INTRODUCTION:

Enzymes are known to be the catalysts for the biochemical reactions and are responsible for bringing about almost all of the chemical reactions in living organisms. Therapeutic enzymes are used most often for enhancing digestive function. The primary focus of this study is on enzymatic action on the protein Fibrin which is an insoluble protein involved in blood clotting^[1]. Fibrinolytic enzyme is a potent serine protease that exists in the human body is also involved in the lysis, or dissolution, of clots as wounds heal. Fibrinolysis is a process where, a fibrin clot, the product of coagulation, is broken down that grows and becomes problematic by preventing blood clots. Its main enzyme plasmin cuts the fibrin mesh at various places, leading to the production of circulating fragments that are cleared by other proteases or by the kidney and liver. In the many steps of the clotting cascade, fibrin is the final product derived from its soluble protein precursor, fibrinogen and may also be responsible for an overzealous propensity to form inappropriate clots in the body^[2].

Fibrin is laid down inside blood vessels that have been compromised by disease or injury. Fibrin forms minuscule strands that eventually dry and harden, capturing blood vessel components effectively. Certainly, fibrin occupies a vital role in health and healing. Inappropriate clotting, of course, is a major risk factor for Cardio Vascular Diseases (CVD) like myocardial infarction and stroke^[3].

Based on their different working mechanisms, thrombolytic agents are classified into two types. One is plasminogen activators, such as tissue-type plasminogen activator (t-PA)^[4] and urokinase^[5], which activate plasminogen into active plasmin to degrade fibrin. The other type is plasmin-like proteins, which directly degrade fibrin, thereby dissolving thrombi rapidly and completely. Expensive prices and undesirable side-effects (internal intestinal haemorrhage) of plasminogen activators and urokinase have prompted researchers to search for cheaper and safer resources^[6]. Microbial fibrinolytic enzymes that have also attracted much more medical interest during recent decades^[7] were successively discovered from different microorganisms, the most important among which is the genus Bacillus from traditional fermented foods^[8]. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases^{[9][10][11][12]}. In recent years, ﬁbrinolytic enzyme has been screened and characterized from various sources including Bacillus thuringiensis IMB B-7324^[13], B. thuringiensis IMV B-7324^[14], Streptomyces sp. MCMB^[15], Virgibacillus sp. SK37^[16] B. thuringiensis IMV B-7324^[17], B. subtilis RJAS19^[18], Proteus penneri SP-20^[19].

Solid-state fermentation (SSF) is deﬁned as the growth of microorganisms on solid materials for the production of biomolecules in the absence or near absence of free water. SSF is a useful technique for utilization of low cost agro-residues in large volumes in biosynthesis of enzymes and metabolites and the use of ideal agro-wastes mainly depend on the cost and the availability of the substrate material^[20]. The agro-industrial wastes such as pigeon pea^[21], green gram husk^[22], potato peel^[23], Jatropha curcas seed cake^[24], sesame oil cake^[25], and groundnut husk^[26] were recently used as the substrate for the production of hydrolytic enzymes. Since, the search for a novel and inexpensive substrate for the production of ﬁbrinolytic enzyme is a continuous process, Cow dung is one of such nutritive-rich feed stocks that was unexploited for the production of ﬁbrinolytic enzymes by Bacillus sp by providing essential nutrients. Cow dung contains ash, nitrogen, carbon, cellulose, hemicelluloses, magnesium, manganese, calcium, zinc, and trace elements^[27]. Cow dung manure is rich in carbon and nitrogen, which indicated that it could be a promising feed stock for the growth of microbes^[28]. In recent years, cow dung which has high moisture-holding capacity, preferred by the bacterial species for their growth and production of biomolecules^[29], was used as the substrate for the production of proteolytic enzymes from Halomonas sp. PV1^[30], Bacillus sp.^[31], Bacillus halodurans IND18^[32] and Shewanella sp. IND20^[33]. Cow dung was utilized for the production of proteolytic enzymes from Bacillus sp., the production of ﬁbrinolytic enzyme from the genus Bacillus using cow dung substrate has not yet been reported.

MATERIALS AND METHODS:

1. Collection of samples:

Four different food samples were collected from a market depending on their nutritional composition. They are Wheat, Maize, Finger millet and Pearl millet.

