Optimal Conditions of Digestive system’s Enzymes
Amy, Argin Haritounian, Lizztte, Zaniadda
11/18/2013
We have three main enzymes in our digestive system that are responsible to
break down and degrade three main source of food that we have such as,
carbohydrates, protein, and fat. These three enzymes respectively are amylase,
pepsin, and lipase. These enzymes need to be in right condition in order to work at
their best rate. Just like many other enzymes and other proteins in our body, these
enzymes need to be in certain environment, temperature, PH, or even presence of
other substances to maximize the yield. Amylase is usually found in our mouth,
pepsin in our stomach and lipase in small intestine. So, their optimal condition will
be different for each one of those. Based on a series of experiment that we have
done on each enzyme, we realized the optimal conditions for each enzyme
separately. For the amylase we mainly need the enzyme and body temperature of
37°C; the pepsin should be in acidic environment, body temperature, and the
enzyme; and lipase need to be around body temperature, and presence of bile as
emulsifier.
Human body has twelve organ systems, which digestive system is one of
those. Digestive system is composed of oral cavity, esophagus, stomach, small
intestine, and colon. Whenever we eat something, the food travels all the way down
to our colon, however, that food needs to be broken down into smaller pieces in
order to be absorbed by our body in the form of energy to do work. In order to
break down the food we have different proteins called enzymes, which help us to
break down the food to smaller pieces. The three main source of food that we
usually have are carbohydrates, proteins, and fats. These enzymes need to be at
certain conditions in order to do their job perfectly and maximally. We want to test
the optimal conditions for our main digestive enzymes that are amylase, pepsin, and
lipase. They break down the carbohydrates, proteins, and fats, respectively. If the
enzymes optimal conditions depends on temperature and PH, I hypothesis that
amylase will work at its best rate when there is amylase, carbohydrates (cooked
food), and body temperature of 37°C; the pepsin will have it’s optimum condition
when we have pepsin, acidic condition (low PH), and body temperature of 37°C; and
finally the lipase will be at its optimum condition when we have lipase, protein,
body temperature of 37°C, and bile.
In order to test our hypothesis for amylase, we first started with four test
tubes. In the first one, we added DI water and cooked starch, which is made of
carbohydrate. For the second one, we added amylase and cooked starch. For the
third one, we added amylase, HCL, and cooked starch, and finally in the last one, we
added boiled amylase and cooked starch. Then we incubate them for one hours
under 37°C, which is a normal human body temperature. For the pepsin’s optimal
condition, we had 5 tubes with one pre-measured pieces of white egg (protein)
inside each tube. Thereafter, we added DI water and pepsin in the first tube, then
HCL and pepsin in the second and third tube, then HCL and DI water in the fourth
tube, and finally NaOH and pepsin in the fifth tube. Again, we incubated them all
under 37° C except the third tube, which we put it in the freezer. At the end, we
tested our lipase for its optimal condition by putting cream (fat) in three tubes. Then
we added DI water and bile in the first tube, only lipase in the second tube, and
lipase and bile in the third one. Then we measured the PH of all the three tubes and
then incubate them for one hour under 37° C. After incubation, we measured the PH
of all the three tubes and record them.
After Incubating, we brought them back to check our result. For the
carbohydrates, we added a reagent called Beneditc’s reagent (light blue), and then
we placed the four tubes into a boing hot water for two minutes. According the
table-1, we got the following results:
1- Starch + distilled water
+
2- Starch + amylase
+++
3- Starch + amylase + HCL
+
4- Starch + boiled salvia
++
The test tube with cooked starch and DI water changed to very dark green, the tube
with cooked starch and amylase changed to very bright orange (carrot color), the
tube with cooked starch, HCL, and amylase turned to creamy orange, and the last
tube with boiling amylase turned to dark orange.
For the protein experiment, we were supposed to check the size of the white
egg and to see whether it had been changed or not. Based on table-2, the first test
tube with pepsin, egg, and DI water only changed in color to light yellow and no
change in shape of the white egg. The second tube with pepsin, HCL, and egg had
color change to light yellow, production of some gas bubble, the size of the egg got
smaller than its original size. The third tube that was incubated in freezer had the
same starting components as the previous tube; however there was no change in
color and shape since the tube got frozen completely. The fourth tube with DI water,
HCL, and egg did not have any change in color and shape, only there was production
of some bubbles. And the last tube with pepsin, NaOH, and white egg, there was
absolutely no change in shape and color.
Table-2
Incubation Conditions
Appearance of egg white after incubation
1-Protein + pepsin @ 37C
Only color change, NO shape change
Color change, gas formed, major shape
2-Protein + pepsin + acid @ 37C
change
3-Protein + pepsin + acid @ 0C
Froze, NO shape change, NO gas formed
4-Protein + acid @ 37C
Only gas formed, NO shape change
5-protein + pepsin + NaOH + @ 37C
Only color change, NO shape change
Finally for the last experiment, which was the fat, we where looking to check
the PH of our tubes to see if there is any change in them after incubation. After
incubating under 37°C, we measured the their PH and for the first one we got PH of
7, for the second was PH 6, and the last was PH 5, as we can see in figure-1.
