Name: NUR ANIS ATIKA BINTI ZAINAL ABIDIN Class: M11N Date : 19th October 2011 Tittle: ENZYME

ACTIVITY

Objective To investigate the effect of different temperature which is 50 C, 200C, 320C, 600C and 1000C the rate of enzymatic reaction of diastase on starch given that it is immersed in water bath with the corresponding temperature or ice for about 5 minute. Introduction Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates sufficient for life. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.Like all catalysts, enzymes work by lowering the activation energy (Ea ) for a reaction, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts in that they are highly specific for their substrates. The way enzymes work is called the Lock and Key mechanism. The enzyme acts as the lock and the substrate acts as the key. The two fit together in the active site of an enzyme, and are said to be complimentary. The substrate and enzyme, when combined together is called an enzyme substrate complex. Diastase is an enzyme that catalyses starch into maltose through hydrolysis process: 

Another properties of enzymes is it can be denatured by heat1. If starch is present thus the colour of iodine will turn from brownish into dark blue.

Research Question What is the effect of different temperature which is 50 C, 200C, 320C, 600C and 1000C the rate of enzymatic reaction of diastase on starch given that it is immersed in water bath with corresponding temperature or ice for about 5 minute? Hypothesis As the temperature increases, the rate of reaction will increase until the optimum temperature is reached, then after this point the rate will decrease as the temperature continues to increase. At low temperatures, the kinetic energy of the molecules is lower, so the rate of effective collisions that can achieve the activation energy needed to start the reaction will be very low. In order to convert substrate into product, enzymes must collide with and bind to the substrate at the active site. Thus the rate of reaction will be very slow. When molecules collide, the kinetic energy of the molecules can be converted into chemical potential energy of the molecules. If the chemical potential energy of the molecules becomes high enough, the activation energy of a reaction can be achieved and the reaction can occur. Thus the greater the kinetic energy of the molecules in a system, the greater is the resulting chemical potential energy when two molecules collide. As the temperature of the mixture of diastase and starch is increased, the kinetic energy of the molecules increases and it is possible that more molecules per unit time will collide and reach the activation energy. Thus the rate of the reaction will increase. Increasing the temperature of a system will increase the number of collisions of diastase and starch per unit time. Thus, within limits, the rate of the reaction will increase. However, when the temperature reaches a certain point, the temperature becomes too high for the enzymes, and the enzymes are denatured. As the temperature of the mixture of diastase and starch is increased, the internal energy of the molecules in the mixture of diastase and starch will increase. Some of this heat may be converted into chemical potential energy. If this chemical potential energy increase is great enough some of the weak bonds that determine the three dimensional shape of the active proteins will be broken. This could cause the protein to denature and thus inactivate the protein. Thus too much heat can cause the rate of an enzyme catalyzed reaction to decrease.
1

http://en.wikipedia.org/wiki/Enzyme

Variables Variables Uni t Temperature

0

Range

Possible effect on result

Independent

50C,200C ,320C,60 0 C,2000C

Dependent

Time taken s for the starch to completely hydrolysed Volume of diastase ml

0-3600 s

Fixed

5ml

Volume of starch solution

ml

5ml

Time interval

min 5 min

Different volume may affect the results as more enzymes will be present to react with starch thus affecting the rate of reaction. Different volume may affect the results as more substrates will be present to react with diastase thus affecting the rate of reaction. A fixed time

Methods on manipulating the variables For 50C melted ice is used .For 200C melted ice with some tap water is used to get the 20 0C temperature. Water bath is used for 320C ,600C and the temperature is adjusted accordingly.For 200 0C the water is heated until boiled. The iodine test for each diastase-starch solution was done for at least an hour at 5 minute intervals. 5ml of amylase solution is used for all the experiments, and the volume measured using a 20ml measuring cylinder.

5ml of starch solution is used for all the experiments, and the volume measured using a 20ml measuring cylinder.

