Reflection Lab Report 3

Reflection by Nurzahidah binti Zamat

 In this experiment, I knew a new term which is saponification number and how to calculate its value. Every sample gave different reading based on the number of fatty acid in it. In this experiment, we used reflux, therefore I can improve my skill by using this technique as we seldomly used this technique in any experiment.
Reflection by Aqmarollah bin Mohd Nasip


Untuk eksperimen kali ini, saya dapat mengetahui maksud istilah “saponification number” dan tahu untuk mengira nilai tersebut. Daripada lima sampel yang diberi, kami dapat mengetahui nilai “saponification number” untuk setiap satu sampel tersebut. Setiap sampel sepatutnya memberi bacaan yang berbeza mengikut nombor asid lemak yang ada dalam sampel tersebut. Saya juga dapat memahirkan diri untuk membuat eksperimen berkaitan “reflux” kerana eksperimen ini melibatkan teknik tersebut.


Reflection by Afifah Syafikah binti Azizan

It’s awesome to know more about lipids. From carry out this experiment, I got new knowledge which is fats and oils are triesters of glycerol, they react with water to form fatty acids and glycerin. When the reaction is carried out in a basic solution, salts of the fatty acids are produced instead of the fatty acids themselves. The salts of fatty acids are soaps and an individual molecule is characterized by an ionic end which is the salt part and a nonpolar end which is the hydrocarbon part. The ionic salt end is water-soluble and the nonpolar hydrocarbon end is water insoluble.Then I also know how to identify them based on their saponification number.
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Lab Report 3: Triglyceride

INTRODUCTION

Triglycerides are fat in the blood. They are used to give energy to our body. If it is excess, they are stored in different places, to be used later. They are important to life and are the main form of fat, called lipids in the body. They are the end product of digesting and breaking down fats in food. Some are made in the body from other energy sources such as carbohydrates. 

But high triglycerides might raise your risk of heart disease and may be sign of metabolic syndrome. Metabolic syndrome is the combination of high blood pressure, high blood sugar, too much fat around the waist, low HDL and high triglycerides. 

 Vegetable oils and animal fats are the main materials that are saponified. These greasy materials, triesters called triglycerides are mixtures derived from diverse fatty acids. triglycerides can be converted to soap in either one or two step process. 

MATERIALS

Triglyceride sample (coconut oil, corn oil, palm oil, margarine, butter) solvent (1:1 ethanol/ether), 0.5M KOH/ethanol solution, phenolphtalein, 0.5M HCl.

PROCEDURE

1. 1.0 g sample triglyceride was placed into a small beaker and dissolved in 4 ml solvent(1:1 ethanol /ether).

2. Dissolved triglyceride was transferred into a small distillation flask and beaker wshed twice with 1 ml solvent to collect all residual material. “wash” also added to the distillation flask.

3. 25 ml of 0.5 M KOH/ethanol solution was added.

4. Exact volume of the mixture was measured.

5. Second system as “control” was set up with 25 ml 0.5 M KOH/ethanol solution + 2 ml of 1:1 ethanol/ether solvent for a final volume identical to our test sample solution.

6. Reflux condenser was set up on each flask and boiling water bath was placed in for 30 minutes. Hydrolysis occur.

7. Flask allowed to cool. Three drops indicators (phenolphthalein) was added to both flasks and titrated with 0.5 ml HCl solution.

RESULT

Sample
B (ml)
T (ml)
Saponification number (mg KOH/1g)
Palm oil
23.00
22.00
28.06 mg KOH/1g
Sunflower
22.20
21.00
33.67mg KOH/1g
Corn oil
23.00
22.00
28.06mg KOH/1g
Margerin
23.00
20.00
84.17mg KOH/1g
Butter
28.00
25.00
84.17mg KOH/1g

