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Juan Pablo Villanueva

ANTWERP INTERNATIONAL SCHOOL


BIOLOGY SL
THE EFFECT OF SALT IN ENZYMATIC ACTIVITY OF AMYLASE

WRITTEN BY: Juan Pablo Villanueva


Date: October 16/2015

Summary
In the investigation that was conducted, maize fruit (Amylase) was subject to
multiple saltine solutes with different salt (NaCl) concentrations and then were
placed in starch based agar in order to determine if the salt concentration of the
different maize grains, had any effect over the enzymatic activity that happens
between Amylase and Starch.

Introduction
Enzymes are proteins that exist in most if not all multicellular organisms, which
allow metabolic chemical reactions, (Such as digestion, absorption, assimilation,
etc.) to happen; in other words, they are the catalysts for most of the chemical
processes that occur in organisms; by degrading or synthetizing substances with
the aim of transforming them into usable energy by the organism. However this is
not the only instance in which Enzymes are present. Fermentation, (such as the
one in processes to manufacture beer, wine, yogurt, etc.)

is in the most part

enzymatic activity.1
Starch (C6H10O5) is an organic compound, a large carbohydrate; it is the most
common carbohydrate in human diet as it is found in potatoes, wheat, maize,
tapioca, etc. When digested, it breaks down into the glucose molecules that
compose it and the organism utilizes it for energy usage or for storage in the form
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Juan Pablo Villanueva


of Glycogen.2
Amylase is the catalysing enzyme for the breaking down of Starch, in the human
body, it is produced and secreted by the pancreas and the salivary glands in order
to aid the process of digestion, however, the conditions and the medium in which
the enzymatic activity happens, can affect the rate and resultant products; for
instance, going back to the fermentation example; when yeast is subject to a
reaction with amylase, the different temperatures of alpha and beta-amylase
resulted in different mixtures of carbohydrates.

It is worth noting that given that Amylase is an enzyme, it is also a protein, and thus
it can be denatured (Loss of quaternary, tertiary and secondary structure) by
different causes, temperature, pH, application of a strong acid or base, application
to a concentrated inorganic salt, among others.
Objective
To understand and assess the effect of salt concentration in enzyme activity.
Research Question
What is the effect of different salt concentrations on enzymatic activity?
Hypothesis
The higher the salt concentration, the less the enzymatic activity will occur, due to
the salt altering the osmotic balance of the medium.

Variables
Independent Variable:
Salt concentration (%)

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Juan Pablo Villanueva


Dependent Variable:
Diameter of iodine indicator (cm)
Controled Variables:

Temperature

Light

Variety of maize

Composition of the Starch-Agar


Control of controlled variables

Temperature: The results for the experiment were obtained at room


temperature (23C +/- 1C)

Light: The results were obtained in a totallly dark shelf.

The species of maize that was used for the investigation was dent corn (Zea
mays var. indentata)

Materials
Table 1: General and Glass Materials

6 Glass Beakers (200mL)

Weighing scale

1 Hot Plate

Cronometer

1 Thermometer

Scalpel

12 Petri Dishes

Tweezers

Masking Tape

Glass Mixer

Millimetred Grid

White Paper

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Table 2: Substances and Reactants
1 cob of Maize (Zea mays
var. indentata)

50 gr of NaCl
550 ml of Water
100 ml of Iodine Solution
Starch based agar 15.8gr

Experimental Procedure
1. The 6 glass beakers were labeled with the masking tape, with the labels:
Control, 0%, 5%, 10%, 15%, 20%.
2. All the beakers were filled with water and the corresponding amount of
NaCl, (Control is 0% as well) e.g. 10% was be 90mL water and 10 gr salt,
15% was 85mL water and 15 gr salt, and so on.
3. The beakers labeled 0%, 5%, 10%, 15%, and 20% were heated in the hot
plate order to speed up the process of NaCl dissolving.
4. When NaCl was totally dissolved the beakers were removed from the hot
plate.
5. With the scalpel, 24 maize seeds were removed from the cob.
6. 4 maize seeds were placed in each of the 6 glass beakers for 4 hours in
order for the saline solution to be absorbed by the maize seeds (Diffusion).
7. The water on the control beaker was brought to a boiling point in order to
denature the amylase in the kernels.
8. The water of all the beakers was disposed.
9. Each of the maize seeds was cut in half longitudinadly using the scalpel and
the tweezers.
10. Using the masking tape; 2 petri dishes were labeled per categorie e.g 2
dishes for control, 2 for 0%, 2 for 5% and so on.
11. Taking the corresponding half maze seeds, 4 halves were placed on each
petri dish with the cut part in contact with the agar.
12. All the petri dishes were covered by the lid, and stored in a pitch dark shelf
at room temperature(23C+/-1C)
13. After 48 hours the petri dishes were removed from the shelf.

