1) This experiment studied the effect of temperature and pH on the enzymatic activity of salivary amylase. 2) The optimal temperature for salivary amylase activity was found to be 37°C, as this is body temperature and the rate of reaction was highest. Activity decreased at higher and lower temperatures. 3) The optimal pH range for salivary amylase activity was pH 6.7-7, as outside this range the enzyme loses its tertiary structure and is denatured, ceasing activity.

1. Experiment 4:
Enzymatic Activity of Salivary
Amylase
Jalipa, Jomari
Mesa, Hannah
Pichon, Jasmine
San Pedro, Anna
Villasenor, Maiko

2. INTRODUCTION

3. Introduction
What are ENZYMES?
• Most diverse group of proteins
• Acts as biological catalysts
– cause or speed up chemical reactions by lowering the
activation energy (Ea)
• Important biological reactions catalyzed by
enzymes:
– Metabolism
– DNA synthesis
– RNA synthesis
– Protein synthesis
– Digestion

4. Introduction
What are ENZYMES?
• Active site
– pocket or cleft
– contains amino acid
side chains that
participate in substrate
binding and catalysis
• Substrate
– Reactant molecules

5. Introduction
Properties of Enzymes
• Large protein molecules
• Reusable (example: 2 H2O2 [catalase]→ 2 H2O + 1 O2)
• Remain unchanged
• VERY specific
• Operate at very high speeds
• Rate of reaction is dependent upon temperature,
pH, [E], and [S]
• Usually work best with 60°C
• Denaturation occurs at very high temperatures

6. Introduction
BINDING MODELS: LOCK & KEY MODEL
• Emil Fischer
• Enzyme specificity
• Substrate has a
complimentary shape to
the active site
• Substrate: key :: Enzyme:
lock
• Wrong key = no reaction

7. • a.k.a. “hand and glove
model”
• substrate induces a
slight change in the
shape of the active site
so that they both
compliments each
other’s shape.
BINDING MODELS: INDUCED FIT MODEL

8. HOW DO ENZYMES WORK?

9. SALIVARY AMYLASE
• Digestive enzyme responsible for catalyzing
starch molecules into simpler sugars (i.e.
maltose and isomaltose)
• Secreted by the salivary glands
• OPTIMUM TEMPERATURE: 37ᴼC
• OPTIMUM pH: 5.6 – 6.7

10. STARCH
• Mixture of two polysaccharides: amylose and
amylopectin
• White, tasteless, solid carbohydrate
• Converted to glucose via hydrolysis
• Detected by iodine (blue-black color)

11. THE BASIC REACTION:
SUBSTRATE enzyme PRODUCT

12. OBJECTIVES:
• to observe the effect of different
temperatures and pH on the enzymatic
activity and specificity of the salivary amylase
• to determine the optimal temperature and pH
values of the salivary amylase

13. Materials
FOR A. EFFECT OF TEMPERATURE:
• Enzyme solution (1 mL saliva + 9 mL distilled H2O+ 30 mL 0.5% NaCl
• Buffered starch (1% starch in phosphate buffer pH 6.7)
• 0.001 M Iodine solution
• 2 Spot plates
• Test tubes
• Medicine droppers
• Constant Temperature bath (4, room temp, 37, 50, 60 and 70 °C)

14. Materials
FOR B. EFFECT OF PH:
• Enzyme solution (1 mL saliva + 9 mL distilled H2O+ 30 mL 0.5% NaCl
• 2% Unbuffered starch
• 0.001 M Iodine solution
• Acetate buffer solutions (pH 4 and 5)
• Phosphate buffer solutions (pH 6.7 and 8)
• Bicarbonate buffer (pH 10)
• 2 Spot plates
• Test tubes
• Medicine droppers
• Water bath set at 37°C

15. Methodology
A. Effect of Temperature
2 mL enzyme solution
In separate test tube, 2 mL of
buffer starch solution
• Incubate in 4°C ice bath for 10
minutes
• Immediately mix 2 mL enzyme
solution to 2 mL buffer starch
solution.
Enzyme- starch
solution
• Immediately take 3 drops and put in
spot plate well.
• Add 2 drops Iodine solution.

