Practical 4: ENZYMES

This summary presents the practical section on how to understand the enzyme properties in reactions
and the effect of temperature on activity of enzyme.

1. AMYLASE:
- Belong to the hydrolase group enzyme.
- Is an enzyme that catalyses the hydrolysis of starch into sugars (act on α-glycosidic bonds of
starch).
- Found in animal saliva, pancreas, intestine…, and in sprouting seeds.
- There are 3 types of amylase:

• α-amylase acts on 1,4-α-D-glucan-glucan glycoside

• β-amylase acts on 1,4-α-D-glucan-malto glycoside linkage

• γ-amylase acts on glucan 1,4-α-glucoside and glucan 1,6-α- glycoside linkages

- Are heat sensitive (affected by heat).

Task 1 - The effect of different temperatures on the activity of amylase:

40-60 green bean sprouts + 20 ml water and blend till homogenous -> filter this mixture and collect the
aqueous phase -> prepare 8 test tubes and mark them as 4-S, 4-E, RT-S, RT-E, 50-S, 50-E, 100-S, 100-E ->
put 2ml amylase suspensions into 4 oC “E” tube, RT “E” tube, 50 oC “E” tube, and 100 oC “E” tube
respectively -> incubate for 5 mins -> add into all test tubes 4 ml of starch suspension -> keep all
reactions for 10 mins in the same condition -> take tubes out -> add 1-2 drops of Lugol solution into
each tube and 2ml water into each “S” tube -> Mix well -> Observe the color of these tubes.

2. PROTEASE:
- Belong to the hydrolase group enzyme (because the enzymes use a molecule of water for
peptide cleavage).
- Are enzymes that break peptide bonds between amino acids of proteins.
- Occur naturally in all organisms and constitute 1-5% of the gene content.

Task 2 - The effect of different temperatures on the activity of protease:

Pineapple pieces + 15 ml water and grind till homogenous -> filter this mixture and collect the aqueous
phase -> put 5ml of pineapple extraction into 2 test tubes respectively -> tube 1 (marked 100) is then
put in boiling water for 15 mins, tube 2 (marked RT) left at room temperature -> add boiled white egg in
each tube -> incubate at room temperature -> check tubes after 2 days (compare 2 pieces of boiled egg
on a petri dish)

3. CATALASE:
- Belongs to the group of oxidoreductase.
- Is an anti-oxidant enzyme to convert peroxide into less toxic substances.
 - When the body is infected with high level of hydrogen peroxide (H2O2), the catalase stored in
peroxisome (in plant cell) or mitochondria (in animal cell) will be activated and contribute in the
process to convert this toxic compound to O2 and H2O.
- Found in plant, animal and microbial cells.
- Is heat inactivated.

Task 3 - The effect of different temperatures on the activity of catalase:

100g potato + 250 ml water and blend till homogenous -> filter this mixture and collect the aqueous
phase -> put 5ml of potato extraction into 4 oC tube, RT tube, 50 oC tube, and 100 oC tube respectively ->
incubate for 5 mins -> add 5 drops of hydrogen peroxide into each tube -> observe what happens in
each tube -> note down the time needed in each tube for the column of oxygen bubbles forming and
reaching to the marked line.