The effect of pH on amylase activity HO KP (2008). How does the degree of acidity / alkalinity affect the rate of enzymatic reaction ?. pH. Germinating Mung bean – source of the enzyme AMYLASE. Amylase is a digestive enzyme that helps break down starch found in (a) saliva
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HO KP (2008)
How does the degree of acidity / alkalinity affect the rate of enzymatic reaction ?
Sprouting mung beans is a rich source of amylase and other enzymes. (Shall we remove the seed coat ?)
Sit down, hold the mortar and pestle tight.Press on the seedlings to release (釋放) cell sap. Grind for a few minutes.
Make an enzyme extract – use only a few ml of water for grinding
Get the top portion of the mung bean 綠豆enzyme extract by using a clean dropper.
Filtering may be done by a muslin cloth.
Less than a few ml of extract is required.
dissolves in hot water
Preparing for the starch - agar suspension
Solidified starch agar
Starch(substrate) is mixed with agar (a jelly like semi-solid)
不是 substance, substrate 指 enzyme reaction 的 reactant.
Which chemical provides for an alkaline medium ?
Into each of six cavities,
Prepare a mixture of “ enzyme + pH medium ”
White cavity tile
Or use a spotting tile
Why is this important ?
Use clean dropper.
Do not dip the forceps into the reaction mixtures. If so, clean the forceps every time !
Put six labels on the side of the Petri dish, in sequence.(Not on the lid 蓋)
At this stage, the “ enzyme - soaked paper discs ” may be put onto the starch agar plate by a pair of clean forceps.
Label the six positions on the starch-agar plate.
Transfer轉移 the disc(s) onto the starch-agar plate by using cleanforceps.
Wash the forceps after every transfer to avoid contamination.
The set-up just before putting into the incubator. Remember to label the group name on the lid.
Label your name
Incubation time : 60 – 120 min.
Incubation temperature – at about 37 deg C
after the plate
has been put
Clearly label each of the conditions Find out the pH of each mixture !!!
Use brown Iodine solution to test for starch. Pour in a little to cover the agar surface.
Measure the diameter of the clear zone and write down the degree of clearing
For each condition, measure (1) the diameter of the clear zone, and (2) how clear the spot is.
What conclusion can you draw ?
The diameter of the clear zoneindicates the degree of enzyme activity.
The diameter of the clear zone in
C5> C1 > C3 > C4 > C2 > C6
Comparing C1 and C6,
It shows that mung bean extract[with enzyme(s)] can digest starch on the agar plate but distilled water alone can not. (If the tap water is slightly alkaline, a little digestion may occur, or the effect is due to contamination.)
Comparing [treatments]C3 and C5, it can be concluded thatdigestion of starch is higher when the enzyme(s) work(s) under an alkaline pH, rather than with an alkali alone.
C6 is a control experiment to let us know that starch does not break down by itself.
The enzyme (or enzymes) in mung bean extract works better under an alkaline condition rather than an acidic condition. Acid inhibits抑制 the activity of amylase.
It is to provide a suitable (or best / optimal) temperature for the enzyme.
At 100 deg C,
Enzymes would be denatured, clear zones could not be seen.
The pH of the reaction medium affects enzyme activity – amylase works better under an alkaline condition.