Following the progress of an enzyme-controlled reaction.


Aim: To follow the progress of an enzyme-controlled reaction using starch solution and amylase.


Hypothesis: It may be assumed that the longer the starch will be in contact with the enzyme the more starch will be converted into maltose. It can also be expected that the more substrate is digested into the break down product the lower color readings will be obtained with the colorimeter confirming the hypothesis that as time goes by the amylase breaks down more starch. It is highly probable that the rate of reaction will be very high at first due to the great abundance of starch and gradually slower as the substrate reduces in quantity and the product increases.


Concerning the starch depletion/enzyme activity rate it is likely to slow down towards the end as the amylase is running out of starch to break down.


Regarding the final Benedicts’ test on the remainder of the solution, it can be confidently stated that it will reveal to be positive due to its high concentration of maltose.

Variables:Aspects modified purposelyAspects maintained the same purposelyUncontrolled aspects

o The amount of time the starch was left to interact with the amylase. · The amount of iodine in each test tube.

o The amount of starch/amylase solution in each test tube
o The ratio of starch solution mixed with amylase (1:2) · Amount of distilled water used to dilute each test tube.

o Each colorimeter sample holder might have varied slightly in transparency although they were not apparent at first sight.
o The exact size of the drops of iodine, amylase/starch solution placed in the sample holders
Apparatus:


· 11 test tubes


· Amylase


· Starch solution


· Pipette


· Syringe


· Beaker


· Colorimeter


· 10 colorimeter sample holders


· Iodine


· Benedict’s solution


· Bunsen burner


· Test tube holder



Diagram:



1 min


2 min


4 min


6 min


8 min


10 min


11 min


12 min


13 min


Method:


Step 1 Place one drop of iodine in 10 colorimeter sample holders


Step 2 Mix amylase and starch solution to a ratio of 1:2 and place two drops of the mixture in the first sample holder at once.


Step 3 Wait a minute before placing to drops into the next sample holder


Step 4 While waiting fill the first sample holder with distilled water to the brim.


Step 5 Repeat steps 3-4 for the remnant sample holders.


Step 6 Measure the color absorption with the colorimeter.


Step 7 Graph the results.


Step 8 With the remaining amylase/starch solution ad benedict’s solution and boil under flame.


Step 9 Note changes of color.


Results and Observations:


When Benedicts’ solution was added to the remaining amylase/starch and boiled an unmistakable change of color occurred. The presence of sugar – in this case maltose – can be confirmed with this test. The solution became a bright orange/yellow.
Absorption readings for iodine tests (colorimeter – 5% uncertainty)


Absorption readings (colorimeter – 5% uncertainty)


Mins


Group 1 ●


Group 2 ■


Group 3 ▲


Group 4 □


1


1.42


0.80


0.96


1.16


2


--


0.58


1.00


0.88


3


--


0.56


0.71


--


4


1.39


0.43


0.50


0.66


5


1.31


0.31


0.48


--


6


1.24


0.31


0.44


0.66


7


1.09


0.25


0.37


--


8


0.44


0.21


0.22


0.43


9


0.42


0.21


0.28


--


10


--


--


--


0.27


11


--


--


--


0.41


12


--


--


--


0.73


13


--


--


--


0.25




Transmission Readings for iodine tests (Colorimeter – 5% uncertainty)


Transmission readings


(Colorimeter – 5% uncertainty)


Mins


Group 2 ■


Group 4 □


1


0.49


0.27


2


0.56


0.34


3


1.00


0.44


4


1.19


0.41


5


1.63


0.53


6


1.44


0.67


7


1.29


0.96


8


1.92


1.16


9


1.98


--


10


2.08


--



Absorption readings for Benedict’s test (Colorimeter – 5% uncertainty)


Absorption Readings for benedicts’ test.


(Colorimeter – 5% uncertainty)


Mins


Group 3


1


0.97


2


0.84


3


1.19


4


1.6


5


1.38


6


0.89


7


1.38


8


1.04


9


1.09


10


1.18


Remainder


0.99


Absorption rate of Starch/Amylase

Mins


Absorption Rate


of Starch/Amylase


1


0.275


2


0.115


3


0.125


4


0.118


5


0.055


6


0.040


7


0.010


8


0.050


9


0.025


10


0.010


11


0.015


12


0.010






Standard Curve


Absorption


Starch Concentration (%)


1


1.20


0.5


0.70


0.25


0.30


0.10


0.10


The standard curve numbers were obtained by Mr. Moore



Reaction Rate (Rate of Starch Depletion) (Assumed enzyme Activity Rate) - results found thanks to the standard curve + absorption results.


Time


Group 1


Group 2


Group 3


Group 4


1


1.55


0.87


1.05


1.27


2


0.63


1.1


0.96


3


0.61


0.78


4


1.53


0.47


0.55


0.72


5


1.44


0.34


0.52


6


1.36


0.34


0.48


0.72


7


1.2


0.27


0.4


8


0.98


0.23


0.35


0.47


9


0.48


0.23


0.24


10


0.46


0.23


0.31


0.29


11


0.45


12


0.8


13


0.27





Conclusion: The most fulfilling results were the absorption readings for iodine. The reasons behind this were that first of all there were more results gathered by all four groups and the numbers were rather advancing in a logical progression. Thanks to the absorption readings for iodine we were able to determine that the longer the amylase is left in contact with the starch the more maltose is produced. This can be calculated by observing the progressive drop of absorption figures in every group. There seemed to be an anomaly in the results of group one (clearly seen in the second graph). The reason for these results is obscure but the figures descend nonetheless suggesting hat the irregularity still demonstrates a progressive decrease of substrate and increase of product.


The transmission results were less satisfying. First of all only two groups made this type of readings and the graph formed was unproductive (at least compared to the absorption readings). But although the data is quite set apart when in comparison with