CPB30103 Biochemical Engineering

Experiment 2: Enzyme Assays and Factors Affecting

Enzyme Activity Page |1

SUMMARY

In this experiment, the main objective was to study the physical characteristics and
properties of enzyme and factors affecting their activity. This experiment had two parts which
were Part 1 (reagents had been completely prepared by the lab assistances) and Part 2. In Part 2,
we were using the UV-Vis spectrophotometer to get the result. In Part 2 (A) the effect of
substrate concentration on enzyme activity and Part 2 (B) the effect of pH on the enzyme
activity, the concentration of the reducing sugar that was obtained were 0.34 g/L and 0.001 g/L,
respectively. There are some factors that affect the enzyme activity of amylase which are the
temperature, pH and the substrate concentration. Thus, the objective of this experiment to assign
in order for the student to comprehend physical characteristic and properties of enzyme and to
study the factors affecting their activity is a success.
CPB30103 Biochemical Engineering
Experiment 2: Enzyme Assays and Factors Affecting
Enzyme Activity Page |2

OBJECTIVES

1. To study the effect of absorbance against standard curve of concentration of glucose (g/L)
by preparing standard glucose solution at the beginning of the experiment.
2. To study the physical characteristics and properties of enzyme and factors affecting their
activity.
3. To determine the optimum temperature for the optimum activity of amylase.
4. To study the effect of different substrate concentration and pH on enzymes activity.
5. To study the effect of pH against concentration of reducing sugar (g/L) by referring the
glucose standard curve.

RESULTS AND DATA

CPB30103 Biochemical Engineering
Experiment 2: Enzyme Assays and Factors Affecting
Enzyme Activity Page |3

Part 1(b) Preparation of the glucose standard curve

Table 1: Data for glucose standard solution

Concentration of glucose (g/L) Absorbance(/)

0 0
0.20 0.100
0.40 0.255
0.60 0.391
0.80 0.539
1.00 0.627

0.7

0.6 f(x) = 0.66x - 0.01

R = 0.99

0.5

0.4
Absorbance (/)
0.3

0.2

0.1

0
0 0.2 0.4 0.6 0.8 1 1.2

Concentration of Glucose (g/L)

Figure 1: Standard curve of Concentration of Glucose against Absorbance

Part 2(a) Effect of Substrate Concentration on Enzyme Activity

Table 2: Data for Substrate Concentration Effect

CPB30103 Biochemical Engineering
Experiment 2: Enzyme Assays and Factors Affecting
Enzyme Activity Page |4

Concentratio Optical Concentration of reducing Amylase activity (U)

n Substrate Density sugar g / min
(% w/v)
0.5 0.140 0.227 7.567x10
1.0 0.227 0.360 1.200x10
1.5 0.326 0.511 1.703x10
2.0 0.418 0.652 2.173x10
3.0 0.569 0.882 2.940x10

0.6
f(x) = 0.17x + 0.06
0.5 R = 1

0.4

0.3
Optical Density

0.2

0.1

0
0 0.5 1 1.5 2 2.5 3 3.5

Concentration Substrate

Figure 2: Concentration Substrate against Optical Density

Part 2(b) Effect of pH on Enzyme Activity

Table 3: Data for pH Effect

pH Optical Concentration of reducing Amylase activity (U)

Density sugar g / min
0 0 0 0
5 0.194 0.310 1.033x10
CPB30103 Biochemical Engineering
Experiment 2: Enzyme Assays and Factors Affecting
Enzyme Activity Page |5

7 0.232 0.368 1.227x10

8 0.173 0.278 9.267x10
9 0.008 0.026 8.667 x10
11 0.009 0.023 7.667 x10

0.25

0.2

0.15

Optical Density
0.1
f(x) = 0x + 0.1
R = 0

0.05

0
0 2 4 6 8 10 12

pH

Figure 2: pH against Optical Density

CPB30103 Biochemical Engineering
Experiment 2: Enzyme Assays and Factors Affecting
Enzyme Activity Page |6

Graph of concentration of reducing sugar (g/L) against pH

0.4

0.35

0.3

0.25

0.2
Concentration of reducing sugar (g/L)
0.15 f(x) = 0x + 0.16
R = 0
0.1

0.05

0
0 2 4 6 8 10 12

pH

Figure 4 Concentration of reducing sugar (g/L) against pH

DISCUSSION
CPB30103 Biochemical Engineering
Experiment 2: Enzyme Assays and Factors Affecting
Enzyme Activity Page |7

The objective of this experiment is assign in order for the student to comprehend physical
characteristic and properties of enzyme and to study the factors affecting their activity. Basically,
this experiment is divided into two parts which are part one and part two. In part one, it is about
the preparation of the DNS reagent and the preparation of the glucose standard curve. For part
two, it basically to give the result from the reaction of the enzyme. The test in part two is two
study about the substrate concentration on enzyme activity and the effect of pH on the enzyme
activity. Enzyme act as the catalyst in an experiment where its function is in order to reduce the
activation energy which needed to start a reaction.

