Enzyme Kinetics
Rain Shokralla
October 11, 2024
Title : Enzyme Kinetics
Aim : To check the effect of Concentration and pH, on the activity of salivary Amylase.
Hypothesis : Salivary Amylase enzymes increase the time of reaction decreases. Amylase is
active at its optimum pH and will have reduced activity in other pH buffers.
Material and Methodology :
Test-‐tube rack
1 calibrated 1-‐ml pipette
10 standard test tubes
2 calibrated
5-‐ml pipettes
wax pencil disposable Pasteur pipettes
test plate pipette bulb
flask of distilled or DI water
buffer solution (pH = 6.8)
beaker distilled rinse water
1% starch solution
5-‐ml graduated cylinder
1% amylase solution
Enzyme Kinetics
Rain Shokralla
October 11, 2024
General introduction: Enzymes are biological catalysts which speed up chemical reactions in
organisms. Proteins in enzymes lower energy production for a reaction to occur. This would help
the Amylase enzymes become reactive to the substances mixed in with the substrates. Substrates
are specific reactants that enzymes react to. As a result, the substrate combines to the enzyme's
active site. In addition to this, active sites are specific regions on enzymes acted upon. The
model and table created below reveal how enzymes and substrates interact.
Specific introduction: Enzyme and Reaction:
● Enzyme: Amylase
● Reaction Catalyzed: Amylase catalyzes the hydrolysis of starch, breaking it down into
simpler sugars like maltose and glucose.
Substrate and Product:
Enzyme Kinetics
Rain Shokralla
October 11, 2024
● Substrate: Starch (a polysaccharide)
● Products: Maltose (a disaccharide) and glucose (a monosaccharide)
Experiment Overview:
Preparation of Dilution Series: Students set up a dilution series of amylase and took the
initial concentration (0.5% in Tube 1). As a result, students prepared dilutions to get
lower concentrations (0.25%, 0.125%, 0.063%, and 0.031%) in Tubes 2 through 5.
Mixing: In each tube, mix the amylase solution with a buffer or distilled water to achieve
the desired concentrations
Purpose: The purpose is to evaluate the effectiveness of amylase at various
concentrations on starch, which is monitored using iodine to check for the presence of
starch.
Conclusion:
This setup allows you to observe how the concentration of amylase influences the rate of starch
breakdown, providing insights into enzyme kinetics and the enzyme-substrate interaction.
Principle :
– Tube 1: Add 5 ml amylase and mix with pipette (dilution 1:1; 0.5% amylase)
– Tube 2: Add 5 ml amylase solution from tube 1 and mix (dilution: 1:3; 0.25%
amylase)
– Tube 3: Add 5 ml amylase solution from tube 2 and mix (dilution 1:7; 0.125%
Enzyme Kinetics
Rain Shokralla
October 11, 2024
amylase)
– Tube 4: Add 5 ml amylase solution from tube 3 and mix (dilution 1:15; 0.063%
amylase)
– Tube 5: Add 5 ml of amylase solution from tube 4 and mix (dilution 1:31; 0.031%
amylase)
– In solution iodine reacts with starch to give an intense blue-violet complex.
Enzyme Kinetics
Rain Shokralla
October 11, 2024
Amylase Test (Starch iodine method) :
Effects of concentration of Amylase on the rate of reaction :
Lower amounts of Amylase increases the rate reaction and causes a faster breakdown of
starch. The text “The Effect of the Concentration Amylase on the Rate of Breakdown of Starch”
states, “If you double the amount of amylase, the breakdown of starch will be quicker because
there are twice as many active sites. Enzymes are made of long protein molecules, which are
folded up so they have an irregular globular shape.” (Bartley Research). The reaction time is
measured by observing and calculating the time it takes for starch to react. Furthermore, using
iodine as an indicator can help reveal the process of the substrate enzymes combining. For
example, when starch is placed in the test tubes, the iodine will turn the substance from yellow to
blue-black. As a result, the starch is broken down, the yellow color fades, and overall indicates a
higher reaction rate.
Amylase concentration : Time (Sec) :
0.50 20 sec
Enzyme Kinetics
Rain Shokralla
October 11, 2024
0.25 20 sec
0.125 30 sec
0.063 50 sec
0.031 90 sec
Enzyme Kinetics
Rain Shokralla
October 11, 2024
Literature Cited :
The effect of the concentration amylase on the rate of breakdown of starch. Bartleby. (n.d.).
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