Effect of Enzyme Concentration on Protein Digestion
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Enzymes were discovered by a German chemist Eduard Buchner near the end of the 19th century. He had been trying to extract a fluid for medicinal use from yeast, however, the yeast extract kept going bad. He then decided to add sugar to the yeast, however, the yeast converted the sugar into alcohol, which is also known as fermentation. Buchner investigated into this and soon found out that living cells were not responsible for this fermentation and that it was caused by the fluid that was trying to be extracted from the yeast. The word enzyme was coined for the active ingredients in the juice that promoted fermentation. Although enzyme literally means "in yeast", it is now however being used as the collective noun for several hundreds of compounds that have shown to have a catalytic action on specific chemical reactions.
Enzymes are biological or organic catalysts made up of protein. They catalyse (increase/decrease the rate of) chemical reactions without themselves being chemically changed at the end of the reaction. It can therefore be used repeatedly and so is effective in small amounts. They essentially work by lowering the activation energy of the reactions and hence allowing the reaction to place at a quicker rate. In enzymatic reactions, the molecules are the start of the process are called substrates, and the converted molecules, the products.
Properties of enzymes:
Enzymes have the following properties:
Enzymes alter the rate of chemical reactions without themselves being chemically changed at the end of the reaction.
Enzymes are very potent. Since enzymes are very specific, a small amount of an enzyme is capable of catalysing a huge chemical reaction.
Enzymes are affected by temperature. Enzymes are inactive at low temperatures. Increasing the temperature increases the activity of the enzymes. There is an optimum working temperature at which certain enzymes work best. This is normally between 37-42 degree centigrades. However, a high temperature, anything above 45 degree centigrades normally destroys the active sites of the enzymes and causes it to denature. This permanently damages the enzyme and they become functionless.
Enzymes are affected by pH. Certain enzymes work best in acidic conditions whereas certain enzymes function better in alkaline conditions. For example, pepsin works best in the stomach where the pH is below 7, however intestinal enzymes work better in coditions of pH of above 7.
Some enzymes may require a compound to be bound to them before they can catalyse chemical reactions. These compounds are called co-enzymes.
Enzymes can work in either directions. Metabolic reactions are reversible and the direction in which the reaction goes depends on the amounts of substrate and products present. The reaction will proceed from left to right until an equilibrium is reached between the substrates and products. Also, if there is a large amount of products, then the reverse reaction starts and hence causes the product to be split up until again equilibrium is established.
Lock and Key & Induced Fit Hypothesis:
Although enzymes have a large size, however, they only have a small region that is functional. This is known as the active site. Active sites can be described as depressions on the surface of the enzyme. Only a few of the amino acids of the enzyme molecule make up this active site; the remainder are used to maintain its overall three-dimensional shape. The active site is the site where the substrate binds onto the enzyme and only substrates with a particular molecular shape will have any chance to bind effectively with the particular enzyme. This is the reason why enzymes are specific in their actions as they can only bind to specific substrate molecules.
Another idea which arose from the lock and key hypothesis is the induced fit hypothesis that suggests that the enzyme alters its shape slightly to ensure that the enzyme molecules bind tightly with the substrate molecule. However, once the product or substrate leaves the active active site, the active sight realigns itself to its original form.
Now, having discussed enzymes generally, we shall move onto discussing the enzymes more specific to this experiment. The enzyme being used in this investigation is Neutrase. Neutrase is a bacterial protease which is produced from a bacterial strain called Bacillus Amyloliquefaciens. Protease is an enzyme which catayses the hydrolysis of proteins into polypeptides/amino acids. In humans, the digestion of protein chiefly starts in the stomach with Pepsin in the gastric juice where the proteins are converted into polypeptides. Protein digestion is represented in this experiment by the fact that when the proteins in the milk are digested, the solution turns from opaque cloudy white to a see-through solution.
In an enzyme controlled reaction such as that of protein digestion in milk, an increase in the concentration of enzyme will lead to an increased reaction rate. This is due to the fact that when there are more enzyme molecules present, there will be a greater chance of more of the enzyme molecules colliding with the substrate and hence increasing the frequency of the collisions. This increased frequency of collisions will help to form an enzyme-substrate complex more rapidly.
