After this step I ended up with a solution containing glucose in different concentrations. In order to measure the glucose concentrations I titrated this solution with quantitative Benedict solution in which sodium carbonate was added this titration was carried out at continuous boiling and stirring of quantitative benedict’s solution the colour change in this titration was from blue to cream colour. I carried out three titrations mostly and their mean was taken in order to represent the final reading.
Subsequently by comparing the different titration readings I was able to find out the effect of change in pH, temperature and starch concentration on the rate of hydrolysis.
EXPLANATION :
Investigation of the end point of titration
To investigate the end point of the titration I made a
0.5% glucose solution in a 100ml measuring flask.
for making a 0.5% glucose solution I added 0.5 grams of glucose in water to make up a solution to 100ml and titrated it with 25ml of quantitative benedict’s to observe the end point. By this titration I was able to recognize the end point of the experiment which was a change from blue to cream colour.
Effect of change in pH on the rate of hydrolysis of starch
To investigate the effect of change of pH on the hydrolysis of starch I made the following solutions.
0.5% starch solution in a 100ml measuring flask.
2% diastase solution in a 100ml measuring flask.
I took out 25ml of each solution in two different beakers and added 5ml of buffer of pH 4 to each of these beakers in order to adjust the pH to 4. I then added both of these solutions into a large beaker and started timing for one minute. After one minute, I added five drops of iodine to this enzyme substrate mixture to stop the enzyme activity. Finally, this solution contains a concentration of glucose, which is obtained by the action of diastase on starch. To measure this concentration I took 25ml of quantitative benedict’s solution in a titration flask and added 10 grams of sodium carbonate in it.
in next step I carried out the titration. I took the starch diastase complex (having a concentration of glucose in it) in a burette and the quantitative benedict’s in which I added 10 grams of sodium carbonate in a titration flask. For this titration I needed a stirrer and a heater which was provided as this titration is carried out only when quantitative benedict’s solution is properly stirred and boiled simultaneously. I carried out three titrations and got the following three results.
Volume of reaction mixture used at ph 4.
Mean volume of reaction mixture used = 10.2 + 10.1 + 10.2 = 10.16 ml
3
The above process was then repeated using the following pHs
Volume of reaction mixture used at ph 4.8 .
Mean volume of reaction mixture used = 9.6 + 9.7 = 9.65 ml
2
Volume of reaction mixture used at ph 5
Mean volume of reaction mixture used = 9.3+ 9.4 = 9.35 ml
2
Volume of reaction mixture used at ph 7
Mean volume of reaction mixture used = 11.0+10.9+11.1 = 11.00 ml
3
Volume of reaction mixture used at ph 9
Mean volume of reaction mixture used = 11.7 +11.7 + 11.6 = 11.67 ml
3
Effect of change in pH on the rate of hydrolysis of starch.
A graph of the rate of hydrolysis of starch against ph is shown below
pH affects the three-dimensional structure of all enzymes. Enzymes are made up of amino acids.
Each amino acid has a -NH2 group and a -COOH group, not to mention certain amino acids that have an extra -COOH group (e.g. aspartame) or an extra -NH3+group (e.g. asparagines). PH is all about concentration of H+ ions. At low pH and high H+ concentration the predominant forms of these groups will be -COOH and -NH3+ or the "protonated forms". At neutral pH the predominant forms will be -COO and -NH3+. At high pH the predominant forms will be -COO- and -NH2. However the actual pH at which each group becomes ionised depends on the particular amino acid and also the environment in which the enzyme is found. The usual way of expressing this is the pK value: this pK is the pH at which half of the groups are ionised.
Interactions between these positive and negative charges are a very important part of what holds the structure together in an enzyme. These links are known as salt links, salt bridges or electrostatic interactions and involve a + to - attraction. Changing the pH therefore alters the properties of these salt bridges. Even a small shift away from optimum pH might mean one of these salt bridges is affected and therefore the shape and activity and stability of the protein will also be affected.
These results shows that the rate of hydrolysis of starch is greater in acidic pH near 4 and 5 and as the pH increases from acidic to basic pH the rate of hydrolysis of starch by diastase decreases.
Effect of change in temperature on the rate of hydrolysis of starch
For investigating the effect of change in temperature on the rate of hydrolysis of starch I prepared the following solutions
1% starch solution in 100ml measuring flask.
2% diastase solution in 100ml measuring flask.
In this experiment I took 25 ml of each of the above solutions in two different boiling tubes. The water bath was set at 20˚c these solutions were kept in the water bath until there temperature reaches to 20˚c. when the temperature reached 20˚c I mixed both of the solutions in a small beaker and allow them to react for one minute then stopped the reaction by adding iodine after one minute. The reaction mixture was then titrated with quantitative benedict’s. Following titration readings were obtained.
