Isolation of amylase from starch

2. Extraction Of Starch From Sweet Potato
In Microbiology LAB
Procedure:
• Take the small pieces of sweet potato of 25g.
• Triturate in pestle and mortar by adding a small amount of
water.
• Filter the potato slurry with a muslin cloth into a beaker.
• Repeat the procedure 2 to 3 times.
• Leave the beaker for 30 minutes.
• Wait till the whiteish material settles down, it takes 20 to 30
minutes.

3. • Discard the decanted water.
• Shift the white retained substance (in the beaker) in the Petri dish.
• The white substance is potato starch, Dry the potato starch.
Identification
• Solubility test
Mixing the dried starch in 50 to 60 ml water, we observe that the starch
remains insoluble in the water until & unless heated and stirred.
• Iodine test
Take 1ml from the solution, and cool under the tap water the cloudy
mass appears. Add 1 to 2 drops of iodine to the solution, the blue color
will appear.

4. Composition Of Starch Agar
• Suspend 15 g of nutrient agar in 100 cm³ distilled water.
• Bring to a boil to dissolve completely.
• Heat 40 g of soluble starch in 100 cm³ of distilled water to form
a suspension.
• Allow to cool and then mix with the nutrient agar solution.
• Dispense and sterilize.

5. Isolation of Amylase Producing Microbes
Procedure:
• Collect soil samples from an
appropriate site.
• Add 1 gm of Soil to 10 ml of
distilled water containing test
tubes.
• Carry out serial dilutions.
• Soil suspension prepared in
10 ml of sterile distilled water 100

6. • Take 0.1 ml of sample from
selected dilutions.
• Spreading on starch agar medium.
• Incubate the plates for
24 hours at 370C.
• Different types of colonies appear.
• Next, the plates were flooded with
Gram’s iodine.
• Again Incubate the plates
for 24 hours at 370C.
• Zone of hydrolysis of starch appears
around the amylase-producing
microbes.

7. The method followed Bhaskara et al. (2011)12. The
three bacteria with the highest Halo: Colony ratios
were chosen for determination of amylase enzyme
activity. Each isolate was subcultured in starch
liquid broth (g/L): 10.0 soluble starch, 10.0 peptone,
20.0 yeast extract, 0.05 KH2PO4, 0.015 MnCl2.4H2O,
0.25 MgSO4.7H2O, 0.05 CaCl2.2H2O, 0.01
FeSO4.7H2O and incubated at 37 ºC, 150 rpm for 1,
2 and 3 days. The clear supernatant (crude
extracellular amylase enzyme) was obtained after
centrifugation at 10,000g for 15 min at 4 ºC. The
crude extract was concentrated using MWCO 10