THEORY

Enzymes are protein molecule and it act as biological catalyst. To act as biological catalyst, it
will increase the rate of reactions without changing the overall process. Enzyme is made by long
amino acid chain that bound together with peptide bond. Enzyme is a living cell and it controls
the metabolic processes in which they will convert nutrients into energy and new cells. Enzyme
helps the breakdown of food material into it’s simplest form and the reactant of enzyme is known
as substrate. Each enzyme is quite specific in character and they act on a particular substrates to
produce a particular products. To determine the rate of reaction and its changes in response, the
central will approach for studying the mechanism of an enzyme-catalyzed reaction with the
changes in parameters such as substrate concentration, enzyme concentration, pH, temperature
etc. This is also known as enzyme kinetics.

Substrate concentration [S] is one of the important parameters that affect the rate of a reaction
catalyzed by an enzyme. The initial velocity, Vo gradually increase during enzyme substrate
reaction with increasing in concentration of substrate. When a graph of substrate concentration
on the X axis is plotted correspond to velocity on Y axis. The observation that can be observed
from the graph is as the concentration of substrate increases, the correspond of Vo will increase.
However, the velocity remained constant without any further increase beyond a particular
substrate concentration. The maximum velocity of an enzyme catalysed reaction under substrate
saturation is called as maximum velocity, Vmax.
Michaelis- Menten Equation

Leonor Michaelis and Maud Menten had postulated that to form an enzyme-substrate complex I
a relatively fast reversible step, enzyme first need to combine reversibly with its substrate.

k1
E+S ES (Eq. 1)
k-1

In the next step, this ES complex will break down into the free enzyme and the reaction of
product P,

𝑘2
ES→ E + P (Eq. 2)

Since the second step is the rate limiting step, the rate of overall reaction must be proportional to
the concentration of the ES that reacts in the second step. The relationship between substrate
concentration, [S] and initial enzyme, Vo has similar shape with most enzymes which is a
rectangular hyperbola. This relationship can be expressed algebraically by Michaelis-Menten
equation and based on the hypothesis for rate limiting step in enzymatic reactions, they can
breakdown the ES complex to free enzyme and product. The derived equation from Micahelis-
Menten is,

𝑉𝑚𝑎𝑥 [𝑆]
Vo= (Eq.3)
𝐾𝑚 +[𝑆]
OBJECTIVES

There are a few objectives that need to reach in this experiment which are,

 To determine the effect of temperature on the enzymatic activity and changes in enzyme
concentration of an enzyme-catalysed reaction.
 To describe the relationship between substrate concentration and the maximum velocity
of an enzyme.
 To estimate the Michaelis-Menten parameters, effect of pH and temperature on enzyme
activity and kinetics of inhibition.
EXPERIMENTAL PROCEDURE

i. Preparation of 2% Starch Solution

1. A weight of 4 g of soluble starch is mixed in approximately 50 ml of cold water.
2. The slurry is added to approximately 100 ml of boiling water while stirring it gently in a
large beaker.
3. A volume of 200 ml is top up to final value and mixed well.

ii. Effect of pH on the activity and stability of amylase enzyme.

1. Five test tubes is labeled with pH 5,6,7,8,and 9. In each test tube, 1ml of 2% starch
solution is placed and 1ml of appropriate buffer (at corresponding pH) is added to each
tube.
2. Get five additional clean test tubes and 2 ml of amylase solution is added in each test
tube.
3. All 10 tubes is placed in the 37oC water bath for about 5 minutes to allow the temperature
to equilibrate.
4. The content of each amylase test tube is poured into each starch test tube and mixed on
vortex mixer.
5. The tubes are returned to the 37oC water bath.
6. Let the hydrolysis reaction proceed for exactly 10 minutes.
7. The amylase activity is determined using the method given in Appendix 1.
8. A graph of pH vs. enzyme activity is plotted.

iii. Effect of temperature on the activity and stability of amylase enzyme.

1. One test tube is labeled with 30 oC. In the tube, 1 ml of 2% starch solution and 1 ml of
pH7 buffer is placed to the tubes.
2. Get additional clean test tube and 2 ml of amylase solution is put in the tube.
3. Both tubes are placed in the water bath for about 5 minutes to allow temperature to
equilibrate.
4. The contents of the amylase are poured into starch test tube and mixed on vortex mixer.
 5. The tubes are returned to the 30 oC water bath.
6. The hydrolysis reaction is proceeds for exactly 10 minutes.
7. The amylase activity is determined using the method given in Appendix 1.
8. Step 1-7 is repeated at 4 different temperatures ranging from 30-70 oC.
9. A graph of temperature vs. amylase activity is plotted.

iv. Effect of substrate concentration on the activity of amylase enzyme.

1. Starch solutions of varying concentration (0.5, 1.5, 2.0, 2.5, and 3.0% w/v) are prepared
as the substrates.
2. Each tube with starch concentration is labeled and 1 ml of each starch solution is placed
into the test tube.
3. 1 ml of pH7 buffer is added to the tubes.
4. Get five additional clean test tubes and 2 ml of amylase solution is added in each test
tube.
5. The contents of the amylase are poured into starch test tube and mixed on vortex mixer.
6. The tubes are returned to the 37oC water bath.
7. Let the hydrolysis reaction proceed for exactly 10 minutes.
8. The amylase activity is determined using the method given in Appendix 1.
9. A graph of starch concentration vs. amylase activity is plotted.

Appendix 1 (Demonstration of Enzyme Activity)

1. After 10 minutes (the time of hydrolysis reaction), the reaction is stop by adding 4 ml of
DNS reagent.
2. Boil for 10 minutes and left to cool to room temperature.
3. The absorbance of the samples are measured at λ=540 mm.
4. The absorbance value is compared with glucose standard cuve prepared to obtain the
glucose concentration.
5. The enzyme activity is calculated.
Note: Enzyme activity is the amount of glucose formed in reaction mixture per unit time.
Appendix 2 (Glucose Standard Curve Preparation)

1. Standard solutions of glucose ia prepared at five different concentrations ranging from 0-

2000 mg/L by serial dilution.
2. Put 1 ml of each glucose solution in test tubes.
3. 1 ml of DNS reagent is added in each tube and mixed for a few seconds on vortex mixer.
4. The test tubes are placed in water bath (T= 100 oC) for 10 minutes and the left to cool at
room temperature.
5. The absorbance of the samples are measured at λ=540 mm.
6. The standard curve of absorbance and glucose concentration is plotted.
Note: The graph is in straight line for absorbance less than 0.7.