Enzyme Kinematics of

Salivary Amylase

PRESENTED BY GROUP 4
PORLUCAS, SISON, VERGARA
Introduction

Enzyme Kinematics of Salivary Amylase

E N Z Y M E S

lower
activation energy

alternative pathway
Human Salivary HYDROLASE

Alpha Amylase
Faciltate the catalysis of
hydrolysis of sugars

SALIVARY
GLANDS
Production by the parotid,
sublingual, and submandibular
glands

STARCH
BREAKDOWN
Preparation for ingestion in the
intestines
FIG.2 STARCH MOLECULE
1,4 glycosidic links of the starch
molecules are hydrolyzed by the
salivary alpha-amylase

FIG.1 ALPHA
AMYLASE
The quaternary structure
of human salivary alpha
amylase
 ENZYME
ACTIVITY
incubation time

pH

temperature

substrate
concentration
 Experimental
in two parts

TESTING
PREPARATION OF FACTORS 
STANDARD CURVE Assesing the effect of
incubation time, temperature,
Preparing a standard curve pH and substrate
to convert absorbance levels concentration on enzyme
into starch concentrations activity
 Starch Standard
Curve

Preparation of Starch Absorbance Reading Graphing of Standard

Solutions Curve
0.2% starch solution in Using a UV/Vis Concentration was
varying concentrations in Spectrophotometer at plotted against
0.1M Phosphate buffer Ph 620 nm absorbance read
6.7
Incubation Time
Preparation of Starch
Solution
9.0 ml 0.1% starch solution
prepared with phosphate
buffer
100 µL 1:20 Human Saliva
Dilution
Transfered 1ml to 10 ml
test tube
Setting of Incubation Absorbance Reading
time
100 µL of HCl is The absorbance is then
immediately added to read using the UV/Vis
stop the solution spectrophotometer at 620
Time is set nm.
Addition of KI and dH2O Plot is graphed
before reading
Repeated for 3,5,7, and 10
minutes
 Temperature

Preparation of Starch Incubation and Absorbance Reading

Solution Heating
The absorbance is then
4.5 ml of 0.1% Starch Preincubation at 10 °C, 40
read using the UV/Vis
Solution in pH 6.7 °C, 60 °C and 80 °C 5
spectrophotometer at 620
phosphate buffer minutes
nm.
0.5 ml 0.9% NaCl 100 µL 1:20 Human Saliva
Plot is graphed
Dilution
HCL, KI dH20 before
reading
 pH
Preparation of Starch
Solution
2.50 ml 0.2% starch
solution,
0.5 ml 0.9% NaCl solution
and 2.0ml 0.1 M
phosphate buffer of
varying pH 2.0, 4.0, 6.7, 7.4
and 10
Enzyme and Absorbance Reading
Incubation
100 µL of 1:20 Human The absorbance is then
Saliva Dilution read using the UV/Vis
5 minutes incubation spectrophotometer at 620
time nm.
Addition of KI and dH2O Plot is graphed
before reading
Done for all pH levels
Substrate Concentration

Preparation of Starch Enzyme and Absorbance Reading

Solution Incubation
The absorbance is then
4.5 ml 2% starch solution 100 µL of 1:20 Human
read using the UV/Vis
in concentrations: 0.10%, Saliva Dilution
spectrophotometer at 620
0.8% 0.05% 0.02% 0.01% 5 minutes incubation
nm.
and 0.005% time
Plot is graphed
Addition of KI and dH2O
Km and Vmax are
before reading
calculated from
Done for all pH levels
Lineweaver burke
equation
 Results and
Discussion
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 HIGHLIGHTS
Discussion

Preparation of Starch Standard Curve

Effect of Incubation time on Enzyme Activity
Effect of Temperature on Enzyme Activity
Effect of pH on Enzyme Activity
Effect of Substrate Concentration on Enzyme Activity
 Enzyme Function

Enzyme with active site Formation of Enzyme Formation of product

compatible with substrate Substrate Complex
Enzyme Function
 UV/VIS
SPECTROMETRY
CONCENTRATION OF A
PARTICULAR SUBSTANCE IN A
SOLUTION HAS A DIRECT
RELATIONSHIP WITH ITS COLOR
 ASSAYS
→
Iodine reacts with iodide dark blue light FUNCTION AND MECHANISM
wavelength

Absorbance of each
solution depends on
starch concentration

Figure 2. The formation of iodine-starch complex

STANDARD CURVE

y = 8.7443x+0.0235
R^2 = 0.9802
STANDARD CURVE

Beer-Lambert Law

sample with the highest concentration of starch must be the one with
the highest absorbance
 SENSITIVITY OF ENZYMES

TEMPERATURE TEMPERATURE TIME SUBSTRATE

CONCENTRATION

Enzymes function most efficiently in optimum conditions.

