17-09-2016, 12:58 PM
Isolation and Screening the Pharmacological activities of Vegetative and Spore crystal Proteins from Bacillus thuringiensis
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ABSTACT
Bacillus thuringiensis, the most successful and most widely used microbial insecticide, produces crystal proteins. The physiological significance of the crystal proteins is poorly understood except for the potent insecticidal activity. In current study, an attempt made to isolate vegetative and spore crystal proteins from Bacillus thuringiensis NCIM2514 and to perform their haemolytic and antioxidant activities. The proteins were separated by salting out methods then by using SDS PAGE the protein molecular weight of the crude protein of vegetative and spore crystal protein identified as presence of low molecular weight protein. Pharmacological activities as antioxidant and haemolytic studies were performed for the crude proteins and found as spore crystal proteins contain more anti-oxidant when compared to vegetative proteins.
Key Words: Bacillus thuringiensis(bt), spore crystal proteins, vegetative proteins.
INTRODUCTION
Bacillus thuringiensis was first isolated in Japan as a pathogen o fthe sotto disease of silkworm bombyx mori by Ishiwata (1901).The organism is a gram positive,spore forming bacterium that produces crystalline parasporal inclusion during sporulation .The inclusion often exhibit strong insecticidal activity against several orders of insects & this makes Bt reliable agent for microbial control of insects pests of agricultural and medicinal importance [Lambert & peferoen 1992: Cannon 1996].the insecticidal parasporal inclusion contains two families of insect toxic molecules, 1.Cry protein, 2.Cyt protein. The cry protein is specifically toxic to insects & is currently classified into 22 geneetically different major groups Cry 1 to 22. [Crickmore et al 1998].Historically it has been believed that Bt as a species is characterized by insecticidal activity with its parasporal inclution. Earlier studies ,however have demonstrated that is natural environments, Bt strain producing Non-insecticidal paraspporal inclusion are more widely distributed than insecticidal ones.[Ohba & A izawa 1986;Hastowo et al 1992; Meadows et al.1992; Ohba 1996,1997; Roh et al 1996]. The non-isecticidal parasporal inclusions have any biological activity as yet or not.Although antibacterial activity has recently has been demonstrated in inclusion proteins of some non-insecticidal strains of Bt [Burtseva 1997] Mizuki and is co-worker reported a new Bt toxin that excreted cytotoxic activity to some cultured human cancer cells from non-insecticide & non-hemolytic Bt strain. It is here for the fist time that a unique activity ,such as anti-oxidant ,is associate with non-insecticde parasporal inclusion of certain Bt strains & 1.the spore crystal proteins have a potent anti-oxidant activity than the vegetative proteins,2.these proteins not having significant hemolytic activity.
METHODS
Bacterial Strains and Growth Conditions
The organism was grown at 28 ℃ on nutrient agar [pH 7.6]consisting of meat extract(10g),poly peptone (10g),sodium chloride(2g),agar(15g) and distilled water(1000ml) until sporulation was completed(approximately 48-72 hours).A loop full of sporulatedBt isolate was placed into 0.5ml of deionised water vortexes vigorously to disperse clumps and heated in water bath at 75 ℃ for 30 mints to kill vegetative and activate spores for germination. The 0.5ml activate spore aliquot was then transferred to 250ml of pre warmed and pre aerated nutrient broth medium at pH 7. This culture was incubated at 30℃ with constant shaking at 250 rpm until greater than 95% free phase bright spores were produced(approximately 48 hours).
Preparation of spore crystal mixtures from cultures of Bt isolates
1M of solid NaCl were added to sporulated nutrient broth cultures of Bt to induce lysis. The cultures were immediately shaken and cooled in ice. The cultures were centrifuged at 6000g for 10 mints at 4℃. The resultant spore crystal pellet was washed once with 1M NaCl,twice with ice cold distilled water and re suspended in an appropriate volume of Tris/KCl buffer. The spore crystal mixturewas aliquot and stored at -20 ℃ until further use.
