Malaysian Institute of Chemical and Bioengineering Technology

BIOCHEMICAL ENGINEERING
CPB 30103
EXPERIMENT 2

ENZYME ASSAYS AND FACTORS AFFECTING ENZYME ACTIVITY

Lecture’s Name: DR. NOR ZANARIAH SAFIEI

NO NAME ID SECTION

1 AZWAN BIN AHMAD 55213115254 L01

2 MAGDALYN EMPINA ANAK SERIT 55213115126 L01

3 SITI ROHANI BT ABDUL RAHIM 55213115136 L01

4 WAN FAIZAH JOHOR BT FU-AD JOHOR 55213115186 L01

5 NURUL FARIHAH BT ABU SAIRI 55213115262 L01

ABSTRACT
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

For the Enzyme Assays and Factors Affecting Enzyme Activity experiment was carried
out with two main objectives were to comprehend physical characteristic and properties of
enzymes and to study the factors affecting their activities. The experiment was done by
following the manual procedures provided. The procedures consist of Part 1 and Part 2. In
Part 1, there were (A) Preparation of the DNS reagent, where needed prepare the DNS
reagent manually, (B) Preparation of the Glucose Standard Curve, where to plot the glucose
standard curve, by the dilutions of a solution of known concentration to determine
concentration of unknown, and, (C) Amylase Activity Assay, that required to calculate
concentration of reducing sugar and determination of amylase activity. In Part 2, there were
(A) Effect of Substrate Concentration on Enzyme Activity and (B) Effect of pH on Enzyme
Activity. Graph 1 shows the glucose standard curve where the glucose concentration is
directly proportional to the absorbance of 575nm. While Graph 2, shows the concentration of
glucose against the concentration of substrate both increasing, and Graph 3, pH against the
concentration of glucose shows fluctuated. In this report have been discussed, the effect of
different temperature on the enzyme’s activity and the temperature for the optimum activity
of amylase. The temperature increases, the rate of reaction also increases until 37⁰C where
the enzyme may denatured. The optimum temperature is different for different enzymes.
Next, the enzyme solution was concluded to be stable at the pH of 7.5. Besides, the results
show that increasing the substrate concentration increases the amylase activity. Hence,
proving the theory, the concentration of glucose increases as the concentration of substrate
increases. As a conclusion, this experiment have achieving both of the objectives.

OBJECTIVES

Page | 2
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

Two main objectives in this experiment were:

To comprehend physical characteristic and properties of enzymes

To study the factors affecting their activities such as concentration, temperature and
pH

RESULT

Page | 3
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

PART 1
Table 1 shows data that provided in the manual for plotting the standard curve as shown at
graph 1. Meanwhile, table 2 shows the amylase assay for standard solution.

Table 1: Absorbance value correspond to the glucose concentration

Concentration of glucose (g/L) Absorbance(/)
0.000 0.000
0.200 0.100
0.400 0.255
0.600 0.391
0.800 0.539
1.000 0.627

Graph 1: Standard Curve Calibration

From the graph, the standard curve equation is Y = 0.6554X – 0.009.

Table 2: Amylase activity assay using UV-visible spectrophotometer

Page | 4
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

Absorbance(/) 0.104
Concentration of glucose (g/L) 0.1724
Amylase activity(µg/min) 5746.6667

PART 2
A. Effect of substrate concentration on enzyme activity

Table 3 shows the data obtained when the substrate concentrations are manipulated while
graph 2 illustrates the effect of different substrate concentration on the glucose concentration.

Table 3: Effect of substrate concentration on enzyme activity

Concentration of Starch Concentration of Amylase

Absorbance(/)
solution (%) glucose (g/L) activity(µg/min)
0.0 0.119 0.1953 6510.0193
1.0 0.253 0.3998 13325.1958
1.5 0.284 0.4471 14901.8411
2.0 0.365 0.5706 19021.4627
3.0 0.410 0.6393 21310.1414

Page | 5
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

Graph 2: Concentration of Glucose against the Concentration of Substrate

B. Effect of pH on enzyme activity

Table 4 shows the data obtained when pH of enzyme solutions are manipulated whereas
graph 3 illustrates the effect of different pH on the glucose concentration. The optimum pH
obtained from graph 3 is 7.6.