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14. The control dishes were open and iodine solution was poured until the
surface was covered so that its colour change would indicate in which
places there was undigested starch.
15. The area of the whitened (Digested starch) agar was measured by using a
grid and white paper, and the resulting area was registered in a table.
16. Steps 15 and 15 were repeated with every petri dish.
17. The left over residues were disposed accordingly.
Raw Data
Table 3: Salt Concentration (%) vs Whitened Iodine Area (cm 2)

Salt
concentration
(%)

Area (indicator) cm2 (+/- 0.25 cm2)


Seed 1

Seed 2

Seed 3

Seed 4

Seed 5

Seed 6

Seed 7

Seed 8

0 (boilt)

0.5

1.5

1.5

0.5

0 (Raw)

4.5

5.5

4.5

3.5

3.5

3.5

4.5

10

3.5

2.5

2.5

15

1.5

1.5

1.5

1.5

20

0.5

0.5

0.5

0.5

Qualitative Observations

When observed after 48 hours; the dishes with the lower salt concentrations
(0%, 5%) showed a white margin in the agar around the maize.

Results Uncertainty

In the experimentation phase when obtaining data for the area, only half
squares from the grid were counted (0.5 cm 2) so the uncertainty in
calculations were of +/- 0.25 cm2

Data Processing
To analyse the data that was obtained from the experiment a correlation test was
performed, given that both variables were quantitative which allowed for obtaining
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a significant conclusion from an R-value or a determination coefficient found in the
correlation, for the error bars in the graph; the standard deviation was used, given
that the data used for the area was a mean from the trials so this allows me to
graphically identify the accuracy of my measurements.
Area Average = (S1+S2+S3+S4+S5+S6+S7+S8)/8
Area Average (5%) = (4+4.5 +4+3.5+3.5+4+3.5+4.5)/8
Area Average (5%) = 3.9375
The data used for the correlation graph is displayed on the following table.

TABLE 3: Salt concentration (%) vs Average of the Area (cm 2)


Salt Concentration (%)

Area Average (indicator) cm2 (+/- 0.25 cm2)

0 (Boilt)

0.875

0 (Raw)

4.875

3.9375

10

2.5625

15

1.75

20

0.25

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Graph 1: Salt concentration (%) vs Area of digested starch (cm)


6
5

f(x) = - 0.23x + 4.96


4 R = 0.99
Area of starch digested (cm)

Linear ()
Linear ()

2
1
0
0

10

15

20

25

Salt concentration (%)

r = -0.9961

Discussion of results
Starting by observing the raw data; it is easy to identify a trend; in which the
amount of salt concentration does exhibit an inverse proportion with the enzymatic
activity.
Further on when submitted to the correlation graph test, and after using the
standard deviation as the y error bars; we can observe graphically the relation
stated previously stated by examining the line of best fit as well; given than when
the error bars are taken into account; the line passes through all the data points.
Also, to show the relation quantitatively; we can take into account the r-value which
describes a very strong negative correlation between both variables. (-0.9961)

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Conclusions
From the results of the data processing it is possible to state that there is a inverse
correlation between the salt concentration and the amount of enzymatic activity
observed; based on this we can say that at least regarding Amylase, salt is a factor
that can slow the metabolic process of digestion; however a total denaturing of the
enzyme was not observed; thus why salt is often used in food conservation.
Evaluation of procedure
Factor
Temperature

Effect on the experiment


have

Improvements
Control the temperature so that it

was

would be optimal regarding the

measured but not controlled,

enzymatic activity (Around 37.5C)

Temperature
fluctuated

could
as

and results were taken over


a significant amount time (2
nights)
Variety of Maize

As I used only one species of Repeat the experiment with


maize in the experiment, I various species of maize
cannot know for sure whether
the results would carry over to
different species.

Time

The digestion process under Repeat the experiment with


suboptimal conditions can take multiple amounts of time given
long, when only given two days for the digestion to occur.
for obtaining results, the data
may have been different than
with other amounts of time
given.

References
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1. Rsc.org,. 'Chemistry For Biologists: Enzymes'. N.p., 2015. Web. 15 Sept. 2015.
2. Users.rcn.com,. 'Carbohydrates'. N.p., 2015. Web. 15 Sept. 2015.
3. Bank, RCSB. 'RCSB PDB-101'. Rcsb.org. N.p., 2015. Web. 15 Sept. 2015.

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