Experiment: enzyme Activity

Objetives:
To be able to hydrolyze starch with the help of enzymes in the samples
To identify prescen of enzymes in the samples y means of qualitative screening
Materials :
12 test tubes
Test tube rack
Tripod
Bunsen burner
Casserole

Wire gauze
Beaker
Graduated cylinder
Pipete
Aspirator
Wooden splint

Reagents
Saliva
Lugols solution
Strach solution
O.1N NaCl

0.05 M HCl
Copper sulfate
Distiled water

Methodology
The students first perpared all needed materials and reagents within the students
vicinity for an orderly conduction of the experimintation and to lessen the
probability of human error.
The experiment was divided into 2 parts. First the students prepared the catalse
enzyme by grating a potato with the potato skin. 50 ml of water was then
communicated to the potato in a beaker which was subjected to 10 minutes with
occassional stirring. The sample was then filtered where the remants were
thrown away by the students. The filtrate was then kept for qualitative testing.
5ml of the catalase sample was mixed with hydrogen peroxide. The students
immediatley inserted a glowing wooden splint.
The students then conducted the part B of the first part of the experiment where
in 5 ml of the sample was added with 5 ml of 3 moles of coodium hyroxide. The
students then mixed the sample by tapping the tewsttube with the palms of the
hand. 5 drops of copper sulfate was then communicated to the mixture.
For the second half of the experiment the students prepared the sample by
cllecting 10-15 ml of saliva from the students. The students then labeled 3 test
tubes where all of the 3 test tubes contained 10 ml of starch colution, 1ml of 0.1
M Na cl solution, and 2 ml saliva. Test tube 1 has an addition of 1ml of 0.05 N
HCl, test tuibe 2 has an addition og 1 ml of distilled water, while test tube 3 has
an addition of 1 ml 0.03 N 0.05 NaOH.

The students then mixed all of the test tubes and subjected the samples to a 37C
water bath where in the students added 2 drops of lugols solution every 3 minute
interval. The students stopped the addition where in the solution turns black.
Lastly for the last part of the experimentation, the students added 10 ml of the
starch solution, 1 ml of 0.1 of the sodium chloride solution. The students then
subject test tube 1 into a beaker of room temperature water, test tube 2 to a 37
degree water bath, and test tube 3 to a boiling water bath. the students then
communicated 2 ml of the saliva sample onto the test tubes and was mixed.
IV Results and discussion
During the first part of the experimentation the students isolated the ctalaase
enzyme residue which was thrown from the sample potato. The students
acquired the said enzyme by means of grating the sample into a fine pulp which
was then mixed occasionally with 50 ml water. The Students then filtered the
enzyme where as it was acquired through the filtrate and not the residue which
was not utilized in the experiment. The following observations were made when
the filtrate was subjected to qualitative testing with the correlation on what the
implications are on a molecular level.
Sample+reagent
Cataalse extract+ hydrogen peroxide

observation
Instant formation of a large foamy
substance.

In the first part of the experiment where as the students tested the presence of
catalase from the sample, 5ml of the filtrate was added with 5 ml of 5% hydrogen
peroxide. Spontaneuosly a glowing wooden splint was communicated to the
mixture. The students observed a lage formation of foam rose at instant upon the
communication of the glowing wooden splint.
The formation of the foam from the mixture concludes a positive result where as
the enzyme catalase reacted with the hydrogen peroxide. According to education
.com (2013), Catalase acts as the catalyzing enzyme in the decomposition of
hydrogen peroxide.The statement above backs up the reaction that happened in
the test tube. When catalase is communicated with hydrogen peroxide ( its
substrate) and is activated by the catalyst of heat :by means of the experiment
the glowing stick, catalase speed up the principle of decomposition to break
down hydrogen peroxide to water and oxide. The chemical equation below
ilustrates the reaction given off by hydrogen peroxide and the enzyme catalase.

sample

Observation

Cataase extract+ 3M NaOH+CuSO4

A black suspended preciptate matrixed

in the solution
During the second art of the 1st half of the experiment, the nature of enzymes
was screened by the students by the addition of 5ml og the catalase sample to 5
ml of 3 moles of sodium hydroxide; which was then shook. 5 drops of copper
sulfate was then added to the test tube with the mixture. The table above denotes
the observations made by the students from the said experiment.
Catalase is very sensitive to inhibition. Even the substrate (H2O2), especially in
higher concentrations, results in the enzyme inactivation.Sulphuric acid
immediately inactivates catalase and stops the reaction of H2O2 degradation. (
University of Massechussets Boston) according to the statement above since
catalase a very sensitive enzyme, addition of the copper sulfate will inhibit the
enzymes activity from the reaction. The students observed in the experiment by
the addition of the copper sulfate to the solution, a black precipitate form this is
due to the binding of the sulfuric acid to the catalase which caused the formation
of black precipitate meaning that the reagent is inhibiting the function of catalase.