EFFECT OF INCUBATION
TIME ON ENZYME ACTIVITY
EFFECT OF
INCUBATION TIME ON
ENZYME ACTIVITY

starch concentration almost reaches zero

(0.00005)Alpha-amylase is a hydrolase
Decrease in the concentration of the substrate
Optimal incubation time of 30 minutes
EFFECT OF
TEMPERATURE ON
ENZYME ACTIVITY

peak (at 40℃), and this is the optimum

temperature at which an enzyme is fully
functional
As temperature increases, the rate of reaction
catalyzed by the enzyme also increases
BUT there is a limit
EFFECT OF TEMPERATURE ON
ENZYME ACTIVITY

THEORETICAL
 OPTIMUM
TEMPERATURE

As temperature
approaches optimum
temperature,KE increases =
collisions of molecules per
unit team increases
 BEYOND OPTIMUM
TEMPERATURE

Some of the bonds of the

3D conformation of
proteins are broken down
= modifications in the
active site = thermal
denaturation of enzymes
DISCREPANCIES

Maintenance of
temperature (range of
50 °C - 60 °C) 
Purity of the sample

TEPERATURES
The instrument (UV-Vis
spectrophotometer)
EFFECT OF PH ON
ENZYME ACTIVITY

THEORETICAL
OPTIMUM PH Activity of enzyme at its
highest

DEVIATION FROM increases the

OPTIMUM PH number of H+ and
OH- ions
interference with the ionic
POSSIBLE EFFECTS interactions that bind an
enzyme together
Change in the shape of the
Note: pH values ideal for
active site of the enzyme
enzymes are related to the
Affect the charge of the
environment wherein it is
substrate molecules
situated
EFFECT OF SUBSTRATE
CONCENTRATION ON ENZYME
ACTIVITY

with an increased
presence of a
substrate, the
salivary alpha-
amylase has a
faster rate in
breaking down
starch.
MICHAELIS-MENTEN PLOT
LINEWEAVER-BURK PLOT

R^2 = 0.9797
Slope = 0.1757
Y-intercept = 51.593
y = 0.1757x + 51.593
LINEWEAVER-BURK PLOT

R^2 = 0.9797
Slope = 0.1757
Y-intercept = 51.593
y = 0.1757x + 51.593
 LINEWEAVER-BURK
PLOT
Can be used to find maximum velocity and
Micahelis constant

Vmax = 0.01938
KM = 3.405 x 10-3
Conclusion and
Recommendations
 Conclusion

incubation time 10 minutes

pH 6.0-8.0

temperature 40° C

substrate concentration Vmax= 0.0139%·min-1

Km= 0.0712%
 Recommendations
1 2 3

PURIFICATION OF BETTER HEATING INCREASE IN

SAMPLE PROCEDURE RANGE OF
PARAMETERS
Recommendations
4 5

EFFECT OF DENATURATION
INHIBITORS
 References
Action of salivary amylase on starch. (n.d.) Retrieved from https://amrita.olabs.edu.in/?
sub=79&brch=18&sim=236&cnt=1

Caprette, D. (2015). Principles of spectrophotometry. Retrieved from

http://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html

Des Gachons, C. P., & Breslin, P. A. (2016). Salivary amylase: digestion and metabolic syndrome. Current
diabetes reports, 16(10), 102.Effect of temperature on enzyme activity. (n.d.). Retrieved
fromhttp://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm

Enzymes and the active site. (n.d.). Retrieved from

https://www.khanacademy.org/science/biology/energy-and-enzymes/introduction-to-
enzymes/a/enzymes-and-the-active-site

Fried, M., Abramson, S., & Meyer, J. H. (1987). Passage of salivary amylase through the stomach in humans.
Digestive diseases and sciences, 32(10), 1097-1103.
Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K., & Chauhan, B. (2003). Microbial α-amylases: a biotechnological perspective. Process
biochemistry, 38(11), 1599-1616.