Initial and FinalPH
8
7
6
5
4
3
2
1
0
Series1
Series2
Series3
Cream + bile Cream + lipase Cream + bile
salts
salts + lipase
Red (initial), Green (final)
Carbohydrates are chained repeated polysaccharides, which when they get
broken down, they become monosaccharaides such as glucose or maltose that we
use them as energy source mainly. For our first test tube we got dark green solution,
which indicated that we did not have a lot of maltose in the solution, thus did not
have amylase. This should be expected since there was no amylase enzyme added in
our starting solution. The second tube turned to light orange color that indicates a
lot of maltose sugar, which means that amylase was present in the solution and it
was successfully broke down the starch into little molecules of sugar. The third tube,
was dark orange, this implies to a little bit of maltose was present in the solution.
This is because we added HCL in the solution at the beginning and since we know
that HCL is pretty strong acid, it can denature the amylase enzyme to the some
extend, which can damage the mechanism of the enzyme and not let it work
properly. The last one turned out to be lighter orange than previous tube. This
means that there is a good amount of maltose in the solution. Theoretically, this
should not have happened since we added boiled amylase. Boiled enzymes should
have denatured the protein (enzyme) amylase completely so it would be inactive in
the solution and could not break down the starch. This could have had many
reasons such as, the amylase did not boil enough to get denatured or the pipet or the
tube that we had used were contaminated some how with un-boiled amylase. Thus,
according to our observation and table-1 the tube number two had the optimal
condition for amylase enzyme which was only the presence of amylase and body
temperature 37° C.
Protein is composed many amino acid which each amino acid is made of
sugar and two other functional groups of amine and carboxylic acid. The pepsin
enzyme, in optimal condition, was supposed to break down the egg to smaller pieces
because it is supposed to break down the big protein into smaller amino acid. The
first test tube did not change in shape, but the color changed a little bit. This is
because we only had egg and pepsin inside the solution. It’s true that we need
pepsin enzyme to beak down the protein, but the environment should be acidic,
however. Protein is being mainly digested in our stomach, which is extremely acidic
almost PH 1 or 2, which plays a main role in digesting protein. The second test tube
on the other hand, had color change, bubble or gas formation, and the most
important thing that the shape of the egg changed to smaller piece. This indicates
that pepsin worked perfectly and the protein got broken down to a smaller piece.
This is mainly because we had both the enzyme pepsin and the acidic condition due
to HCL. According to an experiment done to check the best condition for the phytase
that break down wheat, they used pepsin at different temperature and PH and the
best worked condition was at the PH of 3 and temperature of 38°C (Esmaeilipour).
The third tube that was in freezer got frozen and we did not observe any change or
result and most probably the pepsin enzyme did not work at all because too low
temperature can damage the protein and some how denatures it, so the egg size was
the same. The fourth tube, which only had HCL, did not change the shape of the
protein but it definitely formed some bubble in the solution. This could be because
of the reaction of the HCL with egg or even with the solution that caused the
formation of some bubbles after dissociation of HCL, the Hydrogen gas can form in
the solution and thus formation of the gas bubbles and since we did not add the
amylase we should not be expecting any break down. The last tube in which we had
pepsin, protein and NaOH, only the color changed and the shape of the egg stayed
the same. Therefore, the pepsin did not work. This mainly is because of the basic
solution of NaOH that we added in the solution. NaOH is basic and as we discussed
already, the pepsin enzyme needs to be in an acidic environment not in a basic.
Based on our discussion, we can conclude that the optimal condition for the enzyme
pepsin is when enzyme pepsin is present, the environment is fairly acidic, and the
temperature is almost 37° C.
Fat is one the best sources of our body energy and it’s made of glycerol and a
three fatty acid molecules. When lipase enzyme breaks it down, it releases the fatty
acid molecules into the solution and turn the solution slightly acidic. We can see
from the figure-1 that the first tube’s PH is 7, which is same as its PH before the
incubation. This is because the solution had only cream (fat) and bile salt and it did
not have the main enzyme lipase. Thus, it’s normal to not see any PH change. And
also we can figure out that bile by itself has not the ability to start breaking down of
fat. The second tube’s PH is 6, which means the lipase enzyme broke down some fats
and released some fatty acid in the solution because we had cream and lipase in the
solution. The presence of lipase definitely changed the PH of the solution but not a
lot; however, we cans see that lipase by itself started the breaking down of fat but
did not do much. The last tube’s PH is 5 which we can almost conceder it as acidic.
This is because the solution contains both lipase and bile on top of the cream. Fat by
itself is a huge molecule for the lipase to break down. This is where bile comes in as
emulsifier and breaks down the big molecule of fat into smaller pieces and provides
the lipase the perfect size to break down the fat into glycerol and fatty acids, which
makes the solution acidic. According to an experiment that was done to see the
effects of bile on lipase, the experiment proved that combination of bile and lipase
yield the lowest PH, which means the maximum amount of fatty acid produced
(Borgstrom). Bile by itself cant break down the protein to smaller pieces of fatty
acid, but it helps to emulsify the fat into micelles that lipase can break it down to
smaller fatty acid and glycerol molecules and therefore make the solution acidic. So,
we can see that the optimal condition for the lipase is when there is fat, lipase, bile,
and body temperature of 37° C. So, our hypothesis got supported clearly based on
the experiments that we have done for each enzymes separately.
References
Borgstrom, B., Erlanson, C. 1973. Pancreatic lipase and Co-lipase. European journal
of biochemistry 60-68.
Esmaeilipour O., Krimpen M.M Van., 2010. Effects of temperature, PH, incubation
time and pepsin concentration on the in vitro stability of intrinsic phytase of
wheat, barley and rye. Animal feed science and technology 168-174.