The relationship between the enzyme and temperature is said to be directly proportional
based on the theory. It means that the enzyme activities are increasing as the temperature is
increased but too high in temperature will cause the enzyme to be denature. As the temperature
are increased, it will cause the kinetic energy to be increased at the same time and this will leads
to more energetic collision. Furthermore, more energetic collision will produce large number of
kinetic energy converted into the potential energy thus the activation energy of exergonic
reaction can be achieve. The exergonic reaction is when the free energy of the final state is lower
than the free energy at the initial state. As the temperature of a system is increased it is possible
that more molecules per unit time will reach the activation energy then leads to increase of the
rate of reaction. In addition, in order to perform the reaction, enzyme must collide and bind with
the substrate at the active site. As the temperature increased, the number of collision between the
enzyme and substrate will increased. Besides that the increased in temperature also will
increased the internal energy of the molecules such as the translational energy, vibrational energy
and also the rotational energy. This all of heat sources can then converted to potential energy in
other to increased the rate of the reaction. If the temperature is to be increased to high, the
denature process will happens where it will cause in changing the shape of the active site of
enzyme which cause its reaction to slow down until the shape has changed so much that the
substrate no longer fits. This will cause the enzyme to be permanently damaged and will stop the
reaction.An enzyme will work best at a particular optimum condition. Enzymes usually work in
warm conditions around 35C to 40C unlike chemical catalysts which often work when they are
very hot.
CPB30103 Biochemical Engineering
Experiment 2: Enzyme Assays and Factors Affecting
Enzyme Activity Page |8

In another test, it was assigned to study the relationship between the rate of enzyme reaction
when there are increasing in the concentration of the substrate. I the amount of the enzyme is
kept constant and the substrate concentration is increased, the reaction velocity will increased
until it reaches a maximum point. After it reach that point, any addition of substrate
concentration will not increase the velocity. From the theory, as it reach the maximum velocity,
all of the enzyme has been converted to enzyme substrate complex (ES). By using the maximum
velocity and equation, Michaelis has developed a set of mathematical expression to calculate
enzyme activity in terms of reaction speed from measurable laboratory data.

Km=K-1 + K2 / K+1

From the Michaelis theory, small km (Michealis constant) indicates small amount of substrate
needed in order to achieve maximum velocity. Large km means that need of high number of
substrate concentration. From our results it shows that as the substrate concentration is increased,
the optical density also increased. It means that the logarithmic ratio of the intensity of
transmitted light to the intensity of the incident light passing through the substance. Higher the
number of optical density shows higher number of absorbance of a substance.

From the third test, it is in order to study about the pH effect on the enzyme activity. From
the result obtained, the optimum pH for the enzyme to have maximum enzyme activity is at pH
7. After that pH, the enzyme activity are looking to decreased. This can be explained by as the
pH increased, it will cause the effect towards shape and also change properties of the substrate so
that the substrate cannot be bind with the active site and cannot undergo the catalysis. In
addition, for each enzyme it has the specific pH optimum. From our result, it was proved that the
optimum pH for amylase is 7 based on theory stated.
CPB30103 Biochemical Engineering
Experiment 2: Enzyme Assays and Factors Affecting
Enzyme Activity Page |9

CONCLUSION

This experiment have three parts. The first part is about how to prepare the DNS reagent and to
prepare glucose standard curve. The second part is about the reaction of the enzyme. An enzyme
must have an optimum pH at a certain temperature to reach its maximum activity. If the
temperature too high then the active site will damaged and the substrate cannot bind. Thus, the
reaction cannot occur. If the pH keeps on change, the active site will change as well. Hence, the
reaction cannot occur. Enzyme functioning as a catalyst in an experiment in order to reduce the
activation energy which needed to start a reaction. The third part, was to study the relationship
between the rate of enzyme reaction when there are increasing in the concentration of the
substrate. Increasing substrate concentration increases the rate of reaction. This is because more
substrate molecules will be colliding with enzyme molecules, so more product will be formed.
This will go on until it reached a certain concentration as the enzyme will become fully
saturated. Thus, the objective of this experiment to assign in order for the student to comprehend
physical characteristic and properties of enzyme and to study the factors affecting their activity is
a success.

QUESTIONS

1. Discuss about other applications using enzyme assay. For example, what does the
portable glucometers used by diabetic patiences measure? How do they measure it?
Other application of enzyme assay is for toxicology water testing and as glucose
monitoring. Glucometers use 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. Thats how they monitor their blood sugar levels.
CPB30103 Biochemical Engineering
Experiment 2: Enzyme Assays and Factors Affecting
Enzyme Activity P a g e | 10

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

application of enzymes.
With optimum pH at a certain temperature, an enzyme will shows the maximum activity.
If you changed the pH, these active sites can damaged or changed their shape. The
substrates can no longer fits the active site and reaction cannot happens. Meanwhile, for
temperature, if u set the temperature too high it will cause the frequencies of collisions to
increase as the molecule moves faster when heat is applied. If u increase the temperature
further, the active site will damaged and substrates can no longer fits.

REFERENCES

1. Enzyme. (2001). Wikipedia. Retrieved from https://en.wikipedia.org/wiki/Enzyme

2. The effect of pH on enzyme activity. (2014). Brooklyn College City. University of New
York. Retrieved from
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm
3. Robert. K., S. (2002). Enzyme Activity and Assays. La Trobe University, Bundoora,
Victoria, Australia.
4. Michaelis-Menten kinetics. (2003). Wikipedia. Retrieved from
https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics
5. Dutta, R. (2008). Fundamental of Biochemical Engineering, Springer, New York, pg.312
6. Factors Affecting Enzyme Activity. (2012). Encyclopaedia Britannican. Retrieved from
http://www.britannica.com/EBchecked/topic/189245/enzyme/2123/Factors-affecting-
enzyme-activity

APPENDICES