1.

Abstract
This enzyme assays based on UV-Vis spectroscopy laboratory was conducted to study
the reaction rate of beta amylase ender controlled condition, to determine the standard
curve of the maltose, absorbance versus molarities and to study the purification of
enzymes from sweet potatoes. Sweet potatoes were used to extract starch because it
has high concentration of starch. As the experiment goes by we can concluded that
amylase is optimum at pH 10 to pH 11, maltose works in the range of pH 7 to pH 8
and from the constructed standard curve of maltose we observed that the higher the
volume of maltose the higher the absorbance value.

2. Introduction
The experiment serves as an introduction to the ideas behind the protein purification
and determination of enzyme activity. Enzymes are unstable molecule with a definite
psycho chemical organization. Even a slight change in this organization reduce the
activity of enzyme and sometimes the enzymes is totally inactivated. Therefore the
enzyme have to be isolated under control conditions of pH, ionic strength and
temperature. Since they are proteinacious in nature, standard extraction and
purification procedures for enzymes are such as those used for protein. (Laboratory
Manual, n.d)

The experiment goals is to study the reaction rate of beta amylase under controlled
condition, to determine the standard curve of the maltose, absorbance versus
molarities and to study the purification of enzymes from sweet potatoes.

In this experiment, there will be two parts. First, the preparing of maltose standard
curve. Maltose standard curve is used to determine the reaction rate in micromoles
maltose formed per minute. Maltose standard curve is important as it will be
compared with the result from the second part of experiment. Maltose also known as
maltobiose or malt sugar, is a disaccharide formed from two units of glucose joined
with an (14) bond, formed from a condensation reaction. The isomerisomaltose
has two glucose molecules linked through an (16) bond. Maltose is the second
member of an important biochemical series of glucose chains. Maltose is the
disaccharide produced when amylase breaks down starch. It is found in germinating
seeds such as barley as they break down their starch stores to use for food. It is also
produced when glucose is caramelized. The -amylases are found in plants, sweet
potatoes, soybeans, barley and wheat and are also in bacteria. Theses amylases

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 produce -maltose and -limit dextrins, as a result of the enzymes futility of
hydrolyzing (16) branch linkages.

The second part of this experiment is the preparations of stock enzymes from sweet
potatoes. Enzyme activity is to be view by plotting the graph of absorbance versus
concentration. We are using the sweet potatoes to get the stock of enzyme.
Centrifugation is important in this step as we have to get the very pure supernatant to
get the stock of enzyme. The supernatant is undergone the precipitation by ammonium
sulphate to get the stock of enzyme in this experiment, we should expect there will be
error as the enzyme will decrease in its effectiveness due to some chemical and
physical reaction.

3. Objectives
In this experiment, we are required:
1- To study the reaction rate of beta amylase under controlled condition.
2- To determine the standard curve of the maltose, absorbance versus molarities.
3- To study the purification of enzymes from sweet potatoes.

4. Theory
Enzyme assays are performed to serve different meaning; firstly to identify a special
enzyme, to prove its presence or absence in a distinct specimen, like an organism or a
tissue and secondly to determine the amount of the enzyme in the sample. For the first
purpose, it is the qualitative approach, a clear positive or negative result is sufficient,
the second one is the quantitative approach must deliver data as exact as possible.
Enzyme can be identified by their catalysed reactions contrast with other components
which is one of the advantages of enzymes. (Bisswanger, March, 2014)

A technique that measures interaction of molecules with electromagnetic radiation is

called spectroscopy. Light in the near-ultraviolet (UV) and visible (Vis) have a range
of electromagnetic spectrum has energy about 150 400 kJ /mol. Spectroscopy
absorption is usually performed with the molecules dissolved in a solvent, for
example aqueous buffers. The absorbance of the solute depends linearly on its
concentration. Therefore, absorption spectroscopy is perfectly suited for quantitative
measurements. Not only chemical nature of a molecule but also molecular
environment of its chromophores that the wavelength of absorption and strength of
absorbance dependent on. Spectroscopic measurements are very sensitive not
destructive and need only small amounts of material for analysis. Spectrophotometers
are common laboratory device. They contain 2 light sources which are deuterium

