Isolation of amylase from soil bacteria

Isolation of amylase producing bacteria from soilBy:

Bhagyashree Ganatra

Under guidance of Mr.Uttam Bodake

Department of Biotechnology

Fergusson college, Pune.What are amylases? Amylases are enzymes that break down starch or glycogen.

Amylases are produced by a variety of living organisms, ranging from bacteria to plants and humans.

Bacteria and fungi secrete amylases to the outside of their cells to carry out extra-cellular digestion. When they have broken down the insoluble starch, the soluble end products such as (glucose or maltose) are absorbed into their cells.

What organisms are responsible for amylase production?

Although many microorganisms produce this enzyme, the ones most commonly used for their industrial production are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens and Aspergillus niger General Lab Requirements : Autoclave or pressure cooker Nutrient Agar powder Soluble starch Weighing scales Spectrophotometer or colorimeter Water bath (Temperature controlled)

Materials required : Sterile pipettes (One each of 10 mL, 5 mL and 1 mL) Sterile Glass Petri dishes Wire loop Bunsen burner and matches 95% ethanol. Isolation procedure : Sweep off the debris from the top of the soil, (about 10 grams), into a "Ziploc" bagSuspend about 1 gram soil in 10 mL sterile distilled water, mix properly Allow the soil to settle down.Collect the supernatant in another test tube.Meanwhile, prepare 5 tubes containing 5ml sterile water.

vi. Pipette 0.5ml of the supernatant in the sterile water tubes.

vii. Put the tubes in a beaker on a water bath and let it boil for a few minutes.

viii. Streak on plates containing nutrient agar medium and keep for incubation at room temperature for 24hours.

ix. Starch-producing colonies will have an area of clearing around them.

x. Confirmed by flooding plates with Grams Iodine.

Amylase producing bacteriaxi. Calculate S/R ratio from each plate.

xii. The Selection Ratio is calculated by the given formula : S/R ratio = Diameter of zone of clearance in mm/ Diameter of colony in mm

xiii. The plate having the colony with highest S/R ratio is the best for amylase activity and selected for further use.

xiv. Prepare nutrient broth containing 1% starch. Autoclave for 15mins at 121C.xv. Transfer the colony with highest S/R ratio in the nutrient broth.

xvi. Incubate the above at room temperature for 24hrs.

Xvii. Once growth is observed, centrifuge at 7000rpm for 10mins.

Xviii. The supernatant is the crude amylase. The precipitate is further added in a 100ml flask containing Ammonium Sulphate (76.8gms)

xix. The precipitate obtained from above is mixed with water. This is purified amylase solution.Enzyme assay :

The amylase activity was assayed by DNSA method(Plummer, Practical Biochemistry, 2005) Three tubes were prepared. Tube 1 containing only DNSA reagent was used as blank. Tube 2 contained DNSA reagent + starch + pure amylase Tube 3 contained DNSA reagent + starch + crude amylaseOptical density was checked at 540nm.

O.D was plotted against standard maltose graph and amylase activity was assayed.OBSERVATIONS AND RESULTSOIL SAMPLEDIAMETER OF THE ZONE (mm)DIAMETER OF THE COLONY (mm)SELECTION RATIOSAMPLE 11853.60SAMPLE 21762.83SAMPLE 32054.00SAMPLE 41753.40SAMPLE 51662.66TABLE 1. CALCULATION OF SELECTION RATIOENZYMEO.D. AT 540nmPURE AMYLASE0.493CRUDE AMYLASE0.613TABLE 2. OPTICAL DENSITY OF AMYLASE AT 540nmCALCULATIONS342.3g/mol of maltose in 1lit water = 1M342.3g maltose liberates = 1 unit of enzymeTherefore, 4.7g will liberate = 4.7/342.3 = 0.014 units of crude enzyme and3.8g will liberate = 3.8/342.3 = 0.011 units of pure enzyme.0.014units/ml of crude enzyme is liberated. Hence for 100ml = 0.014x100 = 1.4units0.011 units/ml of pure enzyme = 0.011x100 = 1.1 units.

TABLE 3.DISCUSSION AND CONCLUSIONA total of 40 bacterial strains which produced clear zones in the starch- nutrient agar medium were isolated and purified. Among the 40 bacterial strains, one strain was selected as best amylase producer. As the strain was able to produce maximum amylase activity at 24 h, its activity was assayed using the DNSA reagent and the concentration of enzyme was evaluated using the standard maltose graph.

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