K. Viswanathan*, L. Jeyanthi Rebecca
Department of Industrial Biotechnology, Bharath Institute of Higher Education and Research (BIHER),
Chennai, Tamil Nadu, India
Fibrinolytic enzyme plays an important place in the medical field for cardiovascular disease. Only few studies have reported the fibrinolytic enzyme production from marine actinomycetes, and Streptomyces sp. In this study 114 actinomycetes isolates from different marine soil samples were isolated by using starch casein agar media. These 114 actinomycetes strains were tested for fibrinolytic activity by fibrin plate method. 14 isolates showed fibrinolytic activity amongst the 114 actinomycetes strains, and this was identified based on the clear zone of formation around the colonies. The potent strain of Streptomyces althioticus BN22 was isolated and this was confirmed by 16SrRNA sequencing. The optimization of nutritional and physical parameters for fibrinolytic enzyme production from marine soil was studied for the isolate Streptomyces althioticus. The effects of nutritional and physical parameters were studied for the optimum production of a fibrinolytic enzyme from Streptomyces althioticus BN22 by submerged fermentation. The effects of initial pH, temperature and incubation time were studied. The maximum enzymatic activity was obtained with an initial pH7.5 (45,765 Eu/ml), incubation temperature 50°C (50,827 Eu/ml) and incubation time for (41,175 Eu/ml). The effects of various carbon and nitrogen sources were studied. The results indicated that glucose (51,570 Eu/ml) as carbon source and yeast extract (54,625 Eu/ml) as nitrogen source showed the highest fibrinolytic enzyme activity among them. Thus this study showed its originality by focus, that marine Streptomyces althioticus is an important source for fibrinolytic enzyme production.
Cardiovascular disease like myocardial infarction, stroke and blood clot, the fibrinolytic enzyme plays an important role. Fibrin is formed after enzymatically cleaved from the precursor, Fibrinogen. In infectious diseases high levels of fibrinogen present in the blood, fibrin plays a crucial role in the defense system against micro-organisms.
Blood vessel thrombosis aggravated due to accumulation of fibrin clots which cause major cause for cerebrovascular disease and myocardial infarction. Fibrin formed from the precursor fibrinogen by thrombin, it is lysed by tissue plasminogen activator (tPA) called plasmin.
Drugs acting tissue plasminogen activator system and urokinase have shorter lives, low fibrin specificity, expensive and undesired side effects bleeding complications and allergic reactions.
Fibrinolytic proteases with the risk of thrombolytic effects has been purged through diverse sources such as earthworms, mushrooms, fermented foods and microbial source. Microbial fibrinolytic proteases have attracted medical attention for decades and it is used to cure cardiovascular diseases[5,6]. Many thrombolytic agents have been identified and characterized from different sources[7, 8, 9].
A vast search for an optimal thrombolytic drugs in bacteria, fungi and algae have been extensively studied beside sources. There is a need for an ideal thrombolytic agents in the medical therapeutics for CVD and MI.
Actinomycetes, the prevailing source of antibiotics, were also studied and a few fibrinolytic enzymes have been reported from Streptomyces megaspores, Bacillus[11,12], Streptomyces sp[13,3,14,15], Aspergillusochraceus 513,Fusariumpallidoroseum, Codiumintricatum, Staphylococcus sp. Streptomyces sp[37,38], Streptomyces sp. VITAK1, Streptomycesgancidicus-ASD_KT852565 and Streptomyces sp. VITMK1.
Synthesizing and manufacturing of fibrinolytic enzymes by micro-organisms differs qualitatively and quantitatively depending on the species and strains of microorganisms used as well as on their own nutritional and cultural environment. The overall expenditure of cost effective enzyme production is one of the major challenge.
In the present study, a detail exploration on several strains of actinomycetes was isolated for fibrinolytic enzyme production and most potential strain was Streptomyces althioticus BN22 was chosen for a detailed study on media optimization for the production of the fibrinolytic enzyme.
2. MATERIALS AND METHODS:
2.1 Reagents and Chemicals:
Chemicals and reagents used in the current study were of analytical reagent grade. Thrombin, Fibrinogen, Human Fibrin, Plasma, Glucose, Maltose, Sucrose, Fructose, Galactose, soluble starch, Ammonium chloride, Yeast Extract, Ammonium sulphate, Potassium nitrate, Urea, soya bean and Peptone was purchased from Himedia, Mumbai, India. Other chemicals and reagents were used from Armats Biotek, Chennai.
2. 2 Sample Collection:
The different marine soil samples were collected from Chennai coastal region namely Marina beach, Besantnagr beach and Neelankarai beach.
2.3 Screening of Fibrinolytic enzyme activity:
114 actinomycetes were isolated from the marine soil samples that were collected from chennai coastal region namely marina beach, besantnagr and neelankarai beach, were reported earlier. Actinomycete strains were tested by fibrin plate method, plate containing fibrin 20.0 g/l, peptone 10.0g/l, sodium chloride 5.0g/l, agar 15.0g/l and pH7.0 and the plates were incubated at room temperature for 5-6 days. After incubation, all the fibrin agar plates were observed for the zone of clearance. A clear zone around the actinomycete strain indicated the fibrinolytic activity.
