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ABSTARCT

The main objective of this experiment is to extract the amylase enzyme. The amylase
enzyme was being extracted using natural source which is Bacillus Substilis. The experiment
was divided into two part. At the beginning of the experiment, the medium for the experiment
was prepared. The broth containing 1% of soluble starch, 0.3% of peptone, 0.025% of
magnesium sulphate and 0.025% of potassium chloride. The medium then was sterilized in
autoclave machine at 121C for 3 hours. After it was sterile, it was inoculate and placed in
incubate at 200 rpm and 37C for 24 hours. The media was kept inside the incubator to let the
fermentation occur. After the fermentation, the formation of enzyme will occur. When the
enzyme was extracted, the experiment is being continued by testing the presence of enzyme.
In order to observe the presence of reducing sugar, the DNSA reagent was used. The enzyme
was added with starch in order to observe the enzyme activity. The starch was used because it
is polysaccharide instead of glucose. The presence of amylase enzyme will break down the
starch at let the DNSA change colour. The DNSA will change from yellow to red orange to
indicate the reducing sugar. In this experiment, there will be a control sample. The control
sample was place in the boiled water. The boiled water will let the enzyme denatured. Lastly,
the OD value are measured by using the spectrophotometer. The distilled water was use as a
zero blank in spectrophotometer to help the value of OD to be more accurate. At the end of
experiment, the colour change and the difference of OD value will be observed. Higher OD
value for high concentration of reducing sugar and red orange colour change of DNSA for the
presence of reducing sugar. The average absorbance value that obtained from this experiment
is 0.322.

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INTRODUCTION

Microbial enzymes are widely used in industrial processes due to their low cost, large productivity,
chemical stability, environmental protection, plasticity and vast availability (Burhanet al., 2003).
Amylase is one of the most important enzymes that had been used in various roles in modern
biotechnology era and constituting a class of industrial enzymes having approximately 25-30% of
the world enzyme market (Azad et al. 2009). Corn flour was found to be a suitable natural source
for maximum production of the enzyme amylase. Amylases are important enzymes in nature and
are synthesized microorganisms. There are two types of amylases that identified in microorganism
that is a-amylase and gluco-amylase.
Amylases can form about 25% of the enzyme and it is widely used in pharmaceuticals and
sugar, brewing, textile, distilling and paper industries. The production scale of this enzyme is
necessary at low costs. The production in submerged fermentation is high and the contents of
synthetic media also very expensive and so its better to change to more economically available
agricultural by-product so the cost can be reduced. The most effective amylase is the one that is
thermo stable. It is because in this state, the enzyme that use in application minimize contamination
risk and reduce reaction time, thus enabling considerable energy saving. The application of this
thermo stable amylases enzyme is in liquefaction of starch at high temperature and also in
saccharification of starch in baking.

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AIMS

Objectives of this experiment is to:

1. To extract the amylase enzyme produced


2. To quantify the enzyme activity

THEORY

Amylases are important hydrolase enzymes that have been widely used. These enzymes
break down starch or glycogen. Among the three categories of amylase (Alpha-amylase, Beta-
amylase and Amyloglucosidase, AMG), Alpha-amylase or -Amylase has the highest demand due
to its various range of applications in the industrials. Most of the Bacillus species can synthetics
and produce extracellular alpha-amylase. Due to these reasons, Bacillus subtilisis is used.

There are numbers of techniques which can be used to determine the amylase activity. The
most quantitative procedures generally used involve the measurement of new reducing groups
formed upon amylolytic process. And another objective of this experiment is to quantify the
enzyme, which dinitrosalicylate (DNS) is chosen as it is reliable and simple.

Since DNS is used as the reagent, the glucose reduces the pale yellow-colored alkaline of
DNS to orange-reddish colored salicylic acid. The concentration of glucose presents in the solution
causes the changes in the intensity of the color, which then will be measured using a
spectrophotometer with 540nm wavelength. That specific wavelength is used because it is the
region of where the absorption occurs for orange to red shade.