The aim of this experiment is to see if there is a negative correlation between the enzyme concentration and the rate of digestion of the protein content in the milk. Increase in enzyme concentration leading to decrease in the rate of digestion of protein content.
H1 - There is a positive correlation between the enzyme concentration and the rate at which the protein content in the milk digests. (Increase in rate of reaction).
H0 - There is no correlation between the enzyme concentration and the rate at which the protein content in the milk digests. (No affect on rate of reaction).
Justification For Use
Marvel Powdered Milk
- The source of protein upon which the enzyme to work on.
Enzyme (Neutrase )
- The enzyme which digests the protein content in the milk.
Sodium Phosphate Buffer (pH 6.4)
- In order to ensure the pH of the solution remains constant for optimal working of the enzyme.
Test Tubes And Test Tube Rack
- Test Tube: To hold the enzyme, buffer and milk powder solution.
- Test Tube Rack: To hold the test tubes.
- To measure out the volume of distilled water.
- To measure out the mass of the milk powder.
Pipette And Pipette Filler
- To measure out accurately the volume of the enzyme.
- To hold the water and the test tubes containing the enzyme, buffer and milk powder solution.
- To time the duration it takes for the enzyme to completely digest the protein content in the milk.
- To add the solution of the enzyme, buffer and milk powder into the curvettes.
- To measure the light absorbancy values.
A preliminary experiment was conducted initially in order to test whether the method intended for use was flawless or not. However, through the conduction of this experiment, a number of flaws were noticed and hence dealt with to produce a more flawless and sound experimental procedure.
In the preliminary experiment, it was decided that the temperature of the solution containing the enzyme would be kept constant by using a bunsen burner. However, fluctuation in temeperature were going to be evident and hence causing inaccuracies. Consequently, it was decided to use a thermostatically controlled water bath to keep the temperature constant.
Also, when using the colorimeter, I decided to place the solution inside the curvette and then place it onto the colorimeter and record the values over time until it reached 0. However, this would give inaccurate results based on the fact that when the solution was removed from the water bath and placed inside the curvette, the temperature would decrease over time and hence affect enzyme activity. As a result, it was decided that the solution be removed from the water bath and placed into the curvette at regular intervals. After every 20 seconds, a new curvette would be used with solution removed from the water bath and then placed onto the colorimeter to record the light absorbancy values.
Measure out 10.00 grams of milk powder using an electronic mass balance into a beaker. Then measure out 200 cm^3 of distilled water using a measuring cylinder. Add the water into the beaker containing the milk powder and use a spatula to stir the solution well.
Now, different concentrations of enzymes are to be prepared.
To make a 1% concentration solution of enzyme, add 1cm^3 of the Neutrase into a test tube using a pipette. Then add 99cm^3 of distilled water measured out using a measuring cylinder. Shake the test tube containing the solution (100cm^3) well.
To make a 2% concentration solution of enzyme, add 1cm^3 of the Neutrase into a test tube using a pipette. Then add 98cm^3 of distilled water measured out using a measuring cylinder. Shake the test tube containing the solution (100cm^3) well.
Use the same method as above to make 3%,4% and 5% solutions. Place each of the test tubes into the test tube rack.
Add Sodium Phosphate Buffer (6.4 pH) to each test tube.
Place one of the test tubes containing the enzyme solution and any one of the milk solution test tubes into the water bath for 5 minutes.
After 5 minutes, immediately pour the enzyme solution into the test tube containing the milk solution.
Use a colorimiter to check the amount of light passing through the solution with time. Press 'R' to reset the colorimeter (use the red filter) as red light is transmitted the best and this will give accurate readings.
After every 10 seconds, place some solution from the test tube into the a curvette and the curvette onto the colorimeter and record the result.
The sole independent variable in this experiment is the concentration of the Neutrase solution which ranges from (1-5)%.
Other variables which could affect the investigation have been tabulated as follows:
- How may a change in this variable affect the data?