DISCUSSION

The saponification number (sap) measures the bonded and unbonded acids present in an oil or fat. It defines the exact amount of potassium hydrate in mg necessary to emulsify 1g of fat or oil.  The smaller the molar mass of the fat, the higher the saponification value. For saponification of triglycerides experiment to determine the saponification number of triglycerides, we were using palm oil as a sample for our group. Different group used different lipid which are sunflower oil, corn oil, margarine and butter. Then at the end of the experiment we obtained the final results for each sample. Based on the results obtained, sunflower oil has the higher saponification number between oil which is 33.67 mg KOH/1g compared to corn oil and palm oil which has the same value at 28.06 mg KOH/1g. So it shown that, sunflower oil has shorter fatty acids. However, margarine and butter has the highest saponification number at 84.17 mg KOH/1g. It is because Fats and oils can be characterized by their saponification numbers. One mole of fat requires three moles of potassium hydroxide for complete saponification. If a fat contains fatty acids of relatively high molecular weights, then one gram of the fat will consist of fewer moles. Thus, fats having greater percentages of high molecular weight fatty acids will have lower saponification numbers than fats having greater percentages of lower molecular weight fatty acids.
Theory said that triglycerides containing long fatty acids will have a lower saponification number than triglycerides with shorter fatty acids. Since 1 gram of lipid containing long chains will have less chains in total than 1 gram of lipid containing short chains. Actually, we should get the higher saponification number for palm oil followed by corn oil and the lowest is sunflower oil.  Low fatty acid fats like coconut oil or palm kernel fat have high saponification numbers of 250, whereas most vegetable oils have a saponification number of approximately 190 It means that, palm oil have the shorter fatty acid chain than corn oil and sunflower oil. For the palm oil and corn oil, the saponification number should higher than sunflower oil because they have shorter fatty acid chain than sunflower oil. A few error occur during the experiment that effect the results obtained that are, there is no standard colour of solution when turns colourless. The pinkish colour might still there. So, this affect the titration process. It then, affect the volume of HCl used.

CONCLUSION

 From this experiment, we are able to calculate the saponification number (mg KOH/1g) and can differentiated which fats or oil that containing high fatty acid number based on their saponification number.

 REFERENCES

 1. http://www.chemistryexplained.com/Di-Fa/Fats-and-Fatty-Acids.html#ixzz4gYl8E55s
2. https://en.m.wikipedia.org/wiki/Saponification
3. www.webmd.com/cholestrol-management/tc/high-triglycerides-overview

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Reflection Project 3

Reflection by Nurzahidah binti Zamat

In this project, I can confirmed that based on my readings, green plants and vegetables have higher amount of vitamin C compared to oranges as we always heard. Vitamin C also soluble in water, which, if we consume more than 40mg-70mg per day, it will be removed in our urine. Therefore, there is no need of us taking supplement or any type of vitamin C, as our body will used as much as we need.  In this project, I learned that cooking or keeping food in refrigerator for a long time will reduce the amount of vitamin C in that food itself. Therefore, another method of cooking can be apply to maximize the intake of vitamin C in our body.

Reflection by Aqmarollah bin Mohd Nasip

After done the experiment, I am able to know how to measure vitamin C in food sample. Vitamin C will denatured in a high temperature when be heated. So, if we heat a food that contain vitamin C with a strong heat, the vitamin C in the food will lost. Some recommended way to prepare food with vitamin C contained is by steaming it. I also know that food containing most highest  vitamin C is citrus. I am also know why the reason solution of food sample change colour to blue-black. Vitamin C will react with iodine until no more vitamin C present in the food and it will change the colour to blue-black. This is called the end point of the titration. I hope with this new information, I can improve my knowledge and skill while conducting experiment.

