Biology Internal Assessment 1

Pectinase Enzyme
Aim: In this experiment I will investigate the effect of change of pH on
pectinase enzyme that will act on apple pieces of equal size to produce apple
juice.
Introduction: Enzymes are proteins that catalyze or speed up chemical reactions. In the mass
production of something such as fruit juice, the presence of certain biological catalysts is
extremely vital. Another commonly used enzyme is lipase which is used in cheese ripening.
Pectinase is an enzyme that breaks down pectin, a polysaccharide which is found in plant cell
walls. Pectinases have also been used in wine production in the 1960s. They can be extracted
from fungi such as Aspergillus niger. The fungus produces these enzymes to break down the
middle lamella in plants so that it can extract nutrients from the plant tissues and insert fungal
hyphae.
If Pectinase is boiled, it is denatured, making it harder to connect with the pectin at the active
site. In our experiment, however, by enzymatically breaking down the cell wall, pectinase
releases the juice from within the cells. It was expected that this enzyme would speed up
production, so the effect of pH on the same amount of enzyme was investigated.
Hypothesis: The apples soaked in enzyme solution of pH 4 will yield the highest volume of
juice as pectinase enzyme works best at pH 4. Enzymes are very specific to certain pH.
Variables:
Independent Variable-
The pH of the enzyme solution added was the manipulated variable. Three solutions were
made by adding 3 buffer tablets of pH 4,7 and 9.5 to water. Distilled water was used to dissolve
the tablets.
Dependent Variable:
The amount of juice collected in each case of pH solution is the measured variable, or the
dependent. In every case, all variables and circumstances are controlled, which result in
different volumes of fruit juice produced over the same period of time.


Controlled Variable:
What and How it was Controlled Why it was controlled
The volume of the enzyme added was
constant. A 10ml syringe was carefully
sterilized before adding 10 ml pectinase to
every buffer solution.
It is important to add the exact same
amount of enzyme to every setup, as we are
investigating the effect of the pH, not the
amount of enzyme.
The source of enzyme was kept the same by
creating the enzyme solution from the same
jar of pectin.
If the enzyme was obtained from different
sources, there is a possibility the results are
affected by impurities or other such
particles.
The temperature at which each setup was
carried out was constant. Each beaker was
allowed to soak in a regulated 40C water
bath.
Temperature is an important variable to be
controlled as enzymes denature at a
particular temperature above their
optimum. If temperature was not regulated,
many of the enzymes could become
dysfunctional.
The type of apple used in each trial of the
experiment was the same. This was done by
cutting the small pieces from the same apple
and splitting them into 3 small beakers.
Having the same source of apple is fairly
vital as the amount of juice in different
apples will vary and that may hamper with
results. Hence, the same apple was cut into
pieces and subject to different pH solutions.
The time that the substrate was subject to
the enzyme solution was kept constant by
monitoring along with a stopwatch.
If one set of apple pieces was soaked in
enzyme for slightly longer, we could expect
a slightly anomalous larger reading in juice
produced. Hence, to make it a fair test, they
should all be subject for the same amount of
time.
The same type of gauze or filter paper
should be used.
It is important to ensure that any one gauze
strip isnt less fine and allows tiny particles
of apple to fall through, raising the overall
volume. So, to ensure reliability, same type
is used.






Materials Required:
3 small apples
Pectinase enzyme
3 400ml beakers labeled with pH value
3 100ml beakers labeled with pH value
pH tablets (4, 7, 9.5)
water bath (40C)
gauze
funnel
thermometer
distilled water
knife
weighing scale
10 ml syringe
50 ml graduated cylinder

Method: This experiment required 3 trials, from
which the average of values was taken in order to
ensure the reliability. Same setup and procedure was
repeated separately.
1) Pectinase solution was made by dissolving 1g Pectin
to make 100cm3. An apple is cut into 3 equal pieces
with similar mass. The skin is peeled off and seeds removed.
2) These 3 pieces are then cut into smaller pieces and placed into separate small 100ml
beakers. Important to note, the total mass within the beakers should be more or less similar.
3) Alongside, in 3 separate beakers 80cm3 of distilled water are taken. Each beaker receives the
respective buffer tablet, pH4, pH7, pH9. After mixing the tablet in 80 ml each, further 20ml of
distilled water is added. These buffer solutions are then kept aside.
4) After this, 5ml of pectinase enzyme along with 5ml of the respective buffer solution is added
to each beaker. The apple pieces are allowed to soak in the enzyme solution for about 10
minutes.
5) After soaking at room temperature, the beakers with the enzyme solutions were allowed to
soak in a water bath at 40 C.
6) 3 setups are created with a funnel placed in 3 empty graduated cylinders labeled with the
respective pH solution. The mashed apples are then placed on the filter paper which lines the
funnel. From the point of addition, readings of the volume of juice produced are taken at every
2 minute interval for each cylinder.
Results DCP
Pectinase Solution pH 4
Time
Intervals
0 minutes 4 minutes 8 minutes 12 minutes 16 minutes 20 minutes
Trial 1 10ml 12ml 14ml 15.5ml 16ml
Trial 2 14ml 16ml 17ml 18ml 18.5ml
Trial 3 12ml 13ml 16ml 17ml 17.5ml
Average
juice (ml)
18.3ml



Pectinase Solution pH 7
Time
Intervals
0 minutes 4 minutes 8 minutes 12 minutes 16 minutes 20 minutes
Trial 1 7ml 8ml 8.5ml 9ml 9ml 10ml
Trial 2 14ml 15ml 15ml 17ml 18ml 19ml
Trial 3 12ml 13ml 14ml 14.5ml 15ml 15ml
Average
juice (ml)
17ml.

Pectinase Solution pH 9
Time
Intervals
0 minutes 4 minutes 8 minutes 12 minutes 16 minutes 20 minutes
Trial 1 6ml 7ml 7.5ml 8ml 8ml 9ml
Trial 2 14ml 16ml 16ml 16ml 17ml 17ml
Trial 3 10ml 11ml 12ml 12.5ml 13ml 14ml
Average
juice (ml)
15.5ml



Calculations: The
average juice
produced for every
pH was calculated
by-



Trial 1 total + trial 2 total + trial 3 total/ 3
The characters in blue in ph7 and pH 9 were not used to calculate the average as they were the
anomalous results.
Conclusion & Evaluation: As noted from research, pectinase enzy works at an optimum pH
between 4.5 and 5.5, activated at a temperature of about 45C. For this reason, the regulation of
a water bath at 40C was a possible factor behind the valid results. This experiment didnt
evaluate the effect of the enzyme, assuming that its presence already had an increasing effect
on the amount of juice produced. As observed from the results, the enzyme solution of pH 9
yielded the lowest amount of apple juice. This effectively shows the link between the optimum
conditions of an enzyme and denaturing of enzyme with unfavorable conditions. Pectinase
enzyme is thus most effective in its optimum pH.
This experiment, however controlled in design still had certain extraneous variables that
couldnt be manipulated. A stopwatch was used to monitor all the incubation periods and juice
extraction periods for each pH, and this didnt affect our results. However, in the first trial of
pH7 and pH9 we obtained certain anomalous results in the juice obtained. This was assumed to
be due to some stroke of chance, and not a significant factor. The apples may not have been
peeled efficiently for these two setups. However, in the future, it could be useful to:
Take a few more trials
Have the enzymes soaked in mashed apple(to ensure effective collision)
Test for a wider range of pH to see the extent of optimum conditions
Ensure minimal inaccuracy with less spurting of juice while grinding apple pieces.