Starch Hydrolysis by Amylase | Experiment

Polysaccharides are polymers of carbohydrates. They are made up from monosaccharides which are linked together by glycosidic bonds. Polysaccharides are divided into two main groups: structural polysaccharides and polysaccharides which are used as energy sources. Cellulose and chitin are classified as structural polysaccharides. Cellulose is major component of plant cell walls. It yields glucose when completely hydrolyzed. On the other hand Glycogen and starch are the polysaccharides which used as energy source; glycogen is found in animal cells and starch is found in plant cells.
Plants use starch to use it to store glucose units for energy. It consists of two types of molecules: the linear and helical amylose and the branched amylopectin. Starch generally contains 20 to 25% amylose and 75 to 80% amylopectin. Amylose forms a colloidal dispersion in hot water whereas amylopectin is completely insoluble.
2.2) Hydrolysis of starch/amylase enzyme:
Proteins which catalyze the chemical reactions are called enzymes. Biological catalyzes, enzymes, need specific conditions to be active since they are working in the cells. Temperature must be between 37 and 40 and ph must be neutral. An important metabolic enzyme is amylase that its function is to catalyze the hydrolysis of starch into glucose. Alpha-amylases are found in plants and in animals. Human saliva is rich in amylase, and the pancreas also secretes the enzyme.
SUBSTRATE     ENZYME                       PRODUCTS
starch       Amylase  —->   maltose + maltose + maltose —
starch’s presence can be identified by using the iodine test. Starch and iodine gives blu-black color together and that helps to identify the presence of starch or iodine. The iodine molecule slips inside of the amylose coil. Iodine is not very soluble in water so the iodine reagent is made by dissolving iodine in water in the presence of potassium iodide. This makes a linear triiodide ion complex and this linear complex can easily slip into the coil of the starch. This forms the color. [3]
starch + I2  blue-black color
2.3) Spectrophotometry:
A spectrophotometer is used to find the amount of radiant energy absorbed or transmitted by molecules in a solution as a function of wavelength. The wavelength which a certain molecule can absorb energy is different and therefore it can be used to determine the concentration of a specific type of solution. By comparing the amount of light that is absorbed by the sample with known concentration a calibration curve can be plotted and by using it concentration of the unknown sample can be determined. [5]
3) EQUIPMENT AND CHEMICALS
3.1) equipments:

Beaker
Laboratory scale
Weighing dish
Spatulas
Tubes
Tube rack
Heater
Water bath
Plastic cuvettes
Pipettes
Dropper
pH meter

3.2) chemicals:

Distilled Water
HCl
NaCl
KH2PO4
Na2HPO4
Starch solution
Human salivary
Iodine reagent

4) PROCEDURE
4.1) preparation of starch solution and enzyme solution:

20 g of potato starch was mixed with approximately 50 ml cold water.
This mixture was then added to 900 ml of boiling water.
Mixture was mixed well and it was cooled to room temperature and the total volume was raised to 1 ml by adding sufficient amount of water.
Presence of the starch in the solution was tested by putting one drop from the mixture to glass plate and adding one drop of iodine reagent to it. Blue color means starch is present.
Saliva sample was taken into tube and diluted with 9 ml water then, 60 ml of 0.5% NaCl was added.

4.2) Effect of the pH:

0.1 M KH2PO4 with pH 5, 6, 7 and Na2HPO4 with pH 8, 9 solutions were prepared as buffers, each buffer was prepared 20 ml only the buffer with 7 pH was prepared 100 ml.
5 test tubes were labeled and to each of them 5ml of the starch solution was put and to each test tube solution with different pH was added.
1 ml of the salivary enzyme solution was added to the tubes and it was mixed by shaking.
it was waited for 10 minutes so that the hydrolysis reaction can proceed.
5 ml of HCl solution was added to 5 different tubes and from each of the previous tubes with the starch solution 0.5 ml was taken and added to tubes with HCl.
5 tubes were prepared again for 5 ml of iodine solution this time. From the tubes with HCl 0.5 ml was taken and added to the tubes with iodine.
Absorbance values were measured by using spectrophotometer.

4.3) Effect of temperature:

Water baths with 30 °C, 50 °C, 70 °C and 90 °C were prepared in beakers.
Buffer solution with pH 7 was added to 5 tubes and 5 ml of starch solution was added to these tubes.
All of these tubes were put in different water bath with different temperatures and they were waited in the bath until the temperatures reached the equilibrium.
1 ml of salivary enzyme was added to each tube. After 10 minutes the steps 4-7 in the effect of pH procedure were repeated.

 
5) DISCUSSION
In this experiment our purpose was to hydrolyze starch with amylase enzyme and observe the effect of pH and temperature on this reaction. Before observing the pH and temperature effect first we prepared the solutions that we were going to use in the experiment. First we prepared the starch solution by mixing it first with cold water and then adding it into boiling water. We aimed to get the starch suspension form immediately and without any lumps by this procedure. In the first part of the experiment we observed the pH effect. To do that first we needed to prepare the buffer solutions with different ph values ranging between 5 and 9. We used two different solutions because of the buffering capacity of these two solutions. We made the arrangement of the pH by using HCl and NaOH. To decrease the pH we added HCl and to increase we added NaOH, we detected the pH by using pH meter. Each buffer with different pH values were mixed with starch solution and then salivary solution was added. Which is the amylase enzyme and since it’s from the saliva it hydrolyzes amylose. After waiting for 10 minutes to reaction proceed we needed to stop the reaction, we did it by adding HCl. Then we added iodine solution to detect whether reaction took place or not since if enzyme functions starch in the solution will be hydrolyzed and this will lead to have light color of the solution; absorbance will be low. Since our body is in neutral pH we expect to have light colored solution at pH 7 and dark color at pH 5, 8 and 9. The same logic is valid for the temperature effect. The enzyme won’t work in higher temperature values that can denature it like 90, 70 and maybe 50.
Protein’s absorbance values are expected to increase as the protein denaturizes. This can be explained by the surface of reflection of the light is increased. Denaturized form of protein has higher possibility to be interacted with the light from the spectrophotometer and thus absorbance will increase.
Theoretically we would expect to have both absorbance vs. pH and absorbance vs. temperature graphs to have a min. point where we can say that is the point absorbance is in the min. point at that pH or temperature thus enzyme functions best at that point. In our graphs from the experiment we can observe these min values at approximately expected values. When we look at the temperature graph we see that absorbance is min at around 50 C. normally we would expect that proteins denaturize at that temperature, since human body is 37°C min absorbance at 40°C would be the correct result. This error might be because of that we didn’t measured our test tubes temperatures after we put them in water bath, So maybe what we refer to as 50°C in the data table is actually less than that value.