Reflection by Afifah syafikah binti Azizan

Overall, the lab was an extremely interesting activity as it showcased the delicate procedures in conducting such delicate measurements. It was a great learning experience in trying out a rendition of a titration at its steps. With the help of group mates, it did showed the systematic and patience that is needed in such a lab activity.  Moreover, the lab connected fundamental ideas that involved reactions of different solutions and how to model them through different trials to predict certain concentrations of solutions. To end off, it was a very spectacular lab.
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Project 3: Measuring Vitamin C

INTRODUCTION

There are many foods that are containing vitamin C  such as fruits, drinks and vegetables. Vitamin C, more properly calledby ascorbic acid, is an essential antioxidant needed by the human body. In this experiment, we are going to measure the amount of vitamin C by using the given formula in manual. Different foods have differ amount of vitamin C, so in this experiment, we can know from different food sample, the roughly amount vitamin C on it. As the iodine is added during  titration, the ascorbic acid is oxidised to dehydroascorbic acid, while the iodine is reduced to iodide ions.

Due to this reaction, the iodine formed  immediately reduced to iodide as long as there is any ascorbic acid present. Once all the ascorbic acid has been oxidised, the excess iodine start to react with the starch and then forming the blue-black starch-iodine complex. This is the endpoint of the titration.

MATERIALS

1. Vitamin C standard (0.2 mg/ml)
2. Food sources of vitamin C: for example juices, extraction of plants, flowers, fruits, grains, and vegetables, vitamin C tablet or cooked/treated food sample (boiled/refrigerated/grilled)
3. Starch solution (1%): Mix 1 g starch in 100 ml boiling H2O. Boil for one minute while stirring. Stir until completely dissolved (this solution will be cloudy).
4. Iodine solution: Mix 0.6 g potassium iodide in 500 ml H2O. Mix 0.6 g iodine in 50 ml of ethyl alcohol. These two iodine solutions should be mixed well before combining. Combine the two iodine solutions and add an additional 450 ml of H2O.
5. Hydrochloric Acid (HCl) 1 M, (5 ml).
6. Blender.
7. Filter/ cheesecloth.

PROCEDURE

1. Preparing the vitamin C extracts:
i. Food material was chopped into small pieces and was placed into blender.
ii. 100 ml of distilled water was added to the blender.
iii. Blend was used the highest speed until the material is thoroughly ground.
iv. The ground extract was strained.
v. 30 ml of the strained extract was measured into a 250 ml Erlenmeyer flask or beaker.

2. Measuring vitamin C in the standard and food sample:
i. 30 mL of the Vitamin C Standard was placed in a 250 ml flask or beaker.
ii. 2 drops of the 0.1 M HCl was added to the flask.
iii. 5 ml of the starch solution was added to the flask.
iv. A burette was filled with the iodine solution.
v. The initial volume reading was recorded.
vi. The iodine solution in 1 ml increments was added to the flask while swirling the flask.
vii. Iodine was added until the solution stays blue-black for 15 seconds.
viii. The volume reading on the burette was recorded.
ix. Step i to viii was repeated to measure the vitamin C in the food sample.
x. The amount of Vitamin C in the food sample was calculated using this formula:



3. Comparing cooked food and raw food’s vitamin C
i. Food was prepared according to our creativity. For example, boil or steam or place in a freezer.
ii. Food material was chopped into small pieces and was placed into blender.
iii. Data was obtained using the same method in previous section.
iv. The volume reading on the burette was recorded.
v. The relative amounts of ascorbic acid present in the samples testing was compared.
vi. Our results with those of other members of the class was compared. 

Application: Magic Writing

MATERIALS

Beaker
Iodine
Lemon/Lime juice
Notebook paper
Cup
Art brush

PROCEDURE

STEP A: IODINE SOLUTION
1. 100 ml water was poured into a 500ml-beaker.
2. 10 ml of Iodine was added to the water and stir.

STEP B:
1. A section from the notebook paper was cut.
2. The paper must fit inside a 500ml-beaker.

STEP C: VITAMIN C SOLUTION
1. The juice of the lemon/lime was squeezed into another beaker.

STEP D:
1. The art brush was dipped into the lemon/lime juice
2. A message was writed on the piece of paper.
3. The juice was allowed to dry on the paper.
4. The paper was submersed in the iodine solution in the bowl.