2.4 Genomic identification by 16SrRNA Sequencing:
The PCR amplified product was subject to 16SrRNA sequence analysis at Progen biotech, Salem using electropherogram and the data was obtained. The BLAST sequence was also obtained and it was compared with the database to confirm the identity of actinomycetes species.
2.5 Phylogenetic tree construction:
The sequence alignment was done with the obtained BLAST sequence and it was compared with other species of Streptomyces species from GeneBank. The BLAST sequence helped in the construction of a neighbour tree and phylogenetic tree to assign the isolated actinomycetes to a particular species in taxonomical classification. Thus the sequence establishes the complete genetic identity of the organism. The phylogenetic tree was constructed by MEGA6 software and evolutionary history was inferred using the neighbour-joining method.
2.6 Effect of carbon source on fibrinolytic production:
The sterile production medium was prepared in different 500 ml of Erlenmeyer flasks. Each flask contained different carbon sources. The medium was supplemented independently with 1% of Maltose, Glucose, Sucrose, Fructose, Galactose and Starch. The individual medium was sterilized and the fibrinolytic sample was inoculated at room temperature and it was incubated in the shaker for 48hrs at 200rpm. The culture was filtered by centrifugation process and the filtrate was collected and the enzyme activity was determined.
2.7 Effect of nitrogen source on fibrinolytic production:
The sterile production medium was prepared in different 500 ml of Erlenmeyer flasks. Each flask contained different nitrogen source. The medium was supplemented independently with 1% of Peptone, Soya bean, Urea, Potassium nitrate, Ammonium sulphate, Yeast Extract and Ammonium chloride. The individual medium was sterilized and fibrinolytic sample was inoculated at room temperature and was incubated in the shaker for 48hrs at 200rpm. The culture was filtered by centrifugation process, the filtrate was collected and the enzyme activity was determined.
2.8 Effect of Temperature on fibrinolytic Production:
The sterile production medium was prepared in different 500ml of Erlenmeyer flasks. Test sample were inoculated and individual medium was sterilized. Each flask was incubated at different temperature namely, 35˚C, 40˚C, 45˚C, 50˚C, 55˚C, 60˚C and 65˚C for 48hrs. The culture was filtered by centrifugation process, the filtrate was collected and the enzyme activity was determined.
2.9 Effect of pH on fibrinolytic Production:
The sterile production medium was prepared in different 500ml of Erlenmeyer flasks and pH was adjusted to different pH such as 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 10.0. After sterilization flasks were inoculated with 1% test sample the medium were incubated at 37˚C shaker at 700rpm for 48hrs. The Enzyme activity was estimated.
2.1.1 Effect of Incubation time on fibrinolytic production:
The sterile production medium was prepared in different 500ml of Erlenmeyer flasks with the fibrinolytic culture of activated isolate by adding 1 ml of concentrate and incubated at 37˚C for periods ranged from 24hrs to 96 hrs, and then the medium was extracted and estimated its enzymatic activity.
3. RESULTS AND DISCUSSION:
3.1 Sample Collection:
From the 114 actinomycetes strains were isolated from marine soil samples at chennai coastal region namely marina beach, besantnagr and neelankarai beach. Fibrinolytic enzyme producing actinomycetes strains were streaked on the starch agar plate method. The Streptomyces althioticus BN22 was selected as potent fibrinolytic producing strain and was used in the study.
3.2 Fibrinolytic Activity:
A total 114 actinomycetes isolates were tested for protease activity by using skim milk agar plate method. Amongst that, 40 isolates showed protease activity as measured by clear zone in skim milk agar plate while the rest of them did not showed protease activity. The 40 protease producing actinomycetes strains were tested for fibrinolytic activity by using fibrin plate method. Amongst them 12 isolates showed maximum fibrinolytic activity as measured by clear zone in fibrin plate, while the rest did not show fibrinolytic activity. Based on the maximum fibrinolytic activity, strain BN22 was found to be a potential strain for further investigation. The strain depicting spore chain morphology is shown in (Figure1).
Streptomyces Althioticus strain BN22
Fig 1: Photograph showing spore chain morphology of Actinomycetes
3.3 Identification of Streptomyces althioticus BN22:
Among the different isolates from Chennai coastal regions, the effective fibrinolytic enzyme producing actinomycetes strain was confirmed as Streptomyces althioticus by 16SrRNA Sequencing and by Phylogenetic and evolutionary analysis of the 16SrRNAsequences. Nearly complete 16SrRNA gene sequence of strain BN22 was registered in GeneBank (Accession No: MG999523). Strain BN22 showed 100%, 99% and 98% 16SrRNA gene sequence similarity to the strains Strptomyces althioticus CSSP544, Strptomyces althioticus NBRC12740 and Strptomyces althioticus NBRC 12889 respectively. In a neighbour-joining phylogenetic tree based on 16SrRNA gene sequences, the isolate formed a monophyletic clade with the genetically closest Streptomyces althioticus strain BN22 (Figure 2). Neighbor-Joining method was used for evolutionary, optimal tree with the sum of branch length is shown. The replicating trees of percent in which the related taxa occur together in the bootstrap test (1000 replicates) are shown next to the branches . The maximum evolution tree distances composite like Likelihood method and 21 nucleotide sequences involved in the analysis. All positions accommodate gaps and absent were eliminated. A total of 1282 positions in the final dataset, MEGA6 tool were used for evolutionary analysis.