2. Isolation and Identification of Bacterial Contaminants:

Primary Screening:

The obtained food samples were boiled in water for 1hour approximately. After that, the boiled samples were kept under aerobic fermentation for 48-72hrs. About 1g of the sample was suspended in 9ml distilled water and serial dilution was carried (10^-1 to 10^-9) and plated on skimmed milk agar plates^[34]. These plates were incubated at 37℃ overnight. The same procedure was repeated for other three samples.

Secondary Screening:

The protease-producing (Fibrinolytic enzyme producing) bacterial isolates showing a clear zone were selected and sub-cultured for colony identification. The bacterial cultures were cultured into liquid medium (g/l; casein 10g, peptone 5g, yeast extract 3g, NaCl 5g). A loopful of bacterial isolates were individually inoculated and kept on a shaker water bath for 48hrs at 37℃.

3. Solid-Substrate Fermentation (SSF):

Fibrinolytic enzyme production was carried out in the 500 ml conical flask. Powdered cow dung substrate was moistened with Tris-HCl buffer and sterilized. It was then inoculated with 5% of inoculum at 37ºC for 24-48 hours. After fermentation, the enzyme was extracted by adding 100 ml of distilled water with the fermented medium and placed in shaker water bath at room temperature for 30 mins. After 30 mins, all the cultures were centrifuged separately at 10,000 rpm for 10 mins, and the clear supernatant was used as the source of crude enzyme for determination of fibrinolytic activity on fibrin clots.

4. Purification of enzymes:

Ammonium Sulphate Precipitation:

Purification of the enzyme was achieved using the overnight culture of the isolates. The culture was obtained by performing serial dilution using SSF medium as the inoculum. It was individually inoculated into the nutrient broth and incubated overnight at 37℃. The overnight cultures were centrifuged, and the supernatant was used in the precipitation step. The supernatant was subjected to precipitation using 100% saturated ammonium sulphate solution. The precipitates obtained by centrifugation at 10,000rpm for 10mins were collected and stored at 4ºC^[35].

Dialysis:

Dialysis was performed for each sample by taking 1ml of the precipitates in dialysis tubing. The beaker was filled with 1000 ml of PBS buffer and the dialysis membrane were hung on a glass rod to make the membrane fully immersed. The whole set-up was carried out in the magnetic stirrer for 2 days. The dialyzed sample was stored at 4ºC.

SDS-PAGE:

The SDS-PAGE was performed by loading the dialyzed samples in separate wells for each sample. Protein markers ranging from 245kDa – 11kDa were loaded into the adjacent wells. It was then electrophoresed for 1hour and staining and destaining step were undergone to observe the clear bands. The molecular weight of the samples was estimated using known protein marker.

Efficiency of the Fibrinolytic Enzyme:

The efficiency of the enzyme produced was determined using the standard ‘Streptokinase’ (15,00,000IU/5ml) by allowing the standard and the crude enzyme to react with the fibrin clot^[36]. 100µl of the sample and the standard was loaded onto the wells of ELISA-Reader separately. The OD value was checked and the efficiency of the sample was calculated by plotting the graph against the standard value.

RESULTS AND DISCUSSION:

Screening of Fibrinolytic-enzyme secreting organisms:

Among many dissimilar proteases producing bacterial isolates, potent zone forming isolates with the size ranging from 8mm to 13mm were chosen for secondary screening. Isolates from the primary screening was found to be Bacillus sp. with the colony morphology. Two different colonies were obtained from Corn sample. The identified strain from the samples Corn-1, Wheat, Finger millet and Pearl millet was found to be Bacillus subtilis (Figure 1-3) and it was Gram-positive, motile, casein-hydrolysing, Vogues-Proskaur, citrate, positive, Methyl-red negative (Table 1). The strain identified from Corn-2 failed to give positive results for the biochemical mentioned above but found to be Gram-positive, motile Bacillus sp. (Figure 4) with its colony morphology. It was further confirmed with endospore staining. Many researchers have focussed their efforts on isolating and screening of microorganisms for enzyme production with high fibrinolytic activity from various sources like fermented soy-bean sauce^[37], fermented red-bean^[38], fermented rice^[39], marine sources like fish scales^[40].

