1.

0 ABSTRACT
This production and assay of amylase by bacteria experiment is conducted in order to
evaluate the composition of fermentation medium containing starch in cultivating Bacillus
subtilis. This experiment is also done to extract the amylase enzyme produced, thus quantify the
enzyme activity. The bacterium used in this experiment is Bacillus subtilis. Bacillus subtilis is
known as a Gram positive bacterium and possess the shape of rod and mainly found in soil. It
has the ability to produce -amylase enzyme, thus acting as the carbon source for its growth.
This experiment is started by preparing a liquid medium suited for the bacterias fermentation.
The bacteria is then inoculated from the culture and transferred into the media prepared before
and it is fermented for 24 hours. Dialysis method is also done in order to remove any residual
sugar from the mixture. Through this experiment, the -amylase concentration and enzyme
activity of B. subtilis are able to be identified. Based on the results, the glucose concentration is
0.1563 g/L and the enzyme activity is 0.0313 g/L.min. Besides, the best condition for B. subtilis
fermentation is at 37C, pressure of 1 bar, shaking frequency less than 120 rpm and for 24 hours.

2.0 INTRODUCTION
By possessing rod-shape-like bacteria, Bacillus subtilis is a Gram positive bacterium
which is commonly found in soil. B. subtilis is a bacterium that forms an endospore which allows
it to withstand extreme temperature condition and also dry environments [1]. This bacteria can
also anaerobically functioning if nitrates or glucose are present even they are considered and
obligate aerobe. Same as other bacteria, B. subtilis is present everywhere including in the air and
plant compost though it is assumed that they are mostly inactive through time and are in spore
form [1].
However, many enzymes are produced when they are active. It is known that they are
able to produce and also secrete various substances and compounds such as carbohydrolases like
protease and -amylase [2]. Therefore, production and enzyme activity of -amylase are to be
investigated in this experiment. -1,4-glucan-4-glucanohydrolase or commonly known as amylase is an enzyme that has the ability to catalyze the internal cleavage of -D-(1-4)

[1]

glucosidic bonds in starch. It is also found in other complex polysaccharides and turned them
into smaller oligosaccharides [2].

Alpha-amylase is widely used in paper, pharmaceutical, food and sugar industries as well
as in starch liquefaction. Easy production of this enzyme makes Bacillus subtilis an efficient
experimental tool in laboratory research aside from its easy genetic manipulation [1]. The alphaamylase activity is measured using calorimetric assay by DNS reagent. Due to the semiacetalic
reducer groups of DNS reagent (3, 5-dinitrosalicyclic acid), the catalytic action of amylase is
able to be hydrolyzed to fragments by the starch [4]. The optical density of the mixture then is
taken using spectrophotometer at 540 nm.

3.0 OBJECTIVES
This experiment carries out a few objectives such as listed below:
1. To extract the amylase enzyme
2. To quantify the enzyme activity
3. To find the composition of fermentation medium containing starch to cultivate Bacillus
subtilis
4.0 THEORY
Based on experiment that we have conducted, we found that the objective of this
experiment was to determine at which point of where the enzyme has the highest kinetics.
Kinetics also can be defined as velocity. One equation that relates to the enzyme kinetics is
Michaelis-Menten equation based on Michaelis-Menten model. This model was proposed by 20 th
century that is Leonor Michaelis and also Maud Leonora Menten. According to [5], enzymes are
found in every single living cell and controlling the metabolic procedures in which they changed
over supplements into energy and new cells.
The Michaelis-Menten mechanism for enzyme kinetics can be defined as follows:

[2]

Where E is enzyme and S is substrate. ES is where the substrate attaching the active site
of the enzyme. Next the reaction rates as the rate of formation of products and the kinetics
equations implied by the mechanism,

The transient species that is enzyme-substrate complex ES is set up for an equation for its
rate of change and the steady state approximation is applied as follows:

The equation for ES is solved:

And substitute the equation 3 into equation 1 gives the following new equation:

After rearranging all the equations, Michaelis-Menten constant K M is introduced to give

the following equation:

The value of KM above is then plug in into the equation 5 to give the 7 th equation to
determine rate that is:

[3]

