RESULT

PART 1

B. Preparation of the glucose standard curve

Table 1: Data for glucose standard curve

Concentration [g/L] Absorbance [/]

0 0.000
0.2 0.100
0.4 0.255
0.6 0.391
0.8 0.539
1.0 0.627

Glucose Concentration vs Absorbance

0.7
y = 0.6554x - 0.009
0.6

0.5
Absorbance [/]

0.4

0.3

0.2

0.1

0
0 0.2 0.4 0.6 0.8 1 1.2
-0.1
Glucose Concentration (g/L)

Figure 1: Standard curve of concentration of glucose against absorbance

 Table 2: Amylase Activity Assays

Absorbance [/] Glucose Concentration (g/L) Amylase Activity, (U) μg/min

0.176 0.282 9.400x10-3

PART 2

A. Effect of substrate concentration on enzyme activity

Table 3: Data for substrate concentration effect

Concentration Of Starch Optical Density Glucose Concentration Amylase Activity

Solution [%] (g/L) (U) μg/min
0.5 0.077 0.131 4.377x10-3
1 0.177 0.283 9.433 x10-3
1.5 0.208 0.331 1.103 x10-3
2 0.223 0.354 1.180 x10-2
3 0.283 0.446 1.487 x10-2

Concentration Substrate vs Optical Density

0.35

0.3 y = 0.074x + 0.0752

0.25
Optical Density

0.2

0.15

0.1

0.05

0
0 0.5 1 1.5 2 2.5 3 3.5
Concentration Substrate

Figure 2: Concentration of substrate against optical density

B. Effect of pH on enzyme activity

Table 4: Data for pH effect

pH [/] Optical Density Glucose Concentration (g/L) Amylase Activity

(U) μg/min
5 0.116 0.191 6.367 x10-3
7 0.153 0.247 8.233 x10-3
8 0.075 0.128 4.267 x10-3
9 0.015 0.037 1.233 x10-3
11 -0.001 0.012 4.000 x10-4

pH vs Optical Density
0.18

0.16

0.14

0.12
Optical Density

0.1

0.08 y = -0.0245x + 0.2672

0.06

0.04

0.02

0
0 2 4 6 8 10 12
-0.02
pH

Figure 3: pH value against optical density

 pH vs Glucose Concentration
0.3

0.25
Glucose Concentration [g/L]

0.2

0.15

0.1 Optimum
pH
0.05

0
0 2 4 6 8 10 12
7
pH

Figure 4: pH value against concentration of glucose

DISCUSSION

The enzyme assays and factors affecting enzyme activity experiment were conducted to
study the physical characteristics and the properties of enzymes. Besides that, this experiment
also was run to determine the factors that affecting the enzyme activity. This experiment consists
of two parts.

In part one, the preparation of the DNS reagent was done by dissolving the DNS and
NaOH with distilled water, the preparation of the glucose standard curve from the determining
the optical density of the glucose mixture by using UV-VIS spectrophotometer and also the
determination of the amylase activity assay. In part two of this experiment, the experiment was
conducted to find out the effect of substrate concentration on enzyme activity and to determine
the effect of pH on the enzyme activity.

Based on the result obtained from the table 1 for the glucose standard curve in figure 1, it
showed that the increase the concentration of glucose, the increase the absorbance. This result
obeyed the Beer’s law that indicates that the absorbance is directly proportional to the
concentration (Brubaker, J., 2017). One of the factors that will affect the enzyme’s activity is
temperature. The increase the temperature, the increase the rate of the reaction. However, at very
high temperature, the enzymes will denature. The enzyme activity will steadily increase with
temperature up to around 37℃. If the temperature continues to increase, the rate of reaction will
reduce rapidly because the high amount of heat has denatured the enzyme (Worthington, C.,
2017).

The effect of substrate concentration on enzyme activity in part 2 was studied. From the
result obtained in table 3 and figure 2, it showed that the increase the concentration of the starch
solution, the increase the optical density as proved by the glucose standard curve. The glucose
concentration was determined by using the glucose standard curve from the optical density
(absorbance) obtained for each solution. Then the amylase activity was calculated by using the
1𝜇𝑔
formula 1U (unit) = . The result in table 3 showed that the increase the concentration of the
𝑚𝑖𝑛

starch solution, the increase the amylase activity. During enzyme substrate reaction, the velocity
will increase gradually with the increasing of the substrate concentration. However, beyond a
particular substrate concentration, the velocity will remain constant without any further increase
(vlab.amrita.edu.,2011). Thus, from this it can be concluded that the higher substrate
concentration will undergo higher enzyme activity.

Apart from that, the effect of pH on enzyme activity also was studied by using the pH
from pH 5 until 11. From the result in table 4 and figure 3, it showed that the increased the pH
until pH 7, the increased the optical density. Then, the optical density decreased with the
increasing of pH more than 7. The glucose concentration and amylase activity also increased
until pH 7 then decreased when the pH increased more than 7. Thus, from the result obtained, the
optimum pH for amylase’s activity is 7 as shown in figure 4. Based on the theory, any changes of
pH above or below optimum will cause the decreasing in the rate of reaction (amylase activity)
since more enzyme molecules will have active sites whose shapes are not complementary to the
shape of the substrate. Small changes in pH above or below optimum pH does not caused a
permanent changes to the enzyme because the bonds can be reformed but the extreme change in
pH will cause the enzyme to denature and lost the function permanently (Adam S., 2016).