A standard curve or also known as a calibration curve that is used as a quantitative research
technique. Samples with known properties are measured and graphed, and then the same properties
are to be determined for unknown sample. In this case, it is drawn to describe the absorbance
values into concentration. And based on the graph, it can be seen that the relationship between
concentration and absorbance in this experiment is linear.

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APPARATUS AND MATERIAL

List of material used:


1. Bacillus Subtilis culture in petri dish
2. Bacteriological peptone
3. MgSO4.7H2O
4. KCI
5. Starch
6. Distilled water
7. 1% of soluble starch
8. Citrate-phosphate buffer (pH 7)
9. DNS reagent

List of apparatus used:


1. 500 ml Shake flask
2. Cotton wrapped
3. Aluminum foil
4. Incubator
5. Centrifuge tube
6. Centrifuge machine
7. Dialysis tube
8. Beaker
9. Centrifuge tube
10. Pipette

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PROCEDURE

A. Preparation of 1% Starch Solution.


1. 1 g of starch powder was dissolved into 100 ml of distilled water.
2. The mixtures were stirred on a hot plate to make it easier to dissolve.

B. Preparation for Bacterial Amylase.


1. 3.0 g/L of bacteriological peptone, 0.25 g/L of MgSO4.7H2O, 0.25 g/L of KCl were
dissolved into 100 ml of 1% starch solution in a beaker.
2. Then the medium was poured into a 250 ml shake flask and was capped with a cotton wool.
3. The medium was placed into an autoclave machine at 121C for 3 hours.

C. Inoculation Process.
1. A loop of bacterial culture was added into the amylase medium in the shake flask via
aseptic technique.
2. The shake flask was capped back with a cotton wool.
3. Then, it was incubated at 200 rpm and 37C for 24 hours.

D. Extraction of Enzyme.
1. After 24 hours of incubation process, the bacterial culture were transferred into two 50 ml
of falcon tube.
2. Both of the falcon tubes were centrifuged at 5000 rpm for 20 minutes with a refrigerated
centrifuge that was set at 4C.
3. The crude enzyme was extracted by decant the supernatant into the new falcon tube.
4. The enzyme was put into a refrigerated for the next day experimental procedure.

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E. Demonstration of Enzyme Activity.
1. 1 ml of the enzyme was pipette into a test tube.
2. 1 ml of 1% starch solution was added with pH 6.5 buffer.
3. The mixture was incubated in a water bath at 40C for 30 minutes.
4. 1 ml of DNSA reagent was added into the test tube.
5. After that, the test tube that contains the mixture was placed in the boiling water for 5
minutes.
6. The test tube was then let to cool down at room temperature.
7. Then, the mixture solution were analyzed by measure it optical density by using
spectrophotometer at 540 nm.

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RESULT

Glucose standard curve

No Concentration Absorbance
(g/L) OD
1 0.2 2.168
2 0.4 3.012
3 0.6 3.257
4 0.8 3.34
5 1.0 3.364

Absorbance value vs concentration of


glucose
5

4.5
y = 4.3766x
4

3.5
ABSORBANCE VALUE

2.5

1.5

0.5

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
CONCENTRATION (G/L)

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Absorbance values

First trial : 0.318

Second trial : 0.325

Third trial : 0.322 (average)

From the graph,

For the solution contain glucose, the glucose concentration is 0.03 g/L.

SAMPLE CALCULATION

1. Media Preparation for Bacterial Amylase Production:

a) Bacteriological Peptone

The concentration of the bacteriological peptone = 6.0 g/L

The volume needed = 100 mL

6.0 x 100 mL

= 0.6 g

b) MgSO4.7H2O

The concentration of the MgSO4.7H2O = 0.5 g/L

The volume needed = 100 mL

0.5 x 100 mL

= 0.05 g

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c) KCl

The concentration of the KCl = 0.5 g/L

The volume needed = 100 mL

0.5 x 100 mL

=0.05 g

d) Starch

The concentration of the Starch = 1.0 g/L

The volume needed = 100 mL

1.0 x 100 mL

= 0.1 g

2. Starch Preparation (1% of starch):

Volume of the starch = 1000 mL

0.01 x 1000 mL = 10 g

So, the weight of the starch needed is 10 g to make sure the concentration is 1% of starch.

