1. EXPERIMENT ON ENZYME
UMI ABIBAH BT SULAIMAN D20091034811
SITI RAHAYU BT MOHAMED NOOR
D20091034855
AZMA AMIRA BT MOHAMAD D20091034859
NUR AFIQAH BT MUHAMAD APANDI
D20091034872
DR ROSMILAH
MISNAN

2. Abstract
The main objective of this study was to prepare standard reference
graph for enzyme reaction concentration and to investigate the
effect of substrate concentration, temperature and pH on enzyme
concentration. The value of maximum rate of reaction, Vmax and
Michaelis constant, Km are obtain from Lineweaver- Burke plot.
From the experiment, Low value of Km means high affinity of the
enzyme for the substrate. Increasing the temperature of a system
will increase the number of collisions of enzyme and substrate per
unit time. Extremely high or low pH values generally result in
complete loss of activity for most enzymes. Amount of hydrogen
ion which is related to pH not give effect on the ionic bond of
enzyme stucture and the amino acid sequence of active site in
optimum pH. Changed the pH will breaking down the ionic
bonding that hold the structure of enzyme and the charge within
amino acid and lead to the failure in formation of enzyme-
substrate complexes.

3. Introduction
An enzyme is a biological catalyst that accelerates a
chemical reaction by lowering the energy needed to start
the reaction. It causes reactions to happen much faster
than they would if it wasn't present. In our bodies, many
thousands of chemical reactions are happening every
second. For example, the conversion of sucrose to the
monosaccharide sugars fructose and glucose is a chemical
reaction.

4. METHOD
Part I:
Preparation of
standard
reference

5. RESULT
Part I:
Preparation of
standard
reference

6. Standardgraphs

7. METHOD
Part II:
The effect of
substrate
concentration

8. RESULT
Part II:
The effect of substrate concentration

9. Substrategraphs

10. LB GRAPH

11. Temperature
graphs

12. Discussion
The value of Vmax and Km from Lineweaver-Burke:
1/Vmax = 200 -1/Km = -22
Vmax = 0.005 Km = 0.045

13. Graph of Michaelis-Menten
• the graph at first gradually increase and then
constant at certain values.
• concentration of the starch have reached the
maximum value of velocity
• After this point, increases in substrate
concentration will not increase the velocity.

14. Lineweaver-Burke
• The value of Vmax is 0.005 while the value of
Km is 0.045
• A small Km indicates that the enzyme requires
only a small amount of substrate to become
saturated.
• When this maximum velocity had been reached,
the entire available enzyme has been converted to
ES, which is the enzyme substrate complex.
• Low value of Km means high affinity of the
enzyme for the substrate. This is because Km is
refers to how well substrate binds to the enzyme.

15. METHOD
Part III:
The effect of
temperature

16. RESULT
Part III:
The effect of
temperature

17. DISCUSSION
• Enzymes are proteins that catalyze biochemical reactions.
An enzyme interacts with a molecule, generally called a
substrate, and turns it into a product. The velocity of the
reaction depends on the substrate concentration ([S]). But,
at a certain concentration, the velocity achieves the
maximum value (Vmax).
• m is a Michaelis constant that is the substrate
concentration at which the reaction velocity is half of the
maximum value. Km can be calculated from the
Lineweaver-Burk plot 1/V = (1/Vmax) + (Km/Vmax) x
1/[S].

18. • Lineweaver-Burk analysis is one method of
linearizing substrate-velocity data so as to
determine the kinetic constants
Km and Vmax. The units for Vmax are the
units that you used when entering Y values on
the original data table. The units for Km are
the units you used when entering the X values.

19. • V max for the temperature 8˚ C is 2000, for the
temperature 28 ˚ C is 1250 and for the
temperature 40 ˚ C is 2000. Km for the
temperature 8 ˚ C is 105.00. Then Km for
temperature 28˚ C is 62.00 and Km for
temperature 40˚ C is 100.00.

20. METHOD
Part IV:
The effect of
pH

21. RESULT
Part IV:
The effect of pH

22. Discussion
• From the result, the highest velocity detected at pH 4, 5 and 7. This pH
value gives maximum velocity which provided the suitable
environment for the enzyme reaction.
• From the result, this range is optimum pH for amylase, which the
enzyme is catalysing the reaction with the maximum reaction. The
amount of hydrogen ion which is related to pH not give effect on
the ionic bond of enzyme stucture and the amino acid sequence in
active site. This range make the enzyme is actively bind to substate and
form enzyme-substrate complexes. At the optimum pH, the maximum
rate of reaction can be achieved.
• The range for suitable pH in this experiment is 4 to 7 pH, but the
reading show the decrease velocity at pH 6. Maybe, this is due to
measurement error in handling pipette or absorbance reading

23. • From the theory, the optimum pH for amylase is 6.7 to 7.0 for the
pancreatic and saliva amylase (alpha-amylase). For malt amylase
(beta-amylase), the optimum pH is 4.6 to 5.2. The gamma amylase
need pH 3 for their maximum reaction. For amylase , the difference
pH needed for the difference type of amylase enzyme.
• The lowest velocity is at pH 9. At this pH, the amount of the
hydrogen ion is decreased, so this amount will breaking the ionic
bond of the enzyme structure and the charged in amino acid within
the active site. So, the active site is no longer fit to substrate.
• Changed the pH will breaking down the ionic bonding that hold
the structure of enzyme. So, it lose it functional shape mainly the
shape of active site. Consequently, the substrate will no longer fit
to active site. The enzyme said to be denatured. The amino acid
within the active site will not able to form the enzyme-substrate.

24. Conclusion
From this experiment,we are able to prepare the
standard reference graph for enzyme reaction
concentration.We also determine the effect of
substrate concentration,temperature and pH on
the enzyme reaction.

25. References
• Investigating the effect of enzyme concentration on the
hydrolysis of starch with amylase
(2011). Get on November 29, 2011 from
http://www.123helpme.com/view.asp?id=149093
• Tor Siong Hoo, Bah Hock Guan, Sri Nasariya (2008).
Matriculation biology. Kuala Lumpur:
Oriental Academic Publication.
• Mary K. Campbell (1999). Biochemistry third edition.
United States of America: Saunders and Harcourt
College.

26. APPENDIX

27. Thank you =)