The effect of pH on amylase activity

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The effect of pH on amylase activity
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How does the degree of acidity / alkalinity
affect the rate of enzymatic reaction ?
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Germinating Mung bean source of the enzyme
AMYLASE
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• Amylase is
• a digestive enzyme that helps break down starch
• found in (a) saliva
• (b) some digestive
• juices

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Sprouting mung beans (just remove the seed coat).
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Removal of seed coat dont throw them into the
sink
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Extract amylase from the mung bean seedling.
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Press on the seedlings to release(??) cell sap.
Grind for a few minutes. Sit down, hold the
mortar and pestle tight.
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Mortar
Pestle
Making an enzyme extract use only a few ml of
water for grinding
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Pour the enzyme extract into a clean test tube.
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Agar dissolves in hot water
Preparing for the starch agar suspension
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Petri dish
Solidified starch agar
Starch(substrate) is mixed with agar (a jelly
like semi-solid)
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Enzyme Amylase
Substrate Starch
product Maltose ???
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Chemical in reagent bottle with dropper
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Variable - pH
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Which chemical provides for an acidic medium ?
Hydrochloric acid
Which chemical provides for an alkaline medium ?
Sodium carbonate
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Preparing for the reaction mixture
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C3
C1
C2
C5
C6
C4
Into each of six cavities, Prepare a mixture of
enzyme pH medium
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condition content
C1 Enzyme extract (2 drops) Distilled water (1 drop)
C2 HCl (3 drops)
C3 Dilute Na2CO3 (3 drops)
C4 Enzyme extract (2 drops) HCl (1 drop)
C5 Enzyme extract (2 drops) Dilute Na2CO3 (1 drop)
C6 Distilled water (3 drops)
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Clearly label each of the conditions !!!
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Dropper Hold it vertical !
White cavity tile
Or use spotting tile
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Enzyme extract
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Get the top portion of the mung bean ?? enzyme
extract by using a clean dropper.
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Make sure that each drop of chemical is of the
same size. Why is this important ?
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Soak the paper disc into the reaction mixture for
at least one minute.
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Put six labels on the side of the Petri dish,
in sequence.(Not on the lid ?)
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condition content
C1 Enzyme extract (2 drops) Distilled water (1 drop)
C2 HCl (3 drops)
C3 Dilute Na2CO3 (3 drops)
C4 Enzyme extract (2 drops) HCl (1 drop)
C5 Enzyme extract (2 drops) Dilute Na2CO3 (1 drop)
C6 Distilled water (3 drops)
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Label the six positions on the starch-agar
plate. Soak the 6 paper disc(s) into the 6
respective solutions. Wait for 2
minutes. Transfer ?? the disc(s) onto the
starch-agar plate by using forceps.
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The set-up just before putting into the incubator.
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Incubator ???
Label your name
Incubation temperature at about 37 deg C
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Use brown Iodine solution to test for starch.
Pour in a little to cover the surface.
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Flush away excess Iodine immediately be gentle
!!!
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Look against light
Measure the diameter of the clear zone and write
down the degree of clearing
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Put the plate against a light source to record
results
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For each condition, measure (1) the diameter of
the clear zone, and (2) how clear the spot is.
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A clear zone shows that starch has been digested
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What are your experimental findings ? What
conclusion can you draw ?
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Answer No. 7 Because this is the optimal
temperature of amylase
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1. Pour away Excess Iodine solution
2. Gently wash away the remaining Iodine solution

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At 100 deg C, Enzymes are denatured, clear zones
will not be seen.