Gusakov, A.V., Kondratyeva, E.G., & Sinitsyn, A.P. (2011)., Comparison of two methods of assaying reducing sugars in the determination of
carbohydrase activities. International Journal of Analytical Chemistry, vol. 2011, Article ID 283658. Retrieved from
https://doi.org/10.1155/2011/283658.

Gyémánt G1, Kandra L, Nagy V, Somsák L. (2003). Inhibition of human salivary alpha-amylase by glucopyranosylidene-spiro-thiohydantoin.
Biochemical and Biophysical research communications.  DOI: 10.1016/j.bbrc.2003.10.119

Hachmann, A.S. & Maffezzoli, D. (2017). Human salivary amylase. Retrieved from
http://biology.kenyon.edu/BMB/jsmol2015/3BLKAmylase/index4.html

Horton, H. R., Moran, L., Scrimgeour, K., et. al. (2006). Principles of biochemistry (4th ed.). Pearson Prentice Hall: Upper Saddle River, New Jersey

Kaulpiboon, J. & Rudeekulthamrong, P. (2012). Kinetic inhibition of human salivary alpha-amylase by a novel cellobiose-containing
tetrasaccharide. Journal of the Medical Association of Thailand.  

Khera, D. (2015). How does starch indicate iodine? Retrieved from http://antoine.frostburg.edu/chem/senese/101/redox/faq/starch-as-redox-
indicator.html

Kull, F.J., & Goff, D.J. (1995). The inhibition of human salivary alpha-amylase by type II alpha-amylase inhibitor from Triticum aestivum is
competitive, slow and tight-binding. Journal of Enzyme Inhibition, 9(2):163-70.
Muralikrishna, G., & Nirmala, M. (2005). Cereal α-amylases—an overview. Carbohydrate polymers, 60(2), 163-173.

Nelson, D. L., Cox, M. M., & Lehninger, A. L. (2013). Lehninger principles of biochemistry. New York: W.H. Freeman and Company.

Nielsen, J. E., Borchert, T. V.  (2000). Protein engineering of bacterial alpha-amylases. Biochimica et Biophysica Acta 1543, 253-274.

Ophardt, C.E. (2003). Mechanism of enzyme action. In Virtual Chembook. Retrieved from
http://chemistry.elmhurst.edu/vchembook/571lockkey.html

Payan, F. (2004). Structural basis for the inhibition of mammalian and insect α-amylases by plant protein inhibitors. Biochimica et Biophysica
Acta (BBA)-Proteins and Proteomics, 1696(2), 171-180.

Sky-Peck, H. & Thuvasethakul, P. (1977). Human pancreatic alpha-amylase. II. effects of pH, substrate, and ions on the activity of the enzyme.
Annals of clinical and laboratory science. 

Stiefel, D. J., & Keller, P. J. (1973). Preparation and some properties of human pancreatic amylase including a comparison with human parotid
amylase. Biochimica et Biophysica Acta (BBA)-Enzymology, 302(2), 345-361.

Swanson, K. C., Matthews, J. C., Matthews, A. D., Howell, J. A., Richards, C. J., & Harmon, D. L. (2000). Dietary carbohydrate source and energy intake
influence the expression of pancreatic α-amylase in lambs. The Journal of nutrition, 130(9), 2157-2165.

Tester, R. & Karkalas, J. (2003). Carbohydrates: interactions with other food components. Retrieved from https://doi.org/10.1016/B0-12-227055-
X/00167-

Xvan der Maarel, M.J., van der Veen, B., Uitdehaag J.C., Leemhuis, H., Dijkhuizen, L. (2002). Properties and applications of starch-converting
enzymes of the alpha-amylase family. Journal of Biotechnology, 94(2):137-55.

Zar, M., Ali, S., Shahid, A.A. (2013). The influence of carbon and nitrogen supplementation on alpha amylase productivity of Bacillus
amyloliquefaciens IIB-14 using fuzzy-logic and two-factorial designs. African Journal of Microbiology Research. 7. 120-129. 10.5897/AJMR12.1519.
Thank you!
PRESENTED BY GROUP 4, PORLUCAS, SISON,
VERGARA