The time interval for

interval is necessary to be consistent in performing the iodine test.

each iodine test is set at every 5 minute.

Materials and apparatus Materials Starch solution Amylase Iodine solution Ice cubes Quantity 1 reagent bottle 1 basin Volume/size 15ml 15ml -

Apparatus Measuring cylinder White tile Thermometer Water bath Dropper Glass rods Stopwatch Test tubes

Quantity 1 1 1 1 2 3 1 3

Volume/size 20ml -

Method 1. 3 test tubes are labelled as Trial 1,Trial 2 and Trial 3 for 50C temperature. 2. 5ml of diastase solution is measured and added into each test tube. 3. The test tubes are placed into ice and the temperature is controlled for exactly 5 minutes. 4. 5ml of starch solution is measured and added to each test tube and mixed with a clean glass rod. The temperature is monitored continuously. 5. At intervals of 5 minutes, each test tube is tested for the presence of starch. One drop of diastase-starch mixture is withdrew, placed on a white tile with grooves, and a drop of iodine solution is added. 6. The iodine test is continued for at least 1 hour. 7. Step 1 5 is repeated using a clean test tube for 200C,320C,600C and 2000C.

8. A complete record of the observations are made and presented in the table below DATA COLLECTION Quantitative data Table of temperature and time taken for the iodine test to change the colour Time taken Temperature C (0.5 for the iodine 5C 20 C 32 C test to change 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd the colour(s) (0.1s) 0.0 O O O O O O O O O 300.0 O O O O O O O O O 600.0 O O O O O O O O 900.0 O O O O O O 1200.0 O O O O O O 1500.0 O O O O O O 1800.0 O O O O O O 2100.0 O O O O O O 2400.0 O O O O O 2700.0 O O O 3000.0 O O 3300.0 3600.0 y -iodine colour remain unchange/brownish yellow y O - iodine change from brownish yellow to dark blue Qualitative data 1. The initial colour of the iodine is brownish yellow. 2. The colour of iodine change from brownish yellow to blue black if it is tested with mixture of diastase and starch at certain time and temperature. 3. Some of the iodine remain unchanged after tested with diastase and starch at certain time and temperature. C) 60 C 1st O O O O O O O O O O O O O 2nd O O O O O O O O O O O O O 3rd O O O O O O O O O O O O O 1st O O O O O O O O O O O O O 200 C 2nd O O O O O O O O O O O O O 3rd O O O O O O O O O O O O O

Data processing 1. Average time taken for the starch to be hydrolysed by the enzyme:
               

Eg: For 50C

 2. Rate of enzymatic reaction of diastase on starch:

 Eg: For 50C0.50C

s-1 3. Uncertainty of rate of reaction, R:

 Eg: For 50C0.50C


-9 -1

 s

Temperature (0C)

Average time taken

Rate of reaction,R

Uncertainty of

(0.5C) 5.0 20.0 32.0 60.0 100.0

0.000313 0.000435 0.001250 0 0 Graph of rate of enzymatic reaction

for starch to be hydrolysed (s) (0.1s) 3200.0 2300.0 800.0

(s-1)

rate of reaction, , (s-1)

-9 -8 -7

0 0

against the temperature

0.0013 0.0012 0.0011 0.001 0.0009 0.0008 0.0007 Rate of reaction (s-1) 0.0006 0.0005 0.0004 0.0003 0.0002 0.0001 9E-19 0 -1E-04 -0.0002 -0.0003 Temperature (0C) 20 40 60 80 100 120

Discussion 1. The mixture of diastase and starch solution is immersed into the ice or water bath for 5 minutes to ensure that the temperature is controlled to the specific temperature needed. 2. Iodine test is conducted to detect the presence of starch in the mixture of solution.Iodine s original colour is yellowish brown.When starch is present in the solution that is tested with iodine ,the colour will change from yellowish brown to dark blue.If there is no change in colour of iodine,this notate that starch is completely hydrolysed by the diastase into maltose. Analysis of the graph 1. Initially,a curve showing positive correlation is obtained however at certain point it will drop and shows negative correlation between the rate of enzymatic reaction and the temperature. The graph shows that as the temperature increases, the rate of reaction increases. The highest point on the curve is the assumed the optimum temperature for the diastase enzyme to catalyse the reaction of starch into maltose, as this is the point where the rate of reaction is at maximum.The graph shows that the rate of reaction initially increases slowly but between 200C and 320C the rate of enzymatic reaction increases steeply.

