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Exp:no:2 Extraction of enzyme from bacteria

Two ml of production media culture was transferred into centrifuge tubes and spinned for 20 minutes at 5000 rpm. After 20 minutes, the supernatant portion was decanted, which is the crude enzyme extract.

Demonstration of enzyme activity


One ml of crude enzyme extract from all conical pipetted out into a test tube. To this 1 ml of 1% soluble starch solution was added. The solution was mixed by swirling and incubated for exactly 3 minutes at 20 C. then colour reagent solution, [Solution 1) 12 gms of Sodium potassium tartarate was dissolved in 8 ml of 2M NaOH, by direct heating and constant stirring. Solution 2) 0.1 m of 3,5-dinitro salicylic acid solution was dissolved in 20 ml of deionized water by direct heating and constant stirring. Solution 1 was added slowly with constant stirring to solution 2.The solution was dissolved to 40 ml with deionized water. The solution should be stored in an amber coloured bottle at room temperature] 1 ml was added to all test tubes. The test tubes were capped and placed in a boiling water bath for exactly 15 minutes and cooled on ice to room temperature. Finally add 9 ml of deionized water, mixed well by inversion and determine the colour intensity at 540 nm, using suitable spectrophotometer against blank. The results were tabulated. Or DNS can be prepared as mentioned below : *DNS reagent (1% 3, 5, dinitrosalicyclic acid, phenol 0.2%, sodium carbonate 0.05%, 1% of NaOH and 20 % of sodium potassium tartarate in dis. water)

Blank solution
One ml of 1% soluble starch solution, one ml of distilled water and one ml of color reagent solution heated for 15 minutes, cooled on ice to room temperature. Then 9 ml of deionized water was added.

Amylase assay
Amylase was assayed by adding 0.2 ml of enzyme (crude extract/fermented broth supernatant) to 0.5 ml of 1% soluble starch and incubated for 30 min at 37o C. The reaction was stopped by adding 1 ml of 3, 5 dinitrosalicylic acid, followed by boiling for 10 min and to develop brown color. The final volume was made to 5 ml with distilled water and the absorbancy measured at 540 nm with a spectrophotometer

Determination of enzyme activity Units/ml enzyme = (g of maltose realeased) (f) 1 f dilution factor 1 = volume (in ml of enzyme used)
A calibration curve of absorbancy and concentration of maltose was established with known amount of maltose. One unit (U) of amylase was defined as the amount of enzyme that liberates one Mole of reducing sugars, measured as maltose per min under the conditions of assay.

REFERENCES Abe, J., Bergman, F.W., Obata, K. and Hikuri, S. (1988). Production of raw starch digesting amylase by Aspergillus K-27. Applied Microbiology and Biotechnology 27, 447-450 Bernfeld, P. (1951). Enzymes of starch degradation and synthesis. Advances in enzymology 12, 379-481 Brook, Elizabeth J., Stanton, W.R. and Wallbridge, Ann. (1969). Fermentation Methods for protein enrichment of cassava. Biotechnology & Bioengineering. 11:1271-1284 Rosario, E. J., and Wong, R. L. (1984). Conversion of dextrinized cassava starch to ethanol using cultures of Aspergillus awamori and Saccharomyces cerevisiae. Enzymes and Microbial Technology, 6: 60-64

Dialysis The resuspended precipitate was dialysed extensively using 0.2M sodium phosphate buffer (pH 7.0). Reagents 1. Bicarbonate solution: 100mM Sodium bicarbonate in 1mM EDTA 2. Sodium phosphate buffer, 0.2M, pH 7.0: Thirty nine millilitre of 0.2M Disodium hydrogen phosphate is added to 61.0 ml of 0.2M Sodium dihydrogen phosphate and the final volume was made up to 200 ml with distilled water. The dry dialysis membrane was activated prior to use by rinsing it well in double distilled water and heated to 60C in bicarbonate solution for 15 min. The membrane was then cooled, rinsed in double distilled water. The ammonium sulphate precipitation fractions were loaded in separate membrane tubings. The contents of the tubings were dialysed against 0.2M sodium phosphate buffer (pH 7.0) under constant agitation using a magnetic stirrer. The dialysis buffer was replaced every three hours and the whole process was repeated thrice. The protein content and the amylase activity in the dialysate were determined.

Gel filtration
The protein content in the fraction was determined by measuring the absorbance at 280 nm. The peak fractions were then subjected to enzyme assay. The protein content of the fractions was also estimated. The fractions with enzyme activities were pooled.

Result:
Purification The cell free supernatant was considered as the crude enzyme source and was subjected to various steps of purification. The enzyme activity in the crude source was ???????????. Ammonium sulphate precipitation was the first strategy adopted to purify protease and lipase. Ammonium sulphate purification is a salting out method of purifying a protein by altering the solubility of the protein. Gradient ammonium sulphate fractionation was carried out. Amylase activity was found in the precipitates obtained with 40 60% saturation of ammonium sulphate ???????????/. The enzymes were purified 1.6 fold by this purification step. The specific activity of amylase was ?????????????????U/mg. Nearly 50% of the proteins present in the crude cell free supernatant was precipitated by this salting out process.

The precipitates obtained by ammonium sulphate fractionation were resuspended in 0.2M sodium phosphate buffer, pH 7.0. The resuspended precipitates have ammonium sulphate salt bound to the protein. These excess salts were removed by dialysis. Extensive dialysis of the pooled resuspended precipitates increased the purity of the isolated enzymes. The protease had 3.4 fold purity ??????????? when compared to the crude enzymes. The dialysed enzymes were loaded on a Sephadex G-75 gel filtration column for further purification. Enzyme activity peaked in the ????????????? th fraction while peak amylase activity was observed in the????????????????? th fraction. The fractions ????????????? which exhibited high amylase activity was pooled . The homogeneity of the purified enzyme mixture was studied using SDS-PAGE. The crude enzyme source, namely the cell-free supernatant showed ???????????????? bands on the gel while the partially purified enzyme mixture obtained by ammonium sulphate precipitation and dialysis exhibited three prominent bands. The completely purified enzymes obtained following gel filtration exhibited distinct bands on the gel. Thus purification had removed all protein contaminants that were present in the cell free supernatant and the enzyme mixture was purified to homogeneity.

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