Table 4: Effect of pH on enzyme activity

Concentration of glucose amylase

pH(/) Absorbance(/)
(g/L) activity(µg/min)
5 0.001 0.0153 508.5953
7 0.088 0.1480 4933.3740
8 0.09 0.1511 5035.0931
9 -0.02 -0.0168 -559.4548
11 -0.01 -0.0015 -50.8595

Graph 3: pH against the Concentration of Glucose

Page | 6
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

DISCUSSION

Enzyme Assays and Factors Affecting Enzyme Activity experiment was conducted to
comprehend the physical characteristic and properties of enzymes as well as to study the
effect of temperature, pH and enzyme concentration to enzyme activity.

There are two parts of this experiment altogether. In the first part, two solutions are
prepared as a standard, which are a glucose solution and an enzyme solution. The glucose
standard curve (Graph 1) is prepared from the standard glucose solution to determine the
concentration of glucose at a certain absorbance. Then, an amylase assay is carried out by
using UV-visible spectrophotometer where the absorbance of the prepared enzyme solution is
determined as 0.104 (Table 2). Thus, the concentration of the glucose is calculated using
standard curve equation as 0.1724 g/L while the amylase activity is 5746.6667 (Table 2).

Meanwhile, enzyme solutions with varies pH and substrate concentration are prepared
in part two to study their effect on enzyme activity. Other than that, temperature changes
could also affect the enzyme activity. As the temperature increases, the rate of reaction also
increases ("BBC - GCSE Bitesize: Temperature, pH and enzymes", 2014). However, a very
high temperature would denature the enzyme. Theoretically, amylase’s optimum temperature
is 37oC where it will be more effectively to break down the starch in the small intestine.
Page | 7
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

Therefore, its activity would gradually increase with temperature up to 37 oC but if the
temperature continues to rise, the reaction rate would falls rapidly as the enzyme is denatured.
The optimum temperature is different for different enzymes.

Another factor that significantly affecting enzyme activity is the concentration of the
substrate. Changes in substrate concentration affect the rate of reaction only if it is the
limiting factor ("Factors affecting Enzyme Activity | A Level Notes", 2016). Theoretically,
increasing the substrate concentration increases the rate of reaction. This is due to more
substrate molecules will be colliding with enzyme molecules hence more product is formed
("Factors affecting Enzyme Activity | A Level Notes", 2016). However, after a certain times
of increasing the substrate concentration, there is a point where it stop increasing the rate of
reaction. It is because substrate concentration will no longer be the limiting factor and all the
active sites of the enzyme become saturated with substrate. As shown in Table 3, the
concentration of substrates are 0%, 1%, 1.5%, 2% and 3% whereas the amylase activity are
6510.02 µg/min, 13325.2 µg/min, 14901.84 µg/min, 19021.46 µg/min and 21310.14 µg/min
respectively. The results show that increasing the substrate concentration increases the
amylase activity. Hence, proving the theory, the concentration of glucose increases as the
concentration of substrate increases (Graph 2).

On the other hand, changing pH could change the properties of substrate where the
substrate either cannot bind to active site or cannot undergo catalysis ("pH and enzymes",
n.d.). Generally, different enzyme have different optimum pH values. At optimum pH, the
rate of reaction is at the maximum rate. This is due to the shape of the enzyme’s active site is
the most complementary to the shape of their substrate ("Factors affecting Enzyme Activity |
A Level Notes", 2016). Extremely high or low pH however could cause complete loss of
activity for most enzyme. In other word, the reaction stops because the enzyme denatured and
permanently damaged. From the experiment, the optimum pH of the enzyme solution is
determined as 7.5. In addition, it is found that the enzyme have denatured at pH 9. When the
pH of the enzyme solution are 8, 9 and 11, the amylase activity are 5035.09 µg/min, -559.45
µg/min and -50.86 µg/min respectively (Table 4). Moreover, the amylase activity after pH 9
have falls rapidly (Graph 3). Thus, the enzyme solution is concluded to be stable at the pH
range of 5.0 -8.0 with optimal temperature of 7.5.

