CHE506 Lab 8 Lab Manual

Edited 13 June 2018

Experiment 8: Investigation on enzyme activity and kinetics

Objectives :

 To investigate the effect of pH and temperature on enzyme activity.

 To study the relationship between substrate concentration and the maximum
velocity of an enzyme.
 To estimate the Michaelis-Menten parameters

1. Introduction

Amylases are a family of enzymes that degrade starch (polymers of glucose) into
smaller disaccharides (maltose). A molecule of water is also split during this reaction
and the OH- and H+ ions bind to the exposed ends of the broken starch polymer. This
type of reaction is called hydrolysis (water splitting).Hydrolysis is a common mechanism
used by enzymes to break chemical bonds.

The hydrolysis of starch can be measured through the use of an enzyme test or assay.
An enzyme assay will test for the simple presence of enzyme activity but can also be
used to measure the reaction rate of an enzyme-catalyzed reaction. The assay can
measure either the appearance of one of the products or the disappearance of one of
the substrates over time.

To measure your amylase activity, you will monitor the disappearance of amylase’s
substrate, starch. Starch reacts with iodine (which is yellow) to form a blue compound
(Amax 620 nm). This reaction is the basis of a colorimetric assay for amylase activity.
Broth culture supernatant (which contains the secreted amylase) will be incubated with
starch. After the incubation period, a portion (an aliquot) of this mixture is combined with
acidic iodine. The acid stops the enzymatic reaction and the iodine reacts with the
starch to produce the blue color. Any starch that has not yet been hydrolyzed by the
amylase will turn blue, with the intensity of the blue color being proportional to the
amount of starch remaining. The intensity of the blue color can be quantified
spectrophotometrically by measuring its absorbance at 620 nm. The greater the change
CHE506 Lab 8 Lab Manual
Edited 13 June 2018

in absorbance between a sample containing the initial amount of starch (without

enzyme) and the hydrolyzed mixture containing the enzyme, the greater the amount of
starch degraded by the enzyme, therefore the greater the activity of the enzyme being
measured.

Enzyme activity (reaction rates) is dependent upon the environmental 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-Menton Model:

2. Apparatus and Reagent

1. Alpha Amylase enzyme 9. Falcon tube

2. Starch 10. Micropipet and tips
3. pH buffer solution (pH 4-9) 11. Label sticker
4. DNSA Reagent 12. Schott bottle
5. Beaker 13. Vortex mixer
6. Measuring cylinder 14. Water bath
7. Cuvette 15. Spectrophotometer
8. Falcon tube rack 16. Hotplate
CHE506 Lab 8 Lab Manual
Edited 13 June 2018

3. Experimental Procedures

i. Preparation of 2% Starch Solution

a) Mix 4 g of soluble starch in approximately 50 ml of cold water.

b) While stirring, add the slurry to approx. 100 ml of gently boiling water in a large
beaker.
c) Then top up to final volume of 200ml and mix well.

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

a) Label five test tubes with pH 5, 6, 7, 8 and 9. In each tube, place 1 mL of 2% starch
solution. Add 1 mL of the appropriate buffer (at corresponding pH) to each tube.
b) Get five additional clean test tubes and put 2 mL of amylase solution in each tube.
c) Place all 10 tubes in the 37°C water bath for about 5 minutes to allow the
temperature to equilibrate.
d) Pour the content of each amylase test tube into each starch test tube and mix them
on vortex mixer.
e) Return the tubes to the 37°C water bath.
f) Let the hydrolysis reaction proceed for exactly 10 minutes.
g) Determine the amylase activity using the method given in Appendix 1.
h) Plot a graph of pH vs. enzyme activity.

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

a) Label one test tube with 30ºC. In the tube, place 1 mL of 2% starch solution and 1 mL
of pH=7 buffer to the tubes.
b) Get additional clean test tub and put 2 mL of amylase solution in the tube.
c) Place both tubes in the 30°C water bath for about 5 minutes to allow the temperature
to equilibrate.
d) Pour the contents of the amylase test tube into starch test tube and mix them on
vortex mixer.
e) Return the tubes to the 30°C water bath.
f) Let the hydrolysis reaction proceed for exactly 10 minutes.
g) Determine the amylase activity using the method given in Appendix 1.
h) Repeat step a-g at 4 different temperatures ranging from 30-70 ºC.
i) Plot a graph of temperature vs. amylase activity.
CHE506 Lab 8 Lab Manual
Edited 13 June 2018

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

a) Prepare starch solutions of varying concentration (0.5, 1.5, 2.0, 2.5, and 3.0% w/v) as
the substrate.
b) Label each tube with starch concentration and place 1ml of each starch solution into
the test tubes.
c) Add 1 mL of pH=7 buffer to the tubes.
d) Get five additional clean test tubes and put 2 mL of amylase solution in each tube.
e) Place all tubes in the 37°C water bath for about 5 minutes to allow the temperature to
equilibrate.
f) Pour the content of each amylase test tube into starch test tube and mix them on
vortex mixer.
g) Return the tubes to the 37°C water bath.
h) Let the hydrolysis reaction proceed for exactly 10 minutes.
i) Determine the amylase activity using the method given in Appendix 1.
j) Plot the graph of starch concentration against amylase activity.

Appendix 1 (Demonstration of Enzyme Activity)

a. After 10 minutes (the time of hydrolysis reaction), stop the reaction by adding 4 ml of
DNS reagent.
b. Boil for 10 minutes and then left to cool to room temperature.
c. Measure the absorbance of the samples at λ=540 nm.
d. Compare the absorbance value with glucose standard curve prepared to obtain the
glucose concentration.
e. Calculate the enzyme activity.
Note: Enzyme activity is the amount of glucose formed in reaction mixture per unit
time.

Appendix 2 (Glucose Standard Curve Preparation)

a. Prepare standard solutions of glucose at five different concentrations ranging from 0-

2000g/L by serial dilution.
b. Put 1 ml of each glucose solution in test tubes.
c. Add 1 ml of DNS reagent in each tube and mix for few seconds on vortex mixer.
d. Place the test tubes in water bath (T=100°C) for 10 min and then left to cool at room
temperature.
e. The absorbance of the samples is measured at λ=540 nm.
f. Draw the standard curve of absorbance vs glucose concentration.
Note: The graph is in straight line for absorbance less than 0.7.
CHE506 Lab 8 Lab Manual
Edited 13 June 2018

4. Report: (100 marks)

Plagiarism is highly prohibited.

1 Abstract/Summary (5M)

2 Introduction (5M)

3 Aims/Objective (5M)

4 Theory (10M)

5 Apparatus (5M)

6 Methodology/Procedure (10M)

7 Results (10M)

8 Calculations (10M)

9 Discussion (20M)

10 Conclusion (10M)

11 Recommendation (5M)

12 Reference/Appendix (5M)