RESULT

Standard Vitamin C

Volume of iodine used
Average
I
II
( 15 + 15.3 ) mL
15 mL
15.3 mL
2
= 15.2 mL


Food Sample
Volume of iodine used, mL
Amount of vitamin C in
15 g food sample
Vitamin C in 100 g
food sample
Raw
Treated
Raw
Treated
Raw
Treated
Iced lemon tea
5.6
3.5
0.074
0.047
0.493
0.313
Rice
4.5
4.0
0.059
0.053
0.393
0.353
Broccoli
19.0
11.5
0.250
0.150
1.667
1.000
Papaya
18.0
17.3
0.240
0.230
1.600
1.533
Orange
15.0
14.1
0.197
0.186
1.313
1.240
Flower
9.00
6.00
0.118
0.079
0.787
0.527

Magic Writing


DISCUSSION

In this experiment we perform an oxidation-reduction titration. We mixed vitamin C with the starch, and gradually titrated it using iodine. Iodine reacted with vitamin C and did not reacted with the starch. However, after the vitamin C (ascorbic acid) had been fully reacted with iodine, iodine will reacted with starch producing blue-black colour in the solution. The blue-black colour indicates the amount of iodine needed to react with vitamin C. 

Based on our result, we obtain that the highest amount of vitamin C in our food sample is broccoli, followed by papaya, orange, flower, iced lemon tea and lastly rice. this food sample is also treated to see if there any changes when titrating them with iodine.

Surprisingly, there are changes in the amount of iodine used to react with the vitamin C. The amount of iodine used was decreased in treated vitamin C. This mean that, treated vitamin C produce less vitamin C. In magic writing, we wrote our name using lemon juice, and soak it in iodine solution.

CONCLUSION

In conclusion, we can conclude that broccoli have higher amount of vitamin C compare to other food sample. After that, we can say that, cooking or keeping food in refrigerator can reduce the amount of vitamin C in food. Lastly, this experiment proof that treated vitamin C will resulting in less vitamin C. 

QUESTIONS

1. What juices or drinks had the most vitamin C?
Citric juices have most highest vitamin C

2. Did the drinks have the vitamin C that they advertised on the labels?
The drinks show zero amount vitamin C but we detect a little amount vitamin C in our experiment

3. What food sources had the most vitamin C?
Red peppers

4. What families or groups had the most vitamin C?
Citrus families.

5. Did plants that you do not normally eat have vitamin C?
Yes, vegetables. Some vegetables have a lot of vitamin C.

6. Did heat affect the vitamin C content of food?
Yes.

7. Did heat increase or decrease the vitamin C levels?
Decrease vitamin C levels.

8. What way of food preparation would be the most nutritious ?
Steaming cooking.

9. Do you have any ideas now to get more vitamins from your meals?
Yes. I need to eat more fruits and vegetables to get more vitamin C.

REFERENCES

1. A Text Book of Biochemistry for Medical Students, 9th ed. New Delhi : UBS Publisher’s Distributors Ltd., 2003
2. R. K. Murry, D. K. Granner, P.A. Mayes, V. W. Rodwell, Harper’s Biochemistry, Prentice Hall International Inc., Latest Edition.
3. https://studymoose.com/%EF%BB%BFeffect-of-temperature-on-content-of-vitamin-c-essay 
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Lab Report 2: Enzyme

INTRODUCTION

Enzyme is any of a group of complex proteins or conjugated proteins that are produced by living cells that act as catalyst in specific biochemical reactions. The molecules at the beginning of the process upon which enzymes may act are called substrates and the enzyme converts these into different molecules, called products. Almost all metabolic processes in the cell need enzymes in order to occur at rates fast enough to sustain life. The set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Like all catalysts, enzymes increase the reaction rate by lowering its activation energy. Some enzymes can make their conversion of substrate to product occur many millions of times faster. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules: inhibitors are molecules that decrease enzyme activity, and activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. An enzyme's activity decreases markedly outside its optimal temperature and pH.