16. • Continue incubation for 1 minute
• Quickly take 3 drops of enzyme solution
and 2 drops of iodine and add it to the
second spot plate well.
• Record color
• Label as “1 minute”
• Continue incubation for 1 minute
until a light yellow- colored
solution is observed.
Light yellow colored
solution
• Repeat procedures for other temperatures (RMT, 37, 50, 60 and 70°C)
*Use hot water for high temperatures
• Plot the reciprocal of time (1.t, min -1) versus the temperature (T).
• Determine optimum temp. of Amylase
Blue-Violet colored
solution
• Note time (t)

17. B. Effect of PH
1 mL 2% unbuffered starch
With 1 mL acetate buffer
(pH4)
2 mL enzyme solution
• Incubate for 10 minutes in 37° C
• Immediately take 3 drops and put in
spot plate well.
• Add 2 drops Iodine solution.
• Label as “zero minute”
• Record color

18. Blue-Violet colored
solution
Light yellow colored
solution
• Continue incubation for 1 minute
• Quickly take 3 drops of enzyme solution
and 2 drops of iodine and add it to the
second spot plate well.
• Record color
• Label as “1 minute”
• Continue incubation for 1 minute
until a light yellow- colored
solution is observed.
• Note time (t)
• Repeat procedures for the other pH (5, 6.7, 8, and 10) using the
appropriate buffer.
• Plot the reciprocal of time (1/t, min -1) versus the buffer pH (pH).
• Determine optimum pH of Amylase

19. Results

20. • Effect of Temperature
Temperature Time (min.) Rate (1/time)
4 12 0.083
32.8 1 1
37 1 1
60 5 0.20
70 ∞ 0

21. 0
0.2
0.4
0.6
0.8
1
1.2
Rate
(1/min)
Temperature
4 32.8 37 60 70

22. • Effect of pH
pH Time (min.) Rate (1/time)
4 ∞ 0
5 12 0.0833
6.7 3 0.33
8 4 0.25
10 14 0.07

23. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 2 4 6 8 10 12
pH
Rate
(1/min)

24. Discussion

25. Salivary Amylase
• Alpha amylase
– Form produced by your salivary gland and pancreas
• Hydrolyzes alpha bonds of large, alpha linked oligosaccharides
and polysaccharides
– Acts on linear alpha(1,4) glycosidic linkages
Starch + amylase  maltose + salivary amylase + dextrin

26. Salivary Amylase
• Chloride-dependent enzyme
• AMY1A gene
– encodes human salivary amylase
– Influence salivary amylase concentration and
enzymatic activity

27. Effect of Temperature
• Enzymes work best at a certain temperature
(optimum temperature) where the reaction rate is
at maximum
• Optimum temperature: 37 °C (body temperature)
• Lower than 37°C = slower reaction
• At 40 °C and above, enzymes start to denature
- Destruction of 2° and 3° structures

28. Effect of pH
• pH affects the detailed structure of the active
site of the enzyme
– Requires certain level of acidity and alkalinity
– Too much or too little H+ interferes with electric
charges and disrupts H-bonds
• Extremely high or low pH will result in the
complete loss of enzymatic activity

29. Effect of pH
• Optimum pH: pH 6.7 – 7
• pH 10 is when most enzymes are denatured

30. Conclusion

31. Conclusion:
Effect of Temperature on Enzymatic
Activity
• Enzymes work best at a certain temperature
(optimum temperature) where the reaction
rate is at maximum
• Optimum temperature = 37 °C

32. Conclusion:
Effect of pH on Enzymatic Activity
• Affects the ionization of acidic or basic amino
groups
• Affects the shape of the substrate
• Optimum pH for salivary amylase = pH 6.7 -7