Dianne Faye T.

Manabat
Post Laboratory Report July 5, 2018

Exercise 5
Determination of the Activity of Amylase



In this experiment, the first procedure was starch preparation, followed by standard curve
preparation and incubation of samples. Starch was first prepared by mixing the starch in
phosphate buffer with 6.7 pH. The use of starch solution with phosphate buffer it to prevent any
drastic change in pH during the succeeding steps which can affect enzyme activity and to create
a pH environment that is similar to physiological pH since a-amylase is a digestive enzyme in the
body. The addition of potassium iodide and iodine will provide the color blue in the amylase
solution essential for the reading in the spectrophotometer. The blue color is due to linear
amylose that is comprised of a-1-4 glycosidic bonds, forming complex with iodine. This
mechanism was also responsible for addition of I2/KI solution prior to spectrophotometer
reading. Calcium chloride was added to the salivary amylase and phosphate buffer solution
because calcium is a co-factor of salivary a-amylase and may help stabilize its activity. A study by
Morris (2011) shows that addition of calcium to salivary a-amylase maintained the activity of the
enzyme which is probably due to protection from heat denaturation.

Table 1. Concentration and amount of starch in standard curve.
Test tube Starch (umol/ ml) Amount starch (umol) A620 Adjusted
blank 0 0 0.051 0
0.1 0.146070698 0.292141396 1.32 1.269
0.25 0.365176746 0.730353491 1.72 1.669
0.5 0.730353491 1.460706982 0.339 0.288
1 1.460706982 2.921413964 0.705 0.654
1.5 2.191060473 4.382120946 0.844 0.793
2 2.921413964 5.842827928 1.187 1.136


The standard curve appears to be linear (R2= 0.98956). This can be interpreted as a positive
relationship between addition of starch and absorbance reading.
 Standard curve
1.2
y = 0.1915x + 0.0114
1 R² = 0.98956
Absorbance ( 620nm)

0.8

0.6

0.4

0.2

0
0 1 2 3 4 5 6
Amount starch (umol)

Figure 1. Amount of starch vs. Absorbance for standard curve.



Linear regression:
B a= 0.011351036
M b= 0.191530782
r= 0.994767802
Starch remaining:
x= y-b
M



C1= 0.1 g x 1 mole x 1,000,000umol =2.921413964 umol/ mL
100 mL 342.3 g mole



C1= 0.1 g x 1 mole x 1,000,000umol =2.921413964 umol/ mL
100 mL 342.3 g mole

Concentration Amount starch

C2= c1v1 2.921413964 x 0.1 = 0.146070698 x2 0.292141396
v2 2 mL

C2= c1v1 2.921413964 x 0.25 = 0.365176746 x2 0.730353491

v2 2 mL

C2= c1v1 2.921413964 x 0.5 = 0.730353491 x2 1.460706982

v2 2 mL

C2= c1v1 2.921413964 x 1 = 1.460706982 x2 2.921413964

v2 2 mL

C2= c1v1 2.921413964 x 1.5 = 2.191060473 x2 4.382120946

v2 2 mL

C2= c1v1 2.921413964 x 2 = 2.921413964 x2 5.842827928

v2 2 mL

The amount of starch after incubation of a-amylase with reaction mixture was computed using
the same formula and regression parameters.

Calculations:
Water answer Urea answer NaCl answer
0 0.325 – 1.637590371 0.407-0.01135/0.1915 2.065719986 0.203-0.01135/0.1915 1.000617041
0.01135/0.1915
2 0.214 – 1.005838134 0.184-0.01135/0.1915 0.901416277 0.066-0.01135/0.1915 0.285327319
0.01135/0.1915
4 0.115- 0.541160869 0.129-0.01135/0.1915 0.614256169 0.058-0.01135/0.1915 0.243558576
0.01135/0.1915
6 0.077- 0.447181198
0.01135/0.1915











Table 2. Starch remaining after incubation of a-amylase with reaction mixtures.