Enzymes are generally globular proteins, acting alone or in larger complexes. Like all proteins, enzymes are linear chains of amino acids that fold to produce a three-dimensional structure. The sequence of the amino acids specifies the structure which in turn determines the catalytic activity of the enzyme. Enzymes must bind their substrates before they can catalyze any chemical reaction. Enzymes are usually very specific as to what substrates they bind and then the chemical reaction catalysed. Specificity is achieved by binding pockets with complementary shape, charge and hydrophilic/hydrophobic characteristics to the substrates.

Some enzymes are used commercially, for example, in the synthesis of antibiotics. Some household products use enzymes to speed up chemical reactions: enzymes in biological washing powders break down protein, starch or fat stains on clothes, and enzymes in meat tenderizer break down proteins into smaller molecules, making the meat easier to chew.

PROCEDURE

Part A: Preparation of standard reference

1. A series of dilution for starch solution were prepared by using 1.0 mg/ml stock solution.
2. The starch solution that was prepared were mixed with distilled water and iodine solution.
3. The following table was used as a guide.


Test tube
8 ml starch of x (mg/ml)
Water (ml)
Iodine (ml)
Absorbance at 590 nm
1
0.00
9
1
2
0.01
1
1
3
0.025
1
1
4
0.05
1
1
5
0.10
1
1
6
0.30
1
1
7
0.50
1
1
8
0.70
1
1
9
1.00
1
1

4. Graph of standard curve of absorbance (@ 590 nm) vs. concentration of starch/iodine mixture were plotted. 

Part B: Determination Effect Of Substrate Concentration, Temperature And pH On Enzyme Velocity

a.       The Effect Of Substrate Concentration 

Experiment of starch hydrolysis in different substrate concentration was prepared as the following table:

Test tube
8 ml starch of x mg/ml
Water
(ml)
Amylase (ml)
Incubate each sample at 370C for 10 minutes
Iodine
(ml)
Place all test tubes in an ice bath. Measure the absorbance at 590nm
1
0.00
8
1
1
2
0.01
0
1
1
3
0.025
0
1
1
4
0.05
0
1
1
5
0.10
0
1
1
6
0.30
0
1
1
7
0.50
0
1
1
8
0.70
0
1
1
9
1.00
0
1
1

b.       The Effect Of Temperature
The solution was prepared for the experiment of different temperature:


Test tube
8 ml starch of x mg/ml
Water
(ml)
Amylase (ml)
Incubate each sample at 8, 28, 60, 1000C for 10 minutes
Iodine
(ml)
Place all test tubes in an ice bath. Measure the absorbance at 590nm
1
0.00
8
1
1
2
0.01
0
1
1
3
0.025
0
1
1
4
0.05
0
1
1
5
0.10
0
1
1
6
0.30
0
1
1
7
0.50
0
1
1
8
0.70
0
1
1
9
1.00
0
1
1

b.       The Effect Of pH
The following solution was prepared for the experiment using different pH:
Test tube
Starch of 0.5 mg/ml
2 ml buffer of pH x
Amylase (ml)
Incubate each sample at 370C for 10 minutes
Iodine
(ml)
Place all test tubes in an ice bath. Measure the absorbance at 590nm
1
5
4
1
1
2
5
5
1
1
3
5
6
1
1
4
5
7
1
1
5
5
8
1
1
6
5
9
1
1
7
5
10
1
1
Blank
5
8 ml of dH2O
1

DATA ANALYSIS

 1. Preparation of standard reference
( REFER STANDARD CURVE GRAPH)

2. Determination The Effect Of Substrate Concentration, Temperature And Ph On Enzyme Velocity

a. The effect of substrate concentration

So
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
0.182
0.01
0.01
0.001
0.00
1000.0
0.01
0.147
0.005
0.005
0.0005
100
2000.0
0.025
0.208
0.013
0.012
0.0012
40.00
833.3
0.05
0.239
0.015
0.035
0.0035
20.00
285.7
0.10
0.222
0.012
0.088
0.0088
10.00
113.6
0.30
0.231
0.013
0.287
0.0287
3.33
34.8
0.50
0.155
0.009
0.491
0.0491
2.00
20.4
0.70
0.229
0.012
0.688
0.0688
1.43
14.5
1.00
0.251
0.02
0.98
0.098
1.00
10.2