From all the results obtained, the reading might not accurate due to some errors. The
apparatus used during the experiment might were contaminated by others chemicals that
influenced the inaccuracy of the result. The measurement of the chemicals used also might not
totally accurate that caused the parallax error. However, the data obtained made sense as the
previous experiment operated.
CONCLUSION AND RECOMMENDATION

The objective of this experiment is to study the enzyme assays and factors that affect the
enzyme activity. This experiment had been conducted under two parts. For part 1, it showed that
the increase the concentration of glucose, the increase the absorbance and it obey the Beer’s law.
There are few factors that affect the enzyme activity which are the temperature, pH and the
substrate concentration. Based on the result obtained, the increasing the temperature will increase
the rate of the reaction. However, the enzymes will denature when the temperature reached 37℃.
For Part 2, the effect of substrate concentration on enzyme activity had been observed. As the
concentration of substrate increases, the enzyme activity also increases. However, after period of
time the reaction is decreases due to the saturation of enzyme. Other than that, the effect of pH
on enzyme activity also was studied. Based on the result, the optimum pH for amylase to react is
at pH 7. The pH playing a vital role for the stability of enzyme. Different enzyme have different
rate of reaction at different pH. In this experiment, there are few errors might be occurred which
might affect the result of experiment as some of the result did not follow the theory. Thus some
recommendation had been stated for this experiment. One of the errors is due parallax error
while the reading been recorded. So, the reading of each analysis should be taken three times and
calculate the average. Next, ensure that the apparatus is being washed out with distilled water
before used.
TUTORIALS:

Discuss about other applications using enzyme assay. For example; what does the portable
glucometers used by diabetic patient’s measure? How do they measure it?

 A glucometer is a medical device for determining the approximate concentration

of glucose in the blood especially those people with diabetes
mellitus or hypoglycaemia. The test strips containing glucose oxidase, an enzyme
that reacts to glucose in the blood droplet, and an interface to an electrode
inside the meter. When the strip is inserted into the meter, the flux of the glucose
reaction generates an electrical signal. The glucometer is calibrated so the number
appearing in its digital readout corresponds to the strength of the electrical
current. The more glucose in the sample, the higher the number.

1. Discuss about the importance of optimum pH and temperature in the industrial

application of enzymes.

 Enzyme stability give an important factor to be consider in the application of

enzymes in the industry especially in food industry. The stability and activity
of enzymes is influenced by their physicochemical factor such as pH and
temperature. Therefore, most enzymes exhibit maximal activity at their
optimal conditions that influences enzymes activity. Optimum condition of
enzymes is favorable condition that allows them to perform efficiently. An
enzymes exhibit the optimum condition at which they demonstrate maximum
behavior according to well-establish rules. Enzymes have specific pH and
temperature range or value for their optimal activity. A change in the
physicochemical factors can affects the enzymes activity and can cause
deformity in the structure of enzymes which resulting in loss of action for
most enzymes.
REFERENCES

Brubaker, J. (2017). How to Calculate Concentration Using Absorbance. Sciencing.com.

Retrieved 6 December 2017, from https://sciencing.com/calculate-concentration-using-
absorbance-7153267.html

Worthington, C. (2017). Temperature Effects (Introduction to Enzymes). Worthington-

biochem.com. Retrieved 6 December 2017, from http://www.worthington-
biochem.com/introbiochem/tempeffects.html

vlab.amrita.edu,. (2011). Effect of Substrate Concentration on Enzyme Kinetics. Retrieved 6

December 2017, from vlab.amrita.edu/?sub=3&brch=64&sim=1090&cnt=1

Adam, S. (2016). Factors affecting Enzyme Activity | A Level Notes. Alevelnotes.com. Retrieved
6 December 2017, from https://alevelnotes.com/Factors-affecting-Enzyme-Activity/146
APPENDICES

PART 1

Calculation on the glucose concentration with 0.176 absorbance

Equation from standard curve: y = 0.6554x – 0.009

y: Absorbance x: Glucose concentration

y = 0.6554x – 0.009

0.176 = 0.6554x – 0.009

x = 0.2824 g/L

Calculation on the amylase activity

1𝜇𝑔
1U =
𝑚𝑖𝑛

0.282𝑥10−6 𝑔
U= 30 𝑚𝑖𝑛

𝜇𝑔
U = 9.400𝑥10−3
𝑚𝑖𝑛
PART 2

A. Effect of substrate concentration on enzyme activity

Calculation on the glucose concentration

y 0.077 0.177 0.208 0.223 0.283

x x1 x2 x3 x4 x5

By using standard curve equation, y = 0.6554x – 0.009

Let y = optical density and x = glucose concentration

y = 0.6554 x1 – 0.009 y = 0.6554 x4 – 0.009

x1 = 𝑦 + 0.009 x4 = 𝑦 + 0.009
0.6554 0.6554
= 0.077 + 0.009 x4 = 0.223 + 0.009
0.6554 0.6554
x1 = 0.131 x4 = 0.354