Incubation time Starch Remaining (umol)

(min) Water Urea NaCl
0 1.637590371 2.065719986 1.000617041
2 1.005838134 0.901416277 0.285327319
4 0.541160869 0.614256169 0.243558576
6 0.447181198


Figure 2 shows the amount of starch (umol) remaining after incubation with water, urea,
and NaCl. In this experiment, water is used as a control. However, the curve of urea appears to
be comparable with water. This in contrast with studies showing that urea is an inhibitor of a-
amylase. For example, a study by Torralballa and Eitingon (1966) shows that addition of higher
concentration of urea with longer time resulted in greater inhibition of porcine pancreatic a-
amylase. It can be expected from the experiment that Urea’s inhibitory effect on a-amylase will
result to greater starch retention with time. The mechanism for urea’s inhibition of a-amylase is
attributed to a-amylase’ solubility in urea solution. It is expected that compared to water, urea
will result to greater starch retention since a-amylase is more soluble in urea than in water. The
rate of solubility of a-amylase in urea is 15 mg per mL of 1 M urea solution, while the rate of
solubility of a-amylase in water is only 2 mg per ml of water at 2 0C. Alternative mechanisms
includes exchange in hydrogen bonding, disruption of hydrogen forces, and destruction of ice
berg lattice of the enzyme. On the other hand, the graph shows that incubation of a-amylase
with NaCl resulted in lower amount of starch remaining. This is in agreement with other studies
showing that NaCl is an activator of a-amylase. The activation of a-amylase by NaCl can be
explained by chloride’s polarization of hydrolytic water molecule in the enzyme, which enhances
the rate of the second step in the catalytic reaction (Aghajari et al, 2002). Aside from inbibitors
and activator, other factors that may affect enzyme activity are: temperature, pH, enzyme
concentration, and substrate concentration.










 2.5
Starch remaining (umol)

1.5
Water
1 Urea

NaCl
0.5

0
0 1 2 3 4 5 6
Incubation time (min)

Figure 2. Starch remaining after incubation of a-amylase with reaction mixtures.


The initial velocity corresponds to the slope of the curve at the origin. Table 3 shows the initial
velocity of a-amylase subjected to incubation with different reaction mixtures. This can be
interpreted as the amount of enzyme that will hydrolyze 1 umol starch per minute. As shown in
the table, incubation of a-amylase with NaCl requires the least amount of enzyme (0.1893 units)
to hydrolyze 1 umol of starch per minute. This is because NaCl is an activator of a-amylase. On
the other hand, compared to water, incubation of a-amylase with Urea will require more
enzymes to hydrolyze 1 umol of starch per minute. This is due to higher solubility of a-amylase
enzyme to urea compared to water as discussed above. The inactivation of a-amylase by urea
will require more enzymes to hydrolyze the starch. A non-linear relationship was observed in all
reaction mixtures. This can be explained by error in measurement such as lack of replication of
samples and procedures. Moreover, time is a critical factor in enzyme activity since most of the
factors that affects enzyme activity depends on it. Thus, it may be possible that the procedures
that involves timing such as incubation were not done accurately, and replication of the
procedures can reduce this error. Also, excess addition of Calcium before incubation may have
affected the activity of enzymes since this helps stabilize its activity. Similarly, the amount of
iodine may have confounded the effects. Thus, the measurement of reagents and time should be
well-controlled.

Table 3. Slope, Initial velocity and Correlation coefficient of a-amylase after incubation with
reaction mixtures.
Water Urea NaCl
Slope -0.2018x -0.3629x -0.1893x
Initial velocity 0.2018 0.3629 0.1893
2
R 0.91632 0.89148 0.79125

In conclusion, enzyme activity is influenced by various factors, but is largely dependent on time.
Since enzymes play a role in food production and biochemical reactions, it is worth understanding
how to control for factors that may affect their activity.




References:

Aghajari, N., Feller, G., Gerday, C., & Haser, R. (2002). Structural basis of alpha-amylase
activation by chloride. Protein Science : A Publication of the Protein Society, 11(6), 1435–
41. http://doi.org/10.1110/ps.0202602

Morris, C., Fichtel, S. L., & Taylor, A. J. (2011). Impact of calcium on salivary α-amylase
activity, starch paste apparent viscosity, and thickness perception. Chemosensory
Perception, 4(3), 116–122. http://doi.org/10.1007/s12078-011-9091-7

Toralballa, G. C., & Eitingon, M. (1967). Action of urea and certain other amide reagents on
crystalline porcine pancreatic amylase. Archives of Biochemistry and Biophysics, 119(C),
519–525. http://doi.org/10.1016/0003-9861(67)90486-9