Since the value of [E] that is the concentration of non-complexed enzyme (enzyme that is
not bind to the substrate) is difficult to find, therefore we insert the value of [E] 0 that is known
total enzyme concentration. The following equation shows how [E] and [E]0 relates:

Equation 9 is then substituted into equation 7 and gives rate = v:

Next is if we want to determine the max velocity, v max as we increased the substrate
concentration, to define vmax:

Then,

But since this is an experimental data, we would use the following equation that is:

From equation 14, if we plot graph 1/v vs 1/[S], then we will get a straight line in which
the slope is KM / VMAX and the y intercept that is 1/vmax. The enzyme -Amylase can catalyze the
hydrolysis of inward - 1, 4-glycosidic bond present in starch with the creation of lessening
sugars. In the investigation of substrate concentration on enzyme kinetics, the enzyme is kept
steady whereas the concentration of starch is taken in increasing order. As the substrate
concentration increases, the measure products produced in each progressive tube increases. This
[4]

was clarified by Michaelis and others that an enzyme catalyzed reaction at different substrate
concentrations is diphasic, that means, at low substrate concentration the active sites on particles
(enzyme) are not possessed by substrate and the enzyme rate changes with substrate atoms
concentration. As the quantity of substrate atoms builds, the enzyme accomplishes the immersion
level, subsequent to there is no more reaction sites staying for binding. So the enzyme can work
with full limit and its reaction rate is free of substrate concentration [5].

5.0 APPARATUS AND MATERIALS

Bacteriological Peptone
MgSO4.7H2O
Calcium chloride
Starch
Nutrient agar
Distilled water
DNS reagent
NaOH
Sodium potassium tartarate
Shake flask
Cotton plug
Muslin
Aluminium foil
Centrifuge
Spectrophotometer
Beaker
Universal bottles
Pipette
Test tube
Incubator
Water bath

6.0 PROCEDURE
A. Preparation of liquid medium
1) The ingredients are mixed as below for the preparation for media in 40 mL volumes
of distilled water into 100 mL shake flask.

[5]

Materials
Amount (g/L)
Bacteriological Peptone
6
MgSO4.7H2O
0.5
KCl
0.5
Starch
1
Table 6.1: Amount of ingredients required to prepare medium
2) The mixture is swirled to mix them together and the top of the flask is covered with
aluminium foil.
3) The mixture is autoclaved at 121C for 15 minutes.
B. Inoculation of B. subtilis
1) The loop is flamed to red-hot just in prior to use, burning off any organic material.
The loop is not allowed to touch anything except the specimen itself.
2) The lid of the agar plate is lifted but continued to use it as a cover to prevent
contamination.
3) The loop is cooled by touching the sterile agar surface in prior to touch the culture.
4) 5 loops of grown B. subtilis are taken on agar plates gently by touching the colony
5)
6)
7)
8)

with the wire tip.

The loop is removed from the plate and held it still as replace the lid.
The neck of the flask is passed through the flame.
The loop is put into the sterilized media of 40 mL in 100 mL shake flask.
The neck of the flask is passed again with the culture through the flame before putting

back the aluminium foil.

9) The media is fermented in incubator at 37C for 24 hours.
C. Method to extract amylase enzyme
1) The culture is poured into centrifuge tube.
2) The culture is centrifuged at 8000 rpm at 4C for 15 minutes to separate cell.
3) The supernatant is collected as it contained the crude enzyme.
D. Dialysis method
1) One end of the dialysis tube is tied and the enzyme mixture is poured into dialysis
tube.
2) The other end is also tied securely. The dialysis tube is put in the distilled water in a
beaker.
3) The crude enzyme is poured into clean universal bottles and stored it in the freezer at
about 0C until needed.

[6]

E. Method to measure enzyme activity (DNSA method)

1) 1 mL of the mixture of enzyme amylase that has been extracted and 1 mL of 1%
soluble starch dissolved in 0.1M phosphate buffer at pH7 are incubated for 15 min at
55C.
2) The blank consist of 2 mL enzyme extract that has been boiled is set up for 20
minutes and added it into starch solution.
3) The reaction is stopped by adding 2 mL of DNSA reagent and boiled them for 5 min.
4) The distilled water is added to make up the final volume of 20 mL. Then, the solution
is left to cool at room temperature.
5) The absorbance is measured at 540 nm and the color of intensity is determined.