y = 0.6554 x2 – 0.009 y = 0.6554 x5 – 0.009

x2 = 𝑦 + 0.009 x5 = 𝑦 + 0.009
0.6554 0.6554
x2 = 0.177 + 0.009 x5 = 0.283 + 0.009
0.6554 0.6554
x2 = 0.283 x5 = 0.446

y = 0.6554 x3 – 0.009
x3 = 𝑦 + 0.009
0.6554
x3 = 0.208 + 0.009
0.6554
x3 = 0.331
Calculation on the amylase activity

0.5% starch solution 2% starch solution

U = 1𝜇𝑔 U = 1𝜇𝑔
𝑚𝑖𝑛 𝑚𝑖𝑛
U = 0.131𝑥10−6 𝑔 U = 0.354𝑥10−6 𝑔
30 𝑚𝑖𝑛 30 𝑚𝑖𝑛
U = 𝝁𝒈 U = 𝟏. 𝟏𝟖𝟎𝒙𝟏𝟎−𝟐 𝝁𝒈
𝟒. 𝟑𝟕𝟕𝒙𝟏𝟎−𝟑
𝒎𝒊𝒏 𝒎𝒊𝒏

1% starch solution 3% starch solution

U = 1𝜇𝑔 U = 1𝜇𝑔
𝑚𝑖𝑛 𝑚𝑖𝑛
U = 0.283𝑥10−6 𝑔 U = 0.446𝑥10−6 𝑔
30 𝑚𝑖𝑛 30 𝑚𝑖𝑛
U = 𝝁𝒈 U = 𝟏. 𝟒𝟖𝟕𝒙𝟏𝟎−𝟐 𝝁𝒈
𝟗. 𝟒𝟑𝟑𝒙𝟏𝟎−𝟑
𝒎𝒊𝒏 𝒎𝒊𝒏

1.5% starch solution

U = 1𝜇𝑔
𝑚𝑖𝑛
U = 0.331𝑥10−6 𝑔
30 𝑚𝑖𝑛
U = 𝝁𝒈
𝟏. 𝟏𝟎𝟑𝒙𝟏𝟎−𝟑
𝒎𝒊𝒏
B. Effect of pH on enzyme activity

y 0.116 0.153 0.075 0.015 -0.001

x x1 x2 x3 x4 x5

By using standard curve equation, y = 0.6554x – 0.009

Let y = optical density and x = glucose concentration

y = 0.6554 x1 – 0.009 y = 0.6554 x4 – 0.009

x1 = 𝑦 + 0.009 x4 = 𝑦 + 0.009
0.6554 0.6554
= 0.116 + 0.009 x4 = 0.015 + 0.009
0.6554 0.6554
x1 = 0.191 x4 = 0.037

y = 0.6554 x2 – 0.009 y = 0.6554 x5 – 0.009

x2 = 𝑦 + 0.009 x5 = 𝑦 + 0.009
0.6554 0.6554
x2 = 0.153 + 0.009 x5 = −0.001 + 0.009
0.6554 0.6554
x2 = 0.247 x5 = 0.012

y = 0.6554 x3 – 0.009
x3 = 𝑦 + 0.009
0.6554
x3 = 0.075 + 0.009
0.6554
x3 = 0.128
Calculation on the amylase activity

pH=5 pH=9
U = 1𝜇𝑔 U = 1𝜇𝑔
𝑚𝑖𝑛 𝑚𝑖𝑛
U = 0.191𝑥10−6 𝑔 U = 0.037𝑥10−6 𝑔
30 𝑚𝑖𝑛 30 𝑚𝑖𝑛
U = 𝝁𝒈 U = 𝟏. 𝟐𝟑𝟑𝒙𝟏𝟎−𝟑 𝝁𝒈
𝟔. 𝟑𝟔𝟕𝒙𝟏𝟎−𝟑
𝒎𝒊𝒏 𝒎𝒊𝒏

pH=7 pH=11
U = 1𝜇𝑔 U = 1𝜇𝑔
𝑚𝑖𝑛 𝑚𝑖𝑛
U = 0.247𝑥10−6 𝑔 U = 0.012𝑥10−6 𝑔
30 𝑚𝑖𝑛 30 𝑚𝑖𝑛
U = 𝝁𝒈 U = 𝟒. 𝟎𝟎𝟎𝒙𝟏𝟎−𝟒 𝝁𝒈
𝟖. 𝟐𝟑𝟑𝒙𝟏𝟎−𝟑
𝒎𝒊𝒏 𝒎𝒊𝒏

pH=8
U = 1𝜇𝑔
𝑚𝑖𝑛
U = 0.128𝑥10−6 𝑔
30 𝑚𝑖𝑛
U = 𝝁𝒈
𝟒. 𝟐𝟔𝟕𝒙𝟏𝟎−𝟑
𝒎𝒊𝒏