During the second half part A of the experiment the students then tested the
factors that affect enzyme activity specifically temperature. The students used the
saliva samples and added varrious reagents which was then heated in a
controlled 37C water bath and analyzed when the solution would start to
degraded. The table below denotes the reagents and time spent in order for the
solutions to be gegraded.
Samples +differentiated
Time elapsed
Drops of Lugols
reagent
Test tube 1+1ml 0.05N
18 minutes
12 drops
HCL
Test tube 2+ 1Ml ditsilled 15 minutes
10 dops
H2O
Test tube 3+1ml 0.05 N
0 minutes
0
NaOH
The addition of Lugols solution every 3 minutes was to test whether the sample
starch was completely degraded which only came a positive result only if the test
tube turns purple to a back hue. Test tube 1 garnered the longest time which was
18 minutes until starch was hydrolyzed with 12 drops in order to confirm. Test
tube 2 garnered only 15 minutes and was confirmed after 10 drops. And astly test
tube 3 didnt need any time because even before heating the students noticed
that the starch was already hydrolyzed but to ensure it was still subjected to the
testing for 15 minutes with 2 drops in 3 minutes interval.
The reason why test tube 3 was sontaneuosly hydrolyzed was due to the added
reagent which was sodium hydroxide. Sodium hydroxide reacted with the starch
which caused alkaline hydrolysis. Alkaline hydrolysis is a simple, natural process by
which complex molecules are broken down into their constituent building blocks by the

insertion of ions of water (H2O), H+, and OH- between the atoms of the bonds that held
those building bocks together (alnmag,2010). Where was the water ion is present in the
sodium hydroxide and hyrolusis the glycosidic bond which contain oxygen. Test tube no
2 laso used alkaline hydrolysis but since water is only baseline alkaline, the amylase
present in the saliva had a more contribution
Test tube 1 took the longest because HCL denatures the salivary amylase present in the
test tube cancelling out the reaction. The principle on what hydrolyzed the starch is due to
the water present in the starch solution and also degredation due to heat.
Test tube no.

Type of heat subjected

time

Room temp water bath

2
3

37C water bath

Boiling water bath

until end
time of
experimenta
tion
15 mins
12 mins

Drops of
lugols
10 drops +
until end of
experiment
10 drops
8 drops

The students used the same reagent controls in the experiment but differentiated the type
of temperatures where in they will be subjected at. All of the samples contanied the starch
solution, NaCl, and the saliva sample. Test tube 1 was subected to a room temp water
bath where in the students added 2 drops of the lugols solution until the experimentation
ended; this is due to the fact that the amylase hasnt been reacted with the starch sample
due to the lack with the help of any heat. The students observed that there may be a
definite time frame where in starch will be hydrolyzed in that kind of enviroment but due
to the lack of time, the students have not figured or observed the definit time.
Test tube 2 was subjected onto a regulated 37 C water bath where in it took 15 mins and
10 drops of lugols solution in order for the oncifrmation. The results were similir to the
time frame observed in the previous part of the experimentation. Thus may be due to the
similar heat communicated to the substances.
Lastly for the third test tube, a boiling water bath was communicated to the sample. It
took the sample 12 mins with the help of 8 drops of the lugols solution. Due to the high
heat added, it helped catalyzed the solution in hydrolysis of starch with the amylase. If
more heat and a longer period of time was to be observed amylase would also have
undergo degradation.
According to chem blogspot(2015)Lugol's iodine yields a blue-black color in the
presence of starch. The statement above shows the use of the lugols solution in the 2 parts
of the last half ot he experimentation. The iodine would stick into the chains of the starch
creating a blue-black complex. If when the tested sample would create this reaction, it
means that the strach is still not hydrolyzed as per the experiment. If the solution would
give just a black hue it would mean a positive result for which the starch would be
hydrolyzed and the glycosidic chains are broken. The figure below denotes the

illustration on how the mechanism of lugols solution works

Accorfing to
bbc( 2010) the use
of heat in the
reaction helps the
enzyme achieve its
maximum
potential. This was
observed in part 3
of the experiment
for which the boiling water bath garnered the fastest resulting time in which
starch was hydrolyzed. The heat helped the amylase speed up the reaction. IN
collinear with this the test tube 1 got very ngative results because only room
temp was used as the heat source.
References http://www.education.com/pdf/activator/
http://www.umb.edu.pl/photo/pliki/WL_jednostki/zaklad_biochemii_lekarskiej/pdf/
biochemistry_workbook.pdf
http://www.alnmag.com/articles/2004/08/alkaline-hydrolysis-process
http://generalchemistrylab.blogspot.com/2011/12/iodine-test-for-starch-andglycogen.html
http://www.bbc.co.uk/schools/gcsebitesize/science/add_aqa/proteins/proteinsrev
3.shtml