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DISCUSSION

The experiment is conducted to extract of commercially important enzyme from natural sources
and to analyse the development of enzyme assays quantification of enzyme activity and specific
activity. The bacteria Bacillus Subtilis are being used to produce amylase enzyme. The media used
in the bacterial amylase production are 0.6 g peptone, 0.05 g magnesium sulphate, 0.05 g potassium
chloride and 0.1 g starch. The fermentation media was supplied with starch as carbon source and
also as the enhancer for the amylase production. This is to provide an optimum condition for the
microbial growth. The bacteria is then being cultivated for 24 hours for it to provide sufficient
amylase enzyme for analysis. After 24 hours, the culture is centrifuge to separate the enzyme from
the bacteria and the culture. After the centrifuge process the supernatant is taken to test for any
enzymatic reaction present.

Bacillus subtilis is considered the best in secreted enzyme production and used on an
industrial scale by biotechnology companies. During the fermentation process the enzyme are
supplied with starch solution to enhancing the production of amylase enzyme. Amylase are
enzyme that break down starch to glucose. Thus, providing starch to the fermentation media can
later providing the situation where the bacteria will have to secrete enzyme to convert the starch
to glucose that are needed for their nutrient intake. According to a study conducted by a team in
2014, Bacillus subtilis bacteria has higher rate of amylase secretion if starch are used as carbon
source compared to glucose, fructose, lactose and maltose.

The glucose standard curve is constructed as a reference to find the glucose concentration
based on the absorbance value of the bacteria. From the result, the amount of glucose concentration
could be determined in the glucose standard curves. The average OD value for the sample is 0.322.
From the graph, it can be seen that the glucose concentration is 0.03 g/L. Based on the official
Science school website, it states that the deeper the colour of the solution, the higher the absorbance
measurement. The solution contain glucose has dark yellow colour since the starch has converted
to glucose. The conclusion that can be made from the experiment is that there is an enzymatic
activity and the bacteria is capable of producing the enzyme.

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CONCLUSION

Amylase is the unique class of enzymes, since they are the immense physiological as well
as commercial importance. Studies were carried out to extract the amylase enzyme produced by
Bacillus subtilisis and to quantify its activity using assay method. The activity of alpha-amylase is
investigated using absorbance value from spectrophotometer. The absorbance values of solutions
contain starch for the first, second and third trial is 0.318, 0.325 and 0.322, respectively. By
comparing solution containing starch and glucose, it can be seen that the amount of absorbance
value of glucose concentration is higher to be compared with solution containing the starch. This
is because the enzyme was inactivated in the starch solution, meanwhile in the solution that
consists of glucose, the enzyme is still highly active converting starch into glucose. Overall, the
objectives of this experiment are achieved.

RECOMMENDATION

1) To avoid contamination on the sample and for hygiene purposes, use glove during conducting
the experiment.

2) Weight the sample properly to get the accurate amount needed for preparing every solution anad
to get the accurate concentration of solution.

3) Prepare all the solution needed under laminar fume hood to keep the sterile condition.

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REFERENCES

Azad MA, Bae JH, Kim JS, Lim JK, Song KS, Shin BS, Kim HR (2009). Isolation and
characterization of a novel thermostable alpha-amylase from Korean pine seeds. N
Biotechnology 26:143-149

Burhan A, Nisa U, Gokhan C, Omer C, Ashabil A, Osman G (2003). Enzymatic properties


of a novel thermophilic, alkaline and chelator resistant amylase from an alkalophilic
Bacillus sp. Isolate ANT-6. Process Biochemistry 38:1397-1403

Fogler, Scott H. (2011). Elements of Chemical Reaction Engineering, Englewood Cliffs:


Prentice hall.

Shuler, M. L., & Kargi, F. (2002). Bioprocess Engineering. United States of America:
Prentice Hall.

http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=BEE19E80C95ECAB8A5EB46
75F16A6909?doi=10.1.1.674.8223&rep=rep1&type=pdf

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APPENDICES

Figure 1: Autoclave machine Figure 2: Substances for prepare


the sample

Figure 3: Refrigerated centrifuge


machine

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