SCREENING AND ISOLATION OF FIBRINOLYTIC PROTEASE PRODUCING MESOPHILIC BACTERIA FROM SLAUGHTER HOUSES IN BANGALORE

SCREENING AND ISOLATION OF FIBRINOLYTIC PROTEASE PRODUCING MESOPHILIC BACTERIA FROM SLAUGHTER HOUSES IN BANGALORE

E. Venkata Naga Raju* and G. Divakar

Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur- 522 510, Andhra Pradesh, India

Department of Biotechnology & Microbiology, Acharya & B.M. Reddy College of Pharmacy, Soldevanahalli, Hesaraghatta, Bangalore- 560 090, Karnataka, India

ABSTRACT: This work has been undertaken for the screening and isolation of fibrinolytic protease producing mesophilic bacteria from ten soil samples, collected from slaughter housesvarious regions of Bangalore and used to screen for fibrinolytic protease production by using fibrin plate assay. In  the  present  study,  an  attempt  was  made  to  isolate  efficient  fibrinolytic protease producing  bacteria  from  diverse  environmental samples. Different isolates were screened for possessing the ability to produce fibrinolytic protease. About 5 bacterial isolates were found to be promising to produce fibrinolytic protease. The organisms were tested for various biochemical tests, which leads to their identification as Bacillus cereus, Bacillus circulans, Pseudomonas aeruginosa, Pseudomonas fluorescens and E.coli.

Fibrinolytic Protease, Slaughter houses, Fibrin plate,Bacillus cereus, Bacillus circulans, Pseudomonas aeruginosa

INTRODUCTION:  Enzymes are delicate protein molecules necessary for life. Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation ¹. The extracellular proteases are commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers ². Of these, strains of Bacillus sp. dominate the industrial sector ³.

Fibrinolytic enzyme is well known as a sub class of protease, which has an ability to degrade fibrin ^{4, 5, 6, 7, 8}. Blood clots (fibrin) are formed from fibrinogen by thrombin (EC 3.4.21.5) and are lysed by plasmin (EC 3.4.21.7), which is activated from plasminogen by tissue plasminogen activator ⁹. Deposition of fibrin in blood vessels normally increases thrombosis, resulting in myocardial infarction and other cardiovascular diseases ^{10, 11}.

Fibrinolytic proteases are the single class of enzymes which play an important part in the metabolism of many microorganisms like species of Pseudomonas ¹², staphylococcus ¹³, Alteromonas ¹⁴, Coryneform bacteria ¹⁵, Penicillium ¹⁶, Asperigillus ^{17, 18, 19}, Fusarium ^{20, 21}, Trichotecium ²², Actinomyces ^{23, 24}, Streptomyces ^{25, 26}, Escherichia coli ²⁷and Bacillus ^{28, 29, 30, 31, 32, 33, 34}.

Investigation of fibrinolytic proteases is a central issue in enzymology due to their wide applications in clinical, pharmaceutical, food, and bioremediation process. Among the various proteases, bacterial extracellular proteases are the most significant, compared with animal, Plants, viruses and fungal extracellular proteases.

Extracellular proteases produced by Bacillus species are of main interest from a biotechnological perspective, and are not only in scientific fields of protein chemistry and protein engineering but also in applied fields such as foods and pharmaceutical industries. The genus Bacillus contains a number of industrially important species and approximately half of the present commercial production of bulk enzymes derives from the strains of Bacillus.

In this study, an attempt was made for the screening and isolation of fibrinolytic protease producing mesophilic bacteria from slaughter housesvarious regions of Bangalore.

MATERIALS AND METHODS:

Collection and isolation of sample: Samples were collected from slaughter houses of beef, chicken and fish at Soldevanahalli, Chikkabanavara, Devasandra, K.R. Puram, Tannary road and Yashwanthpur in and around Bangalore, Karnataka, India. The samples were labeled after collected. These were spread onto isolation media (Fibrin plate agar) and incubated at 37^°C for 24 hours after serial dilution of 10^-1 to 10^-6.