Solubilization and activation of parasporal inclusion proteins
Parasporal inclusion proteins were harvested from cultures by solubilizing the spore crystal mixture in 50Mm sodium bicarbonate,10mMDTT,pH 10.5,for 1 hour. The insoluble spore and other debris were sediment by centrifugation at 13000g for 5 mints. The resultant pellet was discarded and the supernatant was then activated with proteolytic enzymes,1mg/ml of trypsin at the ratio of 1:10 proteolytic enzymes to parasporal inclusions for 1 hour at 37 ℃. This was followed by further centrifugation at 13000g for 5 mintsat 4℃ and the total dissolved protein in the supernatant was precipitated in 80% saturated solution of ammonium sulphate anhydrous over night with stirring at 40 ℃. The precipitated protein fraction was dialysed over night against two times distilled water 2L each at 4 ℃.
Purification of vegetative proteins
The vegetative proteins were harvested using the following method including precipitating the vegetative proteins from the nutrient broth supernatant using 70% ammonium sulphate. The precipitated proteins were resuspended in an appropriate volume of 20mM Tris /KCl (pH 7.5) buffer and dialysed overnight at 4 ℃. The partially purified protein stored in deep freezer. All bacterial work was done using sterile techniques under laminar air flow and all buffer solutions were autoclaved before addition of the vegetative proteins.
SEPARATION OF TOTAL PROTEINS BY SALTING OUT METHOD:
Ammonium Sulphate Precipitation
250ml of 24 hours culture of uropathogenicE.coli and normal E.coli was centrifuged at cooling centrifuge for 10,000 rpm for 5 mints. 200 ml of supernatant was taken in the beaker was kept in the ice bath at 4℃. Required amount of powder ammonium sulphate was added. The salt was added slowly with stirring by magnetic stirrer over a period of 30-40 mints and allowed to stand for 30 mints. After 30 mints the solution was centrifuged at 10,000 rpm in cold centrifuge at 9 ℃ for 10 mints. The small amount of precipitate was formed; the precipitate was dissolved in small quantity of 10mm phosphate buffer pH 7.0. the precipitate was stored in refrigerator and used for further examination.
PURIFICATION OF PROTEIN BY DIALYSIS METHOD:
Dialysis
The solution containing the precipitate were dialyzed against 10mm phosphate buffer at pH 7 for 18 hours. A pretreated dialysis membrane was taken at one end it was clamped tightly from open end the fraction precipitate solution was added in to membrane then the open end was closed tightly using lamps. The dialysis membrane bag was kept in the beaker containing 10mm phosphate buffer (pH 7.0) and stirred by magnetic stirrer. The buffer was then tested from ammonium sulphate every one hour initially and then half an hour by nesslers reagent. Nesslers reagent that gives brown colour with ammonium sulphate and buffer was change continuously by changing with buffer solution. After 7-8 hours the equilibrium was achieved when adding dialyzed reagent did not see the brown colour. The dialyzed bag was removed and the partially purified enzymes was used for further studies.
ESTIMATION OF PROTEINS BY LOWRY’S METHOD:
Reagents
Solution A : (100 ml)
0.5g of Copper sulphate pentahydrate
1g of Disodium citrate dehydrate
Add distilled water to 100ml
Solution B: (1L)
20g of sodium bicarbonate
4g of sodium hydroxide
Add distilled water to 1L
Solution C51ml)
1ml solution A
50ml solution B
Solution D20ml)
10ml Folin-ciocalteau reagent
10ml of distilled water
STOCK SOLUTION:
10g of bovine serum albumin was dissolved in 100ml of distilled water.
WORKING STANDARD:
1ml of stock solution was made to 10ml with distilled water (100g/ml)
PROCEDURE:
Different concentrations of working standard was taken and standard graph is plotted. The sample solution was diluted to 2 ml with distilled water, 2.5ml of solution C was added. The solution was vortexes and allowed to stand at room temperature for 5-10 mints. 0.25ml of solution D is added and vortexes. After 20-30 mints, absorbance was read at 750nm.
SDS POLYACRYLAMIDE GEL ELECTROPHORESIS:
Proteins present in the toxin were analyzed by SDS-PHASE according to Lammelie(1970) with same modification. Electrophoresis was peformed at room temperature at an initial voltage of 50mv and later changed to 100mv when dye reached separating gel. When the Bromophenol blue reached 1cm from the bottom of the gel, electrophoresis was stopped. The gel was removed carefully from the glass plates and fixed in 10% Trichloroacetic acid for half an hour. It was then soaked staining solution after 1 hour the gel was rinsed briefly in water to remove excess stain and immersed in distaining solution. Detaining stopped when the gel background was colourless.