Fig.2: Neighbour-joining phylogenetic tree based on 16SrRNA gene sequence
3.4 Effect of Carbon source:
Effect of various carbon sources like maltose, glucose, sucrose, fructose, galactose and starch were studied for the maximum production of fibrinolytic activity. The results indicated maximum activity with glucose which is the optimum carbon source for the fibrinolytic enzyme production (Figure 3). Although, a lower enzyme yield was observed in the presence of sucrose, fructose, galactose and starch, results of the used different carbon source in the present study for fibrinolytic enzyme production were in disagreement with the others found in literature. The optimal carbon source for fibrinolytic enzyme production was found as glucose. 1% of glucose concentration was used for fibrinolytic enzyme production from Penicillium chrysogenum and Streptomyces sp. DPUA1576 used glucose while used maltose as carbon source for fibrinolytic enzyme production. The carbon medium components play an important role in enhancing the enzyme production. Hence the optimum carbon source varies with the source of the enzyme.
Fig 3: Effect of carbon source on enzyme activity
3.5 Effect of Nitrogen source:
Effect of various nitrogen sources like peptone, soya bean, urea, potassium nitrate, ammonium sulphate, yeast extract and ammonium chloride were studied for the maximum production of fibrinolytic activity. The results indicated maximum activity with yeast extract which is the optimum nitrogen source for the fibrinolytic enzyme production (Figure 4). Similarly, nitrogen source was reported [29, 30]. Hence the yeast extract was found to be best nitrogen source for fibrinolytic production.
Fig. 4: Effect of nitrogen source on enzyme activity.
3.6 Effect of temperature:
Effect of various temperatures like 35°C, 40°C, 45°C, 50°C, 55°C, 60°C and 65°C were studied for the maximum production of fibrinolytic activity. The results indicated maximum activity at 50°C which is the optimum temperature for the fibrinolytic enzyme production (Figure 5). The optimum temperature was 35°Cfor FP84 (45°C) . Similarly a temperature of 55°C was reported for thermophilic Streptomyces sp  for fibrinolytic enzyme production.
Fig.5: Effect of temperature on enzyme activity.
3.7 Effect of Ph:
Effect of various pH like 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 10.0 on maximum production of fibrinolytic activity was analyzed. The results indicated maximum activity at pH 7.5 which is the optimum pH source for the fibrinolytic enzyme production (Figure 6). While at pH 7.5 majority of the species showed maximum growth rate on most types of media. In addition, this optimum pH was correlated with highest growth yield. The optimum pH of 7.5 in accordance with our result for the production of fibrinolytic enzyme was reported in the case of Streptomyces sp. XZNUM00004 and Streptomyces megaspores SD5 [32, 10], Streptomyces sp CS684 . Anti-Diabetic Metabolites from Marine Streptomyces sp. VITJS8 .
Fig. 6. Effect of pH on enzyme activity
3.8 Effect of Incubation period:
Effect of various incubation periods namely, like 2, 4, 6, 8 and 9 was studied for the maximum production of fibrinolytic activity. The results indicated maximum activity at 4 or 6 days of duration which is the optimum incubation time for the fibrinolytic enzyme production (Figure 7). These results were in accordance with , who reported that the suitable incubation time period for fibrinolytic enzyme production from Streptomyces sp. NRC 411 was 7 or 6 days. Also, it was similarly reported from actinomycetes . The best incubation time period, 7 days was reported  for fibrinolytic enzyme production.
Fig. 7: Effect of incubation Period
Fibrinolytic enzymes have important role in the medical field for cardiovascular related disease. There are many types of fibrninolytic enzymes like a nattokinase, serratiopeptidase, papain, DNase, bromelain and honokiol available in the market. Only few of researchers had reported fibrinolytic enzyme production from marine actinomycetes.
Streptomyces althioticus BN22 is a suitable microorganism for fibrinolytic enzyme production. The maximum predicted fibrinolytic enzyme production (54,625 Eu/ml and 51,570 Eu/ml) could be achieved with the medium consisting of yeast extract 1% and glucose 1% concentration. Therefore, the fibrinolytic enzyme production by Streptomyces althioticus BN22 emerges as a good alternative for further therapeutically application using pharmaceutical process.
CONFLICT OF INTEREST:
Conflict of Interest declared none.
We thank Bharath Institute of Higher Education and Research for their support and thanks to Armats Biotek Training and Research Institute for the laboratory facilities provided to us for the required research work.
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Received on 28.03.2019 Modified on 21.04.2019
Accepted on 20.05.2019 © RJPT All right reserved