2.

3.

At 5.0 0.5 C, the rate of reaction is at lowest point because at low temperatures, the kinetic energy of the molecules are low, thus the rate of effective collisions that can achieve the activation energy needed to start the reaction will be very low. In order to convert substrate into product, enzymes must collide with and bind to the substrate at the active site. At this low temperature, the rate of collisions will be very low, therefore, the rate of reaction will be very slow.Therefore the time taken for the starch to be hydrolysed is longer for 50C 0.5 C. 0 At 20 0.5 C the rate of reaction increases more than 5 C 0.5 C because the kinetic energy of the molecules are slightly higher,thus the rate of effective collisions that can achieve the activation energy needed to start the reaction will be slightly higher. At 20 0.5 C the rate of collision will be slightly higher .Therefore the time taken for the starch to be hydrolysed is shorter than that of 50C 0.5 C and the rate of reaction of diastase on starch in 20 0.5 C is higher than 50C 0.5 C.

4.

5.

At 32 0.5 C temperature the time taken for the iodine colour to remain unchanged is the lowest thus it indicates that the rate of reaction is the highest because at this temperature,the temperature is assumed the most optimal for the enzyme to work as the rate of reaction is the highest.The kinetic energy of the molecules are the highest, thus the rate of effective collisions that can achieve the activation energy needed to start the reaction will be very high. At 600C to 1000C there is no reaction occurring.It is assumed that the temperature is too high for the activity of enzyme.Denaturation might occur that cause the enzyme to dysfunction and become inactive thus it will change the shape of the active site of the
enzyme. Starch can no longer be hydrolysed as it cannot fit into the active site of the enzyme.Therefore throughout the experiment the colour of iodine will always turn from yellowish brown to dark blue.

Evaluation
Limitations The heat from our hand might affect the temperature of the mixture of starch and diastase in the test tube. Parallax error might occur while taking the measurement of starch solution and diastase solution. Iodine might be oxidized if left open during the experiment The dropper used to drop the iodine might be contaminated with the mixture of diastase-starch solution Impurities in the apparatus might affect the result. Suggestion Hold the beaker using test tube holder when conducting the experiment. Ensure that the eyes is parallel to the meniscus point so that precise reading can be taken. Close the iodine reagent bottle with it s cap and only open it when needed. Rinse it after every iodine test is conducted.

Rinse all the apparatus using distilled water before it is used.

Conclusion

It can be concluded that as the temperature increases, the rate of reaction of diastase (enzyme) increases, until the optimum temperature which is about 320C; after the optimum temperature, the rate of reaction decreases as the temperature continues to increase and at one point the reaction will stop because the too high of temperature causes the enzyme to denature. This is because as the temperature increases, the kinetic energy of the molecules increase, and the molecules collide more frequently with high energy, so more molecules can achieve the activation energy needed for the reaction, hence increasing the number of reactions per second, which is the rate of reaction. However, this apply only till a certain point, where highest rate of reaction is

reached. This is the optimum temperature, and after this point the gradient of the graph will slope downwards, as the rate of reaction decreases, because the temperature is too high and the diastase are denatured, losing its three-dimensional form, becoming inactive and unable to catalyse the reaction of starch to maltose.

References 1. Biology for the IB Diploma, CJ Clegg, Hodder Education, London, 2007. 2. Biology IB Study Guide, Oxford University Press, 2007. 3. Wikipedia (http://en.wikipedia.org/wiki/Enzyme)