TABLE 1: TABULATION FOR SAMPLES DESCRIPTION

Screening of Fibrinolytic proteases production by plate assay: The isolates were screened for fibrinolytic protease activity in triplets. This was done by inoculating the organisms on the modified fibrin plate agar ³⁵. Containing 1.2%w/v agarose, 0.4%w/v human fibrinogen and 20U/ml human thrombin in a petridisc and incubated at 37^oC for 24 hours. A clear zone around the growth of the bacteria was indicated to fibrinolytic proteases activity.

TABLE 2: TABULATION FOR RESULTS OF COLONY CHARACTERISTICS WHICH SHOW FIBRINOLYTIC PROTEASES ACTIVITY          

ISOLATES ON FIBRIN PLATE AGAR

PURE CULTURE IN PETRIDISH

PURE CULTURE IN SLANTS

FIGURE 1, 2, AND 3: FIBRINOLYTIC PROTEASES PRODUCTION AND PURE CULTURES

Identification of Bacteria: The isolated bacteria were identified based on cellular morphology, growth condition, grams staining, endospore staining, capsule staining and biochemical tests ^{36, 37}.

TABLE 3: TABULATION FOR RESULTS OF STAINING TECHNIQUES

TABLE 4: TABULATION FOR RESULTS OF VARIOUS BIOCHEMICAL TESTS    

 RESULTS AND DISCUSSION:  Five bacterial isolates were obtained (Table 3) from soil samples of ABMRCP 1 to ABMRCP 10 (Table 1), identified morphologically and biochemically as Bacillus cereus, Bacillus circulans, Pseudomonas aeruginosa, Pseudomonas fluorescens and E. coli. The colonies were subjected to grams staining, capsule staining and endospore staining.

The colonies which were positive and negative for grams staining, capsule and endospore staining were considered for further studies (Table 3 & 4). The selected colonies were streaked on fibrin plate agar. The plates were subjected to incubation for a period of 24 hoursat 37^oC. The plates which showed clear zone around the streaked area of test organism were selected as fibrinolytic proteases producing strain.

The organisms named (Table 2) showed the inhibition zone and were subjected to various biochemical tests (Table 4).

GD isolates (Table 2) showed the following results for the biochemical tests. These were positive for methyl red test, starch hydrolysis, citrate utilization test, oxidase test, gelatin hydrolysis test, urease test and nitrate reduction test and few isolates were shows negative for Voges Paskauer test, indole test and catalase test. After biochemical tests these organisms were confirmed to belong to the Bacillus species (Bacillus cereus, Bacillus circulans, Pseudomonas aeruginosa, Pseudomonas fluorescens and E. coli) which shows the capability of producing fibrinolytic protease.

CONCLUSION: Keeping in view the industrial importance of fibrinolytic proteases, researchers are more and more focusing on discovering fibrinolytic proteases with novel properties to meet the industrial requirements as well as increasing demand of global enzyme market. The search for promising strains of fibrinolytic proteases producers is a continuous process. The isolates which show higher fibrinolytic proteases activity were selected for biochemical characterization and identification. The organisms were identified as Bacillus cereus, Bacillus circulans, Pseudomonas aeruginosa, Pseudomonas fluorescens and E.coli. On the basis of data obtained in the present work it can be concluded that Bacillus species isolates can be employed in the production of fibrinolytic proteases.

ACKNOWLEDGMENTS: The authors would like to thank the chairman Acharya Institutes, Sri B. Premnath Reddy, for providing laboratory facilities and supporting this research work.

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    How to cite this article:

    Raju EVN and Divakar G: Screening and isolation of Fibrinolytic protease producing Mesophilic bacteria from slaughter houses in Bangalore. Int J Pharm Sci Res 2013: 4(9); 3625-3629. doi: 10.13040/IJPSR. 0975-8232.4(9).3625-29

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