7.0 RESULTS
[7]

Glucose Concentration

Optical Density (OD)

Real OD

0.2

0.107

0.107

0.4

0.134

0.134

0.6

0.596

0.596

0.8

0.600

0.600

1.0

0.790

0.790

(g/L)

Table 7.1: Glucose concentration on the activity of -amylase enzyme

Optical Density vs. Glucose Concentration

0.9
0.8
0.7

f(x) = 0.92x - 0.1

R = 0.89

0.6
0.5
Optical Density (OD) 0.4
0.3
0.2
0.1
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1.1

Glucose Concentration (g/L)

Figure 7.1: Graph of optical density versus glucose concentration

Fungi / Bacteria

Optical Density
(OD)

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Glucose
Concentration (g/L)

Enzyme Activity
(g/L.min)

B. subtilis

0.039

0.1563

0.0313

Table 7.2: The production of -amylase and enzyme activity of B. subtilis

Method Used

Fermentation Condition

Temperature = 37C
Pressure = 15 psi (1 bar)
Shaking frequency = less than 120 rpm
Duration of inoculation = 24 hours
Temperature = 4C
Shaking frequency = 8000 rpm
Duration of extraction = 15 min

Dialysis

Temperature = 0C
Duration of dialysis = 24 hours

DNSA

Temperature = 100C
Duration of boiling = 5 min

Inoculation of B. Subtilis

Extraction of amylase enzyme

Table 7.3: Fermentation condition of B. subtilis in each method used

8.0 CALCULATIONS
The ingredients used in liquid medium preparation are as follows:

Volume of distilled water = 40 mL

= 0.040 L

For Bacteriological Peptone:

Weight of Bacteriological Peptone ( g )=6

g
0.040 L
L
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 0.24 g
For MgSO4.7H2O:
Weight of MgS O4 .7 H 2 O ( g )=0.50

g
0.040 L
L

0.02 g
For KCl:
Weight of KCl ( g )=0.50

g
0.040 L
L

0.02 g
For starch:
Weight of starch ( g )=1.0

g
0.040 L
L

0.04 g

From Figure 7.1 (Graph optical density vs. glucose concentration):

y=0.916 x0.1042

Thus, to calculate for real OD of 0.039:

0.039=0.916 x 0.1042
0.916 x=0.1432

x=0.1563

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Therefore, the glucose concentration is 0.1563 g/L.