HAEMOLYTIC ASSAY:
Haemolytic activity of the vegetative and spore inclusion protein were assayed by the quantity of haemoglobin released from the cytoplasm of erythrocytes. Various concentrations of vegetative proteins were incubated with 2%v/v human or rabbit erythrocytes for 1 hour at 27 ℃. The absorbance of the resultant supernatant at 550nm was measured and compared to the absorbance of 100% lysis to determine the percentage of haemolysis induced by the vegetative proteins. Erythrocytes incubated in protein buffer and protease inhibitors, excluding the vegetative proteins were
used as negative controls. All experiments were performed in triplicates.
Percentage of haemolysis = (Absorbance measured)/(Absorbance of total haemolysis)×100
ANTIOXIDANT ACTIVITY:
Determination of scavenging activity against hydrogen peroxide
The hydrogen peroxide scavenging activity of extract was determined by the method of Ruch et al. The different concentrations of spore protein and vegetative proteins was dissolved in 3.4ml of 0.1M phosphate buffer solution with pH 7.4. It is mixed with 600µl of 43mMsolution of hydrogen peroxide. After 10 mints the absorbance of reaction mixture was determined at 230nm. The reaction mixture without sample was used as blank. Ascorbic acid was used as a reference compound. The percentage inhibition activity was calculated as:
Percentage inhibition =((Absorbance of control)-(Absorbance of sample))/(Absorbance of control)×100
RESULT AND DISCUSSION:
PURIFICATION OF PROTEIN BY DIALYSIS METHOD
The isolated vegetative and spore crystal proteins from Bt were purified by dialysis method.the purified proteins used for further protein estimation and biological activity.[Fig.1]
ESTIMATION OF PROTEIN BY LOWERY’S METHOD
The concentration of protein in the crude extract of the Bt of the vegetative and spore crystal protein was found to be 1700µg/ml and 3100µg/ml respectively.[table 1,graph 1]
HEAMOLYTIC ASSAY
The heamolytic activity of the vegetative and spore crystal protein was performed.these vedetative proteins shows heamolytic activity then spore crystal protein.[Table 2,Graph 2]
SDS POLYACRALAMIDE GEL ELECTROPHORESIS
Protein present in the toxin was analysed by SDS-PHASE according to Lammeli (1970) with same modification.
Lane 1 M-Marker protein (molecular weight 14-97)
Lane 2- vegetative protein
Lane 3- spore crystal protein[Fig.3]
ANTI-OXIDANT ACTIVITY
The spore crystal protein from Bt shows more antioxidant activity than vegetative protein.[Table 3,Fig 5,Graph 3]
SUMMARY AND CONCLUSION :
Bacillus thuringiensis, the most succeeessfully and most widelyused microbial insecticide produces crystal proteins. The physiological significants of the crystal proteins its poorly understood except for the potent insecticidal activity. In the last five years, human cancer cell-killing Bt toxins, parasporin were discovered and they are characterized at molecular level.
In this study,an attempt was made to isolate vegetative protein and spore crystal proteins from the non-insecticidal bacterial species Bt NCIM2514. Then the vegetative crystal protein content of the crude extract was found to be 3100µg/ml and 7200µg/ml respectively according to Lowery’s method.
The crude protein was then characterized by performing SDS Polyacrylamide gel electrophoresis having concentration of 12% with the aim to characterize low molecular weight of the proteins 14-97 confirm the presence of different molecular weight proteins in crude extracts.
The activities studies were formed with the crude proteins and were found that the spore crystal contain more anti-oxidant when compared to vegetative proteins.
Haemolytic assay was also carried out with the crude proteins and it was found that these proteins not having significant haemolytic activity.
From these above studies we have concluded that the spore crystal proteins have potent anti-oxidant activity than the vegetative proteins.
In future, work has to be done to purified & isolate the individual protein by column chromatography, followed by HPTLC and mechanism of action of proteins is also necessary to study.All the above techniques may lead to the development of a wonderful new protein drug in future from Biotechnology.