Volume of reaction mixture used at 20˚c
Mean volume of reaction mixture used = 6.5+6.4+6.4 = 6.44 ml
3
this experiment was repeated at temperatures of 30˚c , 40˚c and 70˚c. following sets of titration results were obtained.
Volume of reaction mixture used at 30˚c
Mean volume of reaction mixture used = 5.6+5.6+5.5 = 5.57 ml
3
Volume of reaction mixture used at 40˚c
Mean volume of reaction mixture used = 6.3+6.2+6.2 = 6.23 ml
3
Volume of reaction mixture used at 70˚c
Mean volume of reaction mixture used = 8.6+8.6+8.5 = 8.57 ml
3
Effect of change in temperature on the rate of hydrolysis of starch.
A graph between the temperatures and the rate of hydrolysis of starch is shown below
Below optimum up to optimum temperature:
Like all enzymes, diastase catalyses a (bio)chemical reaction. A fundamental of chemical thermodynamics is that all reaction rates will increase as the temperature increases. With a typical amylase, therefore as you increase the temperature to the optimum, the rate at which an enzyme catalyses the breakdown of starch increases. The first reason for this is because you increase the rate at which the enzyme and the starch substrate collide (faster Brownian motion). Also the amylase has an optimum shape or flexibility and will hold this ideal shape at the optimum temperature. For pancreatic amylase this will be at body temperature. Enzymes that do well at very high temperatures tend to have a more rigid, inflexible structure that may be held together by covalent linkages, known as disulfide bridges, between different regions of the structure. Enzymes from organisms that live at low temperatures (e.g. fish) will have a much more open structure.
Temperatures above optimum:
As you go over the optimum temperature of the enzyme, although chemically you are increasing the chances of starch breakdown, you are also increasing the chances of the breakdown of the three-dimensional structure of the enzyme. As the heat in the system increases, the vibrational energy of the entire distase molecule also increases. This puts a strain on the weak interactions that hold the enzyme together. At temperatures just above optima, there may be a situation where the enzyme is in a sort of equilibrium where it temporarily loses some of it's structure and then regains it to work again. At higher temperatures these bonds literally get shaken apart and the three -dimensional structure of the protein destabilises. This is called denaturisation. Other forces that also disrupt these bonds will have the same effect: extremes of pH, mixing a detergent, extreme concentrations of salt and so on. I like the analogy of a car built for road use that you take off-road. The car is not designed for these conditions and will quickly break down due to being bumped around.
My results shows the rate of hydrolysis of starch is highest at near 30˚c and slower at 20, 40 and 70˚c. At 20˚c the collisions between the substrate and enzyme molecules were less therefore the rate of reaction was slow. As most of the enzymes works best near 30˚c the maximum number of collisions occurring between enzyme and substrate molecules without any denaturisation so worked best at 30˚c. the results at 40˚c and 70˚c shows that the enzymes had started denaturing and the reaction rate went slow.
Effect of change in concentration of starch in the rate of hydrolysis of starch.
For investigating the rate of hydrolysis of starch I prepared the following solutions.
100ml of 1% starch solution.
100ml of 2% starch solution.
100ml of 3% starch solution.
100ml of 1.5% enzyme solution.
To start this experiment 25ml of 1% starch solution and 25ml of 1.5% diastase solution were taken. These solutions were mixed in a beaker and allowed to react for one minute after one minute the reaction was stopped. The same is done with the other two solutions of starch 2% and the 3%. The reaction products(glucose) that were obtained were titrated with quantitative benedict’s solution in order to find out the concentration of glucose present in the solution and to find out the rate of hydrolysis of starch. Following results were obtained from these titrations.
Titration readings using 1% starch solution.
Mean volume of solution used = 12.6+12.7+12.7 = 12.67ml
3
Titration readings using 2% starch solution.
Mean volume of solution used = 12.3+12.3+12.2 = 12.27 ml
3
Titration readings using 3% starch solution.
Mean volume of solution used = 12.3+12.2+12.3 = 12.27ml
3
Effect of change in the concentration of starch on the rate of hydrolysis of starch
A graph of concentration of starch against rate is shown below
These results shows that increasing the concentration of starch increases the rate of the reaction. This is because there are more molecules of starch present to be broken down, increasing the rate. However there comes a certain point whereby increasing the concentration of starch will no longer affect the rate of the reaction. From my results I can observe that this optimum stage at where this happens is when the concentration of the starch is at 2 and 3 percent. The reason for this is because all the active sites of the diastase enzyme were busy in breaking down the starch molecules, leaving no available active sites for new starch molecules. Therefore increasing the concentration of starch did not effect the rate of the reaction.
BIBLIOGRAPHY