Hydrolysis reaction = 5 minutes

Enzyme activity=

Amount of glucose formed

time

Enzyme activity=

0.1563 g /L
5 min

Enzyme activity=0.0313

g
.min
L

9.0 DISCUSSION
In this experiment, there are several main methods are conducted which are inoculation
of B. subtilis, dialysis, extraction of amylase enzyme and DNSA method. All the procedural steps
have their own methods used in order to conduct them. The results obtained are tabulated into
three tables which are Table 7.1, 7.2 and 7.3. Table 7.1 represents the glucose concentration on
the activity of -amylase enzyme consisting glucose concentration (g/L), optical density (OD)
and the real OD. The optical densities of the glucose concentration are obtained from five
different glucose concentrations with 0.2 scales from 0.2 to 1.0 g/L. The optical densities
obtained at 0.2, 0.4, 0.6, 0.8 and 1.0 g/L are 0.107, 0.134, 0.596, 0.6 and 0.790 respectively. In
this case, the values of the real optical densities are the same as optical densities obtained. From
the values obtained, the graph of optical density versus glucose concentration is plotted and the
graph is linear as the optical density is directly proportional to glucose concentration.
The second table which is Table 7.2 represents the production of -amylase and enzyme
activity of B. subtilis. In this table, we can see that the optical density for the bacteria B. subtilis
obtained is 0.039 and the enzyme activity value is 0.0313 g/L.min. The third table which is Table
7.3 represents the fermentation condition in each method used. For inoculation of B. subtilis
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method, the fermentation condition which consists of temperature, pressure, shaking frequency
and the duration of inoculation are obtained. The bacteria are inoculated at the temperature of
37C and pressure at 15 psi (1 bar), with the shaking frequency lower than 120 rpm for 24 hours.
As the for the extraction of amylase enzyme fermentation condition, it is centrifuged at 8000 rpm
shaking frequency for 15 minutes at 4C to separate the cell. The next method which is dialysis
took 24 hours at 0C to be done. The last method in this experiment is the DNSA method in
which to measure the enzyme activity. The fermentation condition for this method is boiled at
100C for 5 minutes and the absorbance is measured at 540 nm.
There are several calculations were done to obtained the weight of the materials involved.
One of the calculations is done for the substance bacteriological peptone with the weight
obtained of 0.24g. Next, the weight of magnesium sulfate heptahydrate, MgSO 4.7H2O after the
calculation is made is 0.02g. For potassium chloride, KCl, the weight obtained is the same as the
magnesium sulfate heptahydrate meanwhile the weight of starch obtained after being calculated
is twice more than the MgSO4.7H2O or KCl which is 0.04g. From the plotted graph of optical
density versus glucose concentration, the linear equation obtained which is y = 0.916x - 0.1042,
the value of x calculated at y = 0.039 is 0.1563. Finally, the enzyme activity value is obtained by
dividing the amount of glucose formed which is 0.1563 g/L over time of hydrolysis reaction at
which is 5 minutes and the enzyme activity value calculated is 0.0313 g/L.min.

10.0

CONCLUSION
The objectives for this experiment are to extract the amylase enzyme, to quantify the

enzyme activity and to prepare the standard graph of optical density versus glucose
concentration. Based on the result obtained, we can see that as the concentration of glucose
increase, the absorbance optical density also increases. We also able to find out the suitable
condition for fermentation in every method we used and it was listed in Table 7.3. According to
the calculation shown above, we also can determine the enzyme activity using the formula. In
this experiment, the enzyme activity produced is 0.0313 g/L.min for glucose concentration at
0.1513 g/L and absorbance optical density at 0.039. As a summary, this experiment can be
considered as success because we able to achieve the objectives.
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11.0

RECOMMENDATION

There are some recommendations that can be done in order to improve the results such as:
a) Make sure that while measuring the amount or quantity of chemical, the position of eyes
are right. This is to avoid parallax error.
b) Avoid using the contaminated apparatus because it will affect the result obtained.
c) Make sure that all the contaminated materials are disposed after used it in appropriate
container.
d) While taking the absorbance optical density reading, make sure that the cuvette was
wiped cleanly to prevent any scratch that will affect the spectrophotometer reading.
e) Wear gloves while handling the experiment.

12.0

REFERENCES

1. Kirk, E. (2009). Bacillus subtilis. Retrieved on November 12, 2015 from

http://web.mst.edu/~microbio/BIO221_2009/B_subtilis.html
2. Inman, F.L., Walters, H., Holmes, L.D. (n.d.). Using -amylase from bacillus subtilis as a
catalyst for the production of sweet potato biofuels. Retrieved on November 12, 2015
from
https://www.uncp.edu/sites/default/files/Images_Docs/Departments/biotech/files/Using aamylase from Bacillus subtilis as a Catalyst for the Production of Sweet Potato
Biofuels.pdf
3. McEm, L. (July 25th, 2013). Bacteria identification help. Retrieved on November 12,
2015 from http://www.scienceforums.net/topic/77745-bacteria-identification-help/
4. Alina, C., Vasilica, P., Eugen, M., Sanda, F. (2012). Spectrophotometric studies about
amylase activity in starch hydrolysis reaction. Retrieved on November 12, 2015 from
file:///C:/Users/Calum%20Hood/Downloads/Amylase.pdf
5. Vlab.amrita.edu. (2011). Effect of substrate concentration on enzyme kinetics. Retrieved
on November 15, 2015 from http://vlab.amrita.edu/?sub=3&brch=64&sim=1090&cnt=1
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13.0

APPENDICES

Figure 13.1: DNSA reagent

Figure 13.2: Mixture in dialysis tube

[14]

Figure 13.3: Preparation of cotton plug

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