Page | 8
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

As a conclusion, this experiment have conducted properly. All the objectives of the
experiment were achieved although there are a few errors has happened throughout the
experiment.

CONCLUSION AND RECOMMENDATION

This experiment was conducted to the physical characteristic and properties of enzymes
as well as to study the effect of temperature, pH and enzyme concentration to enzyme
activity. As the conclusion from this experiment, temperature for enzymes need to be
maintain at 37 oC to the enzymes form denature. Other than that, optimum pH is needed in
this experiment to avoid enzyme from denature because loss of activity. Substrate
concentration can increase the rate of reaction which means increasing of amylase activity.

There were several errors occurred during conducting the experiment and some
recommendation need to do to avoid any error happen. Firstly every beaker or test tube needs
to label in different concentration as an action for comparison. Use tong to hold the test tube
while boiled it to avoid any accident happen. Pipette need to be changed for substrate in
different concentration. Use uniform time scale to add DNS reagent with the mixture which is
in 10 minutes.

Page | 9
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

QUESTIONS
1. 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?
Enzyme assays are performed to serve two different purposes:
(i) to identify a special enzyme, to prove its presence or absence in a distinct
specimen, like an organism or a tissue and
(ii) to determine the amount of the enzyme in the sample.

In the application using enzyme assay, the glucometer is one of the application. Many glucose
meters employ the oxidation of glucose to gluconolactone catalyzed by glucose oxidase.
Others use a similar reaction catalyzed instead by another enzyme, glucose
dehydrogenase (GDH). This has the advantage of sensitivity over glucose oxidase but is more
susceptible to interfering reactions with other substances.
Most glucometers today use an electrochemical method. Test strips contain a capillary that
sucks up a reproducible amount of blood. The glucose in the blood reacts with an enzyme
electrode containing glucose oxidase or dehydrogenase. The mediator in turn is re oxidized
by reaction at the electrode, which generates an electric current. The total charge passing
through the electrode is proportional to the amount of glucose in the blood that has reacted
with the enzyme. The coulometric method is a technique where the total amount of charge
generated by the glucose oxidation reaction is measured over a period of time. Both methods
give an estimation of the concentration of glucose in the initial blood sample.
Page | 10
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

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

application of enzymes.
Living cells use chemical reactions to produce new materials. Living things produce catalysts
called enzymes which allow chemical reactions to occur quite quickly at ordinary
temperatures and pressures. Enzymes are powerful 'biochemical catalysts' and are widely
used in the food industry and are being used more and more to manufacture many other
chemicals. These biological catalysts promote most of the reactions in living tissue. Cells
contain protein molecules that act as biological or biochemical catalysts, they are known as
enzymes.
 The chemical reactions brought about by living cells are quite fast in conditions that
are warm rather than hot.
 This is because the cells use these enzyme catalysts. The key and lock mechanism is
important. If the temperature are high, the active site of the enzyme will be damage,
thus the enzyme will be dysfunction.
 Enzymes are protein molecules which are usually damaged by temperatures above
about 45º C. Although not damaged by lower temperatures, the reactions may be too
slow to be of any use.
 Different enzymes work best at different pH values.

Page | 11
 Experiment 2 - Enzyme Assays & Factors Affecting Enzyme Activity

REFERENCES

 BBC - GCSE Bitesize: Temperature, pH and enzymes. (2014). Bbc.co.uk. Retrieved 6

December 2017, from
http://www.bbc.co.uk/schools/gcsebitesize/science/add_aqa/proteins/proteinsrev3.sht
ml

 Factors affecting Enzyme Activity | A Level Notes. (2016). Alevelnotes.com. Retrieved

6 December 2017, from https://alevelnotes.com/Factors-affecting-Enzyme-
Activity/146

 pH and enzymes. Academic.brooklyn.cuny.edu. Retrieved 6 December 2017, from

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm

 Dutta.R, Fundamental of Biochemical Engineering, Springer, New York, 2008, pg 31

 Factors Affecting Enzyme Activity, 2012, Encyclopedia Britannican , (Online).

(Accesses on 17th.September 2012). Available from World Wide
Web:http://www.britannica.com/EBchecked/topic/189245/enzyme/2123/Factors-
affecting-enzyme-activity

Page | 12