From Michaelis-Menten graph,
Vmax=  0.1
Km  = 0.32 mg/ml
From Lineweaver-burk graph,
1/ Vmax = 40
Vmax= 1 / 40
 = 0.025 mg/mlmin
-1/ Km= -2
Km       = 0.5 mg/ml


b. The effect of temperature          



for 8  ͦC:

SO
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
1.058
0.10
0.10
0.01
0.00
100.00
0.01
0.980
0.09
0.08
0.008
100.00
125.00
0.025
1.060
0.015
0.01
0.001
40.00
1000.00
0.05
0.991
0.095
0.045
0.0045
20.00
222.22
0.10
1.490
0.14
0.04
0.004
10.00
250.00
0.30
2.231
0.24
0.06
0.006
3.33
166.67
0.50
3.235
0.35
0.15
0.015
2.00
66.67
0.70
3.580
0.39
0.31
0.031
1.43
32.26
1.00
3.732
0.41
0.59
0.059
1.00
16.95


for 28  ͦC:


So
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
0.812
0.0700
0.0700
0.0070
0
142.9
0.01
0.317
0.0200
0.0100
0.0010
100.00
1000.0
0.025
0.246
0.0055
0.0195
0.00195
40.00
512.8
0.05
0.293
0.0053
0.0447
0.00447
20.00
223.7
0.10
0.239
0.0050
0.0950
0.0095
10.00
105.3
0.30
0.297
0.0057
0.2943
0.02943
3.33
34.0
0.50
0.348
0.0250
0.4750
0.0475
2.00
21.1
0.70
0.327
0.0230
0.6770
0.0677
1.43
14.8
1.00
0.374
0.0300
0.9700
0.0970
1.00
10.3

for 60  ͦC:

S0
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
1.189
0.11
0.110
0.0110
0.00
90.00
0.01
1.324
0.13
0.120
0.0120
100.00
83.30
0.025
1.100
0.015
0.010
0.0010
40.00
1000.00
0.05
1.141
0.013
0.037
0.0037
20.00
270.30
0.10
1.100
0.015
0.085
0.0085
10.00
117.60
0.30
1.070
0.100
0.200
0.0200
3.33
50.00
0.50
1.336
0.135
0.365
0.0365
2.00
27.40
0.70
1.360
0.138
0.562
0.0562
1.43
17.80
1.00
1.444
0.140
0.86
0.0860
1.00
11.60

for 100  ͦC:

S0
Absorbance
s.curve
∆S
V= ∆S/t
1/ SO
1/V
0.00
1.014
0.09
0.09
0.009
0.00
111.10
0.01
1.181
0.11
0.1
0.01
100.00
100.00
0.025
1.171
0.015
0.01
0.001
40.00
1000.00
0.05
1.417
0.14
0.09
0.009
20.00
111.10
0.10
1.252
0.12
0.02
0.002
10.00
500.00
0.30
1.197
0.115
0.185
0.0185
3.33
54.00
0.50
1.384
0.13
0.37
0.037
2.00
27.00
0.70
1.723
0.171
0.529
0.0529
1.43
19.00
1.00
1.612
0.161
0.839
0.0839
1.00
12.00

Vmax and Km values for each temperature are shown below:

Temperature
Vmax (mg/mlmin1)
Km(mg/ml -1)
8  ͦC
0.01667
0.4
28  ͦC
0.0125
0.125
60  ͦC
0.05
1
100  ͦC
0.025
1

c. The effect of pH

S0
Absorbance
S.curve
∆S
V= ∆S/t
0.5
1.470
0.15
0.35
0.0350
0.5
1.230
0.12
0.38
0.0380
0.5
1.395
0.14
0.36
0.0360
0.5
1.390
0.137
0.363
0.0363
0.5
1.390
0.137
0.363
0.0363
0.5
1.500
0.151
0.349
0.0349
0.5
1.189
0.10
0.10
0.040


Standard Curve
Graph of Substrate Concentration A
Graph of Substrate Concentration B
Graph of Temperature

 DISCUSSION

From this experiment, we can discuss that the activity of enzyme is affected by its environmental conditions. Changing this alter the rate of reaction caused by the enzyme. In nature, organisms adjust the conditions of their enzymes to produce an optimum rate of reaction, where necessary, or they may have enzymes which are adapted to function well in extreme conditions where they live.
                