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 lamp that emits light in the UV region and a tungsten-halogen lamp for the visible
region. The light that passes through a monochromatic illuminator or through optical
filters, the light is focused into the cuvette and the amount of light that passes through
the sample is detected by a photomultiplier. (Schmid, 2001)

Figure 4.1: Ultraviolet-visible Spectroscopy

5. Apparatus and Materials

Apparatus:

a- Meat grinder

b- Sorvall SS-34 rotor at 12000 rpm

c- Centrifugal bottle
d- Spectrometer
e- Cuvette
f- Universal bottle
g- Penimbang electric
h- Beaker

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Materials:

a- Sweet potato

b- Acetic acid buffer (pH 3, 5, 7, 9, 11)

c- Ammonium sulphate
d- Maltose
e- Distilled water

6. Methodology

A) Maltose standard curve

1. 0.1 g of maltose was dissolve in approximately 80 mL of distilled water and
heaved the final volume up to 100mL with distilled water so that each mL of
the solution contained 1,0 mg of maltose.
2. In 2 mL of distilled water, ten appropriate dilution were made in the range of
100-1000 L mL-1 .
3. 2 mL of DNS reagent was added in each test tube containing dilutions.
4. The mixture were boiled for 5 minutes at 100C in water bath and then was
cooled at room temperature.
5. The volume is then raised up to 20 ML with distilled water.
6. A blank was run parallel with 2 mL distilled water (without maltose) and 2 mL
of DNS reagent.
7. The absorbance was measured at 540 nm on spectrophotometer.
8. A graph was plotted between absorbance and maltose concentrations.
9. The slope of the variable was calculated and used for the measurement of
reducing sugars released by the action of amylase.

B) Getting stock of active enzyme fom sweet potatoes.

1. Two sweet potatoes is grinded in a meat grinder and then mixed with a
quantity of water equal to half of mass of the pulp in the blender for two
minutes.
2. Insoluble debris is removed by centrifugation for 15 minutes in Sorvall SS-34
rotor at 12000 rpm.
3. The enzyme is separated from the supernatant by precipitation using
ammonium sulphate.
4. Ammonium sulphate is added in the amount of 0.47 g per mL of supernatant
and the precipitate is harvest by centrifugation at 12,000 rpm for 10 minutes.
5. Pallets are suspended with 1 mL of water for each 8 mL of original pulp and
recentrifuged (12,000, 10 minutes)

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 6. Supernatant were saved cold and should contain active enzyme.

C) Determination of the amount of amylase activity

1. 0.1 mL of the preparation is mixed with 0.1 mL of 1% starch and 0.1 mL of
pH 5 buffer (around 0.1 M acetic acid)
2. The mixture is Incubated at 37 degrees for 15 minutes.
3. 0.3 mL DNSA reagent is added and then heated in boiling water bath for 10
minutes.
4. Then, the mixture is diluted with 4 mL of water and the absorbance is
determine at 540 nm.

7. Results
A. Analysis of Amylase Activity

Bottles Absorbance pH

1st 0.811 10.95

2nd 0.790 11.05

3rd 0.572 11.05

Average 0.724 11.02

Table 7.1: Readings of absorbance and pH for analysis of amylase activity

Amount of ammonium sulphate per 15 ml of

supernatant,

0.47 g
15 ml=0.141 g
50 ml

B. Analysis Maltose solution

Volume Absorbance pH
0.2 0.108 7.57
0.4 0.139 7.58

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 0.6 0.152 7.66
0.8 0.161 7.73

