and Research for their encouraging support and our special thanks to ArmatsBiotek Training and Research Institute for providing us with the laboratory facilities required for this research work.
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Received on 04.04.2018 Modified on 20.05.2018
Accepted on 29.06.2018 © RJPT All right reserved
Research J. Pharm. and Tech 2018; 11(10): 4365-4369.
DOI: 10.5958/0974-360X.2018.00799.0