First factor that affected the activity of enzyme is substrate concentration. Increasing substrate concentration increases the rate of the reaction. This is because more substrate molecules will be colliding with enzyme molecules, so more product will be formed. Based on our result, our Vmax on Michaelis-Menten graph is 0.10 µM/min and Km is 0.32 µM, while Vmax on Lineweaver-Burke graph is 0.025mg/mlmin and Km is 0.50 mg/ml. After a certain concentration, any increase will have no effect on the rate of reaction, since substrate concentration will no longer be the limiting factor. The enzymes will affectively become saturated, and will be working at maximum possible rate.

Second factor that affected the activity of enzyme is temperature. Increasing temperature increase the kinetic energy that molecules possess. In a fluid, this means that there are random collisions between molecules per unit times. In this experiment, we needed to incubate each sample in 4 different temperature which are at 8, 28, 60 and 100 oC for 10 minutes. Based on our result, the Vmax  for 8 oC   is 0.0167mg/mlmin-1and Km is 0.4mg/ml-1,Vmax  for 28 oC   is 0.0125 mg/mlmin-1 and Km is 0.125 mg/ml-1, Vmax  for 60oC   is 0.05 mg/mlmin-1 and Km is 1.0 mg/ml-1 and Vmax  for 100oC   is 0.025 mg/mlmin-1 and Km is 1.0 mg/ml-1. Our result is not accurate because of different hand during making a different concentration of starch.

Since enzymes catalyze reactions by randomly colliding with substrate molecules, increasing temperature increases the rate of reaction, forming more product. However, increasing temperature also increases the vibrational energy that molecules have, specifically in this case enzyme molecules, which puts strain on the bonds that hold them together. As temperature increases, more bonds, especially the weaker hydrogen and ionic bonds, will break as a result of this strain. Breaking bonds within the enzyme will cause the Active Site to change shape. This change in shape means that the active site is less complementary to the shape of the Substrate, so that it is less likely to catalyze the reaction. Eventually, the enzyme will become denatured and will no longer function. As temperature increases, more enzymes' molecules' active sites' shapes will be less complementary to the shape of their substrate, and more enzymes will be denatured. This will decrease the rate of reaction. In summary, as temperature increases, initially the rate of reaction will increase, because of increased Kinetic Energy. However, the effect of bond breaking will become greater and greater, and the rate of reaction will begin to decrease.

Last factor that affected the activity of enzyme is pH.Based on our experiment, the values of velocity every pH are 0.0350 mg/mlmin-1, 0.0380 mg/mlmin-1,0.0360 mg/mlmin-1,0.0363 mg/mlmin-1, 0.0363 mg/mlmin-1,0.0439 mg/mlmin-1 and 0.040 mg/mlmin-1.

CONCLUSION

 Knowledge of basic enzyme kinetic theory is important in enzyme analysis in order both to understand the basic enzymatic mechanism and to select a method for enzyme analysis. The conditions selected to measure the activity of an enzyme would not be the same as those selected to measure the concentration of its substrate. Several factors affect the rate at which enzymatic reactions proceed - temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators.

 REFERENCES
1. Alton Meister (1979), Advances In Enzymology And Related Idea Of Molecular Biology, Interscience ® Publication, Cornell University Medical College, New York.
2. John T. Moore and Richard H. Langley (2011), Biochemistry for Dummies ®, 2nd Edition, Wiley Publishing, Icn. , Indianapolis, Indiana.
3. http://www.chemguide.co.uk/organicprops/aminoacids/enzymes2.html

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