1.0 0.194 7.77

1.2 0.275 8.22

Table 7.2: the readings of absorbance and pH for analysis of maltose

solution

Standard Curve of Maltose

0.3

0.25

0.2

Absorbance 0.15
0.1

0.05

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4

Volume of Maltose Solution, ml

absorbance Linear (absorbance)

Graph 7.1: Graph absorbance versus volume of maltose

C. Calculation Serial Dilution of Maltose

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 342.3 g/mol is the amount of maltose that used in this
experiment.
Amount of maltose of maltose needed to make 10 ml of maltose
solution.
1L
342.3 g/mol 1.2 mol/L 1000 ml 10 ml = 0.6846 g of

Maltose
The dilution of maltose with water is done to make the
concentration 0.2 mol/L to 1.2 mol/L by using the equation,
M 1 V 1=M 2 V 2

a) 1.2M of Maltose Solution

(1.2 M) (V) = (1.2 M) (10 ml)
V = 10.0 ml

b) 1.0 M of Maltose Solution

(1.2 M) (V) = (1.0 M) (10 ml)
V = 8.3 ml
10 ml 8.3 ml = 1.7 ml of water is added to the dilution of
maltose solution.

c) 0.8 M of Maltose Solution

(1.0 M) (V) = (0.8 M) (10 ml)
V = 8.0 ml
10 ml 8.0 ml = 2.0 ml of water is added to the dilution of
maltose solution.

d) 0.6 M of Maltose Solution

(0.8 M) (V) = (0.6 M) (10 ml)
V = 7.5 ml
10 ml 7.5 ml = 2.5 ml of water is added to the dilution of
maltose solution.

e) 0.4 M of Maltose Solution

(0.6 M) (V) = (0.4 M) (10 ml)
V = 6.7 ml
10 ml 6.7 ml = 3.3 ml of water is added to the dilution of
maltose solution.

f) 0.2 M of Maltose Solution

(0.4 M) (V) = (0.2 M) (10 ml)
V = 5.0 ml

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 10 ml 5.0 ml = 5.0 ml of water is added to the dilution of
maltose solution.

pH versus Concentration
12
10
8
pH Values 6
4
2
0
0 0.2R =0.4
0 0.6 0.8 1 1.2 1.4 1.6

Concentration of Maltose Solution, M

pH Linear (pH)

Graph 7.2: Graph pH values versus concentration of maltose

solution.

D. Rate of Reaction in Micromoles of Maltose Formed per

Minutes
For example, when the concentration of maltose solution is 0.2 M
mol 1000 moles 1L
0.2 0.2 ml=0. 04 moles
L 1 mole 1000 ml

Reaction rate = 0.04 moles / 15 minutes

moles
-3
= 2.667 10 min

Concentration, Amount of Maltose in 2.0 ml of Reaction Rate,

mg/mL Maltose Solution, moles moles/min
0.2 0.04 2.667 10 -3
-3
0.4 0.08 5.333 10
-3
0.6 0.12 8.0 10
-2
0.8 0.16 1.067 10
-2
1.0 0.20 1.333 10
-2
1.2 0.24 1.6 10

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 Table 7.3: the readings of reaction rate in micromoles of maltose
per minutes.

8. Discussion
The experiment is conducted to observe the characterization of beta amylase in sweet
potato. Beta amylase is an enzyme that can produce beta-maltose from starch and
glycogen. The enzyme works by the process of hydrolysis. Sweet potato has high
concentration of starch. However the process does not occur in neutral condition. The
optimum condition of the enzyme to work is pH between 4 and 5. We try to induce
this condition by adding acetic acid. Acetic acid gives a slightly acidic condition for
the enzyme to work on. From the graph 7.2 we can see that as the pH increases the
concentration of maltose solution increases. There is an abundant amount of starch in
sweet potato. The concentration of maltose solution also affected the reaction rates per
minutes. As the concentration of molecule increases, the reaction rates also increases.
Reaction rates indicate the amount of micro molecule of maltose formed per minutes.
In enzyme kinetics we know that the rates of enzyme activity increases as the
concentration of substrate increases. This is because, when there is more substrate is
available for the reaction, more enzyme-substrate complex can be formed thus will
increase reaction rates. The amount of enzyme available for the reaction is also
important in enzymatic reaction. Increasing the amount of enzyme would also
increase the rate of reaction. Without any disturbance enzyme will remain unchanged
after the reaction with substrate. By increasing the amount of enzyme, there is higher
platform for attachment for the substrate to form enzyme substrate complex. Then,
there will be higher production of micro maltose formed per minutes.