- How will it be controlled?
Type of milk used
Some milks will have more or less protein molecules present than others which alters the amount of substrate molecules being available to form enzyme-substrate complexes.
This will be controlled by ensuring that the same milk powder is used throughout the experiment.
pH of the solution
Enzymes work better in solutions of different pH. The activity of the enzyme depends upon the pH of the solution; if optimal pH solution is used, the enzyme would work better than if non-optimum pH was used.
This will be controlled by ensuring that a pH buffer ( Sodium Phosphate - pH 6.4) is used. This will minimise any changes in the pH of the solution and ensure that the optimum pH is kept constant for efficient working of the enzyme.
Volume of Neutrase solution used
A larger volume would of enzyme ( Neutrase ) soltuion would result in a greater amount of enzyme-substrate complexes and hence increasing the rate of the reaction.
Neutrase solution volume will be kept constant by precisely measuring the volume required using a pipette and pipette filler.
Temperature of the solution
Temperature alters the rate of enzyme activity and a higher temperature would result in greater enzyme activity. However, anything normally above 45 degree centigrades would lead to denaturing of the enzyme.
Using a thermostatically controlled water bath will maintain a constant temperature. The temperature would be kept constant at the optimum working temperature for the enzyme.
Volume of milk used
A larger volume of milk would result in a greater amount of enzyme-substrate complexes being formed as there is a greater number of protein present and thus increasing the rate of the reaction.
Milk volume will be kept constant by precisely measuring the volume required using a pipette and pipette filler.
Health & Safety Regulations:
- Wearing a lab coat inside the laboratory.
- Wearing safety goggles to protect the eyes from chemicals.
- Wearing plastic gloves when handling the enzyme and milk solution and to prevent contamination.
- Keeping the test tubes in a rack to prevent accidental breakage or spilling.
- Bunsen burners will be ensured that they are kept on yellow flame when not in use.
I have chosen to use the Spearman's Rank Correlation to use as a statistical test.
The Spearman's Rank Correlation is a technique used to test both the direction and strength of the relationship between two variables. It uses the statistic R's which fall between the range of -1 and +1.
The results of the experiment prove that the H1 hypothesis is correct. There is a positive correlation between the enzyme (Neutrase) concentration and the rate at which the protein in the milk digests. In other words, increasing the concentration of the enzyme increases the rate of reaction.
The results show that an increase in the concentration of the enzyme leads to a descrease in the time taken for the solution to go clear. This is due to the fact that when the concentration of the enzyme is increased, there are more active-sites present on the enzymes onto which the substrate binds onto. This creates more and more enzyme-substrate complexes. The higher concentration of enzyme increases the kinetic energy of the molecules and increases the frequency of the collisions between the enzyme and substrate molecules. A lower concentration of enzymes means there would be fewer active sites and hence, a slower rate of reaction.
However, if we kept increasing the concentration of the enzyme, one would notice that the line would start to level off because all of the active sites on the enzymes would be occupied and hence, increasing the concentration would have no effect on the rate of the reaction.
Observing the results, one can easily generate the conclusion that there is a positive correlation between the enzyme concentration and the digestion of the protein content. Even though, the set of results achieved are not fully accurate and hence, there are anomalies. However, improvements can be made to minimise inaccuracies and unreliability in the experiment.
One of the factors that could have affected the experiment could have been human reaction error in timing. Also, the starting of the stop-clock for each of the tubes could have been different and this could have resulted in unreliable results. To avoid this, however, an automatic stop-clock could be used.
Another factor that could have affected the experiment could have been the movement of the reacting mixture constant. The mixing between the substrate ( Protein in the milk ) and the Enzyme ( Neutrase ) could not have been same in each tube. This could have resulted in inaccuracies in the result as the molecules in the tubes with the better mixing would have greater kinetic energy and hence, the reaction rates would be quicker as more and more enzyme-substrates would be formed quicker. To avoid the extent of mixing in each tube from affecting the results, I will ensure that next time, there will be no stirring of the mixtures when solutions are added in any way so that this way, it will be constant ( no mixing ) for each tube.
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