UNIKL

 MICET   1  
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EXPERIMENT  6:  MEASUREMENT  OF  ENZYME  ACTIVITY  

INTRODUCTION

Enzyme activity (reaction rate) is dependent upon the environment conditions either in nature or
in the laboratory (e.g. temperature, pH, etc). This is because these conditions can alter the amino
acid side chains in a protein, affecting protein structure and folding and sometimes the enzyme’s
active site. The effects of some of those conditions will be explored in this exercise. Just as in
any chemical reaction, the concentration of reactants (substrates) will affect enzymatic reaction
rates. In regards to substrate concentration, enzyme kinetics follows the Michaelis-Menten
model:
k1   k3  
E + S <--------->ES-------->E +P

k2  

Vo = Vmax x[S]/Km + [S]

Where: Km = Michaelis constant

[s] = substrate concentration

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We can estimate Km and Vmax from a curve shown above. However, Vmax is never really attained,
we can’t really measure it. Then, because we plot Km as Vmax/2, it too has error. Therefore, many
investigators have manipulated the Michaelis-Menten equation to give a linear plot. The most
common is the well-known Lineweaver-Burk plot with the equation below:

1 𝐾𝑚 1
=   +
𝑉 𝑉𝑚𝑎𝑥  [𝑆] 𝑉𝑚𝑎𝑥

Lineweaver-Burk plot

OBJECTIVES

• To determine the effect of substrate concentration on enzyme activity.

• To plot Lineweaver-Burk
• To determine the Michaelis constant (Km)

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PART I ENZYMATIC HYDROLYSIS

Materials and Apparatus

Starch solution

Amylase solution (1%)

0.2 M phosphate buffer, pH 7

Waterbath

Procedure

Effect of substrate concentration on the activity of amylase enzyme

a) Prepare a starch solution of concentration 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 %
(w/v) as the substrate. Place 0.5 ml of the substrate of various concentrations, amylase
and phosphate buffer (Table 1) into the test tubes.
b) Incubate the reaction mixture for 30 min at 30◦C with shaking of 150 rpm.
c) Determine the amylase activity using DNS method (Part II).

Table 1

Amylase Phosphate 0.25-3.0 %

Test tube no solution, 1% buffer (0.2 M), Starch solution,
(ml) ml ml
1 0.2 0.3 0.5 (0.25 %)
2 0.2 0.3 0.5 (0.5%)
3 0.2 0.3 0.5 (0.75 %)
4 0.2 0.3 0.5 (1.0 %)
5 0.2 0.3 0.5 (1.5 %)
6 0.2 0.3 0.5 (2.0 %)
7 0.2 0.3 0.5 (2.5 %)
8 0.2 0.3 0.5 (3.0 %)

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Part II: MEASUREMENT OF ENZYME ACTIVITY:DINITROSALICYLIC (DNS)
ASSAY

This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars.
This involves the oxidation of the aldehyde functional group present in, for example, glucose and
the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is
reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions:

oxidation
aldehyde group----------> carboxyl group

reduction
3,5-dinitrosalicylic acid ----------> 3-amino,5-nitrosalicylic
acid

Because dissolved oxygen can interfere with glucose oxidation, sulfite, which itself is not
necessary for the color reaction, is added in the reagent to absorb the dissolved oxygen. The
above reaction scheme shows that one mole of sugar will react with one mole of 3,5-
dinitrosalicylic acid. However, it is suspected that there are many side reactions, and the actual
reaction stoichiometry is more complicated than that previously described. The type of side
reaction depends on the exact nature of the reducing sugars. Different reducing sugars generally
yield different color intensities; thus, it is necessary to calibrate for each sugar. In addition to the
oxidation of the carbonyl groups in the sugar, other side reactions such as the decomposition of
sugar also compete for the availability of 3,5-dinitrosalicylic acid. As a consequence,
carboxymethyl cellulose can affect the calibration curve by enhancing the intensity of the
developed color.

Although this is a convenient and relatively inexpensive method, due to the relatively low
specificity, one must run blanks diligently if the colorimetric results are to be interpreted
correctly and accurately. One can determine the background absorption on the original cellulose
substrate solution by adding cellulase, immediately stopping the reaction, and measuring the
absorbance, i.e. following exactly the same procedures for the actual samples. When the effects

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of extraneous compounds are not known, one can effectively include a so-called internal
standard by first fully developing the color for the unknown sample; then, a known amount of
sugar is added to this sample. The increase in the absorbance upon the second color development
is equivalent to the incremental amount of sugar added.

Materials Required

1. Distilled water
2. Glucose
3. 3,5-dinitrosalicylic acid
4. Potassium sodium tartarate
5. Sodium hydroxide
6. Sodium carbonate
7. Measuring cylinders
8. A balance
9. Spatulas
10. 2 L beakers
11. Hydrolysate (From Part I)

Procedure:

1. Prepare the DNS reagent by dissolving 5g of DNS and 8g of NaOH in 100ml of distilled
water and top the volume to 300ml. Add 150g of potassium sodium tartarate slowly until
all the DNS dissolve and top up the volume to 500ml.
2. Add 1.5ml of hydrolysate (from Part I) in a test tube and add 1.5ml of the DNS reagent.
3. Boil the reaction mixture for 5 min and allow to cool down. Add 10ml of distilled water.
4. Read the absorbance at 540nm.
5. Prepare a standard curve for glucose using the concentration of pure glucose in the range
0-100mg/L by repeating steps 2-4.
6. Use the standard curve to determine of unknown glucose concentration.

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RESULTS AND DISCUSSION

1. Determine the velocity of each substrate concentration.

2. Plot velocity over substrate concentration
3. Determine Km.
4. Plot a Lineweaver-Burk equation
5. Explain how the factors affect the kinetics of the enzyme and why substrate concentration
would have minimal changes in velocity at zero order with respect to substrate
concentration.
6. How does Lineweaver-Burk plot be useful in explaining enzyme inhibition?

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