Fig 1: B.subtilis from Fig 2: B.subtilis from

Finger millet Pearl millet

Fig 3: B.subtilis Fig 4: Bacillus sp.

from Corn-1 from Corn-2

Fibrinolytic enzyme production in SSF using agro-residues:

In the present study, cow dung was used for the production of fibrinolytic enzyme which can spearhead the enzyme bioprocess in industrial scale. Other agro-residues used for similar studies were Wheat bran, Indonesian tempeh, Fermented red bean, Corn husk, Soybean meal. In SSF, selection of an ideal substrate is a key factor with the consideration of its availability and cheap cost. Based on the results obtained and the studies on cow dung for the similar purpose, the cheap cow dung is a substrate of choice for the production of fibrinolytic enzyme for this isolate.

Purification of Fibrinolytic enzyme:

In the present study, the fibrinolytic enzyme was precipitated with ammonium sulphate (100%) and further purified by SDS-PAGE. The purified enzyme migrated as a single band with the apparent molecular weight of 20kDa (Figure 1) by comparing with the protein marker of known molecular weight ranging from 245kDa – 11kDa. Various studies on purification and characterization of fibrinolytic enzyme from different bacterial sources like Bacillus thuringiensis IMB B-7324^[13], Streptomyces sp. MCMB^[15], Virgibacillus sp. SK37^[16] was reported that the purified fibrinolytic enzyme has a molecular weight within the range of 20-45 kDa^{[41][42][43][44][45][46][47]}.

Figure 5: Electrophoretic analysis of the dialyzed samples using SDS-PAGE

Efficiency of the fibrinolytic enzyme:

In the present study, the efficiency of the produced enzyme was determined by the defibrination of the fibrin clot. To the fibrin clot, fibrinolytic enzyme produced from 5 different samples were allowed to react for 1hour and Streptokinase as known standard. The concentration of the unknown samples was calculated by plotting against the known standard (Table 2). Based on the results obtained, B.subtilis from finger millet showed excellent fibrinolytic activity with highest concentration (2,58,000) comparatively (Fig 2). The crude enzyme of Bacillus sp digested fibrin clot directly and this kind of in vitro blood clot lytic studies was recorded previously with various organisms^{[48][49][50][51][52]}.

Table 2: Efficiency of Fibrinolytic enzyme production from different samples by Bacterial isolates

SL. NO.	VOLUME OF SAMPLE (µl)	CONCENTRATION (IU/ml)	OD VALUE
STANDARD (STREPTOKINASE)
1.	100µl	30,000	0.117
2.	200µl	60,000	0.200
3.	300µl	90,000	0.342
4.	400µl	1,20,000	0.431
5.	500µl	1,50,000	0.524
TEST SAMPLES
1. (Corn-1)	100µl	2,34,000	0.818
2. (Corn-2)	100µl	2,22,000	0.767
3. (Finger Millet	100µl	2,58,000	0.899
4. (Pearl millet)	100µl	2,40,000	0.840
5. (Wheat)	100µl	2,13,000	0.742

Figure 6: Evaluation of the Efficiency of different samples by Extrapolation

CONCLUSION:

In the present study, the production of fibrinolytic enzyme from Bacillus sp was enhanced by the cow dung substrate under SSF. The efficiency of the fibrinolytic enzyme produced by Bacillus subtilis from the finger millet sample showed the good clot lytic activity with the yield of highest concentration. This study clearly implies that fermented food-grade microorganisms could produce potent fibrinolytic enzyme by utilizing agro-residues and helpful in developing vast thrombolytic agents.

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Received on 17.12.2018 Modified on 21.01.2019

Research J. Pharm. and Tech. 2019; 12(4): 1963-1966.

DOI: 10.5958/0974-360X.2019.00328.7

ORGANISM	SAMPLE	BIOCHEMICALS
ORGANISM	SAMPLE	MR	VP	CITRATE	GLUCOSE	MANNITOL
Bacillus subtilis	Wheat, Corn-1, Finger millet, Pearl millet	-	+	+	+	+
Bacillus sp.	Corn-2	-	-	-	-	-