Moreover, from the experiment we are able to prepare the standard curve for maltose
based on different concentration. From the standard curve we are able to determine
the volume of maltose formed based on the absorbance reading that we get. We can
match the value of absorbance reading from the value of the absorbance from the
standard curve to determine the value of micro maltose formed.

Based on the UV-Vis spectroscopy, have a concept of absorption spectroscopy or

reflectance spectroscopy in the ultraviolet-visible spectral region. The UV-Vis is used
to identify the amount absorption of ultraviolet light thus the amount of absorbance is
higher as the concentration increases. In this case we know that the amount of micro

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 molecule of maltose formed per minutes will determined the amount of absorbance
reading. Higher amount of concentration of maltose results in higher amount of
absorption readings. From the experiment we get to make the conclusion that it is
true, the amount of concentration affect the absorbance reading.

When conducting the experiment, there is several steps that is need to be taken an
extra care, so that it will not affect the results. The process to determine the enzyme
activity should be done with short time interval because the unreacted enzyme can be
denatured if it is left in room temperature. Thus, it can affect the amount of micro
molecule of maltose formed.

9. Conclusion
Based on this experiment, we already learned the reaction of beta amylase to produce
maltose under optimum condition. From the result gained during the experiment, we
can see that the amylase is working at optimum pH around pH 10 to pH 11.
Meanwhile the maltose works in pH 7 to pH 8. Other than that, we constructed the
standard curve for the maltose based on the data we gained, and we can observe that
the higher the volume of maltose solution the higher its absorbance value.
Furthermore, we can also see that the higher the concentration, the higher the reaction
rate of the maltose. Besides, we also gained the experience and skill on how to purify
enzymes from food.

10. Recommendation
In this experiment, we use sweet potatoes to get the starch needed.
Other than potatoes, we can actually use rice, corn and other staple
food. Furthermore, we used buffer solution at pH 5 in this
experiment, yet we can use buffer solution at different ranges
between 3 and 12, so that we can see experimentally the optimum
pH of the reaction rate of the enzymes. Other than that, the
readings of the absorbane is repeated three times to get the

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 averange the absorbance. Next, before taking absorbance values
the cuvette must blanks first using distilled water

11. Reference

1. Bisswanger, H. (March, 2014). Enzyme sssays.

2. Laboratory Manual. (n.d).

3. Schmid, F.-X. (2001). Biological Macromolecules: UV-visible

Spectrophotometry.

4. Stephen L. (2000). Ultraviolet/ visible light absorption spectroscopy

in clinical chemistry. Encyclopaedia of Analytical Chemistry, 1699-
1714.

5. Kettling U., Koltermann A., Schwille P., & Eigen M. (1998). Real-time
enzyme kinetics monitored by dual-colour fluorescence cross-
correlation spectroscopy. Department of Biochemical Kinetic, 1416
1420.
a. Retrieved from: http://www.pnas.org/content/95/4/1416.full

1) Mesias M., Defiesta J. Enzyme assays. Retrieved from:

http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Complex_R
eactions/Enzymes/Enzyme_Assays

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 12. Appendix

Figure 12.1: Weighing scale Figure 12.2: Sweet potato

after blended.

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Figure 12.3: Blender Figure 12.4: Ammonium
sulphate is used in

Analysis of amylase
activity.

Figure 12.5: Centrifugal chamber

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Figure 12.6: Supernatant in centrifuge bottle.

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