Investigating The Activity Of The Enzyme Lipase On Milk

Plan

An Enzyme is any one of many specialized organic substances, composed of polymers of amino acids, that act as catalysts to regulate the speed of the many chemical reactions involved in the metabolism of living organisms.
Enzymes are classified into several broad categories, such as hydrolytic, oxidizing, and reducing, depending on the type of reaction they control. Hydrolytic enzymes accelerate reactions in which a substance is broken down into simpler compounds through reaction with water molecules. Oxidizing enzymes, known as oxidizes, accelerate oxidation reactions; reducing enzymes speed up reduction reactions, in which oxygen is removed. Many other enzymes catalyze other types of reactions.
Individual enzymes are named by adding ASE to the name of the substrate they react with. The enzyme that controls urea decomposition is called urease; those that control protein hydrolyses are known as proteinases. Some enzymes, such as the proteinases trypsin and pepsin, retain the names used before this nomenclature was adopted.
Enzymes are large proteins that speed up chemical reactions. In their globular structure, one or more polypeptide chains twist and fold, bringing together a small number of amino acids to form the active site, or the location on the enzyme where the substrate binds and the reaction takes place.

A catabolic enzyme splits up the substrate molecule. This process is shown below:

An anabolic enzyme combines two substrate molecules. This process is shown below:

Enzyme and substrate fail to bind if their shapes do not match exactly. This ensures that the enzyme does not participate in the wrong reaction. They are constructed of proteins composed of long chains of amino acids folded into a specific 3D shape held together by weak hydrogen bonds. On the enzyme there is a place called the active site and due to the way in which the protein chain is folded has a specific shape into which the substrate molecule is able to fit. This is called the Lock and Key Hypothesis because the enzyme (lock) and substrate (key) only fit into their own match.

The enzyme itself is unaffected by the reaction. When the products have been released, the enzyme is ready to bind with a new substrate.

However, if an enzyme meets an inappropriate heat or pH level, it is denatured which results in a crucial change in shape so that the key (substrate molecule) can no longer fit into the lock (denatured enzyme). This process is shown below:

As a class, enzymes are extraordinarily efficient. Minute quantities of an enzyme can accomplish at low temperatures what would require violent reagents and high temperatures by ordinary chemical means. About 30g of pure crystalline pepsin, for example, would be capable of digesting nearly 2 metric tons of egg white in a few hours.
Although an increase in temperature may accelerate a reaction, enzymes are unstable when heated vigorously. The catalytic activity of an enzyme is determined primarily by the enzyme's amino-acid sequence and by the tertiary structure-that is, the three-dimensional folded structure of the macromolecule. Many enzymes require the presence of another ion or a molecule in order to function.
Enzymes do not attack living cells. As soon as a cell dies, however, enzymes that break down protein rapidly digest it. The resistance of the living cell is due to the enzyme's inability to pass through the membrane of the cell as long as the cell lives. When the cell dies, its membrane becomes permeable, and the enzyme can then enter the cell and destroy the protein within it. Some cells also contain enzyme inhibitors, which prevent the action of an enzyme upon a substrate.

Aim

To find the effect of temperature on enzymes, using full fat milk as a substrate. Full fat milk provides the lipid substrate for the enzyme (lipase) to break down and digest.

Possible Variables That Can Affect Enzyme Activity

Temperature
pH level
Substrate concentration
Enzyme concentration

I have chosen to vary the temperature of the milk rather than the other variables. This is because I know that the rate of a chemical reaction should double after every 10˚C. I also know the optimum temperature at which human enzymes work (37˚C- body temperature), so I expect the ...

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Aim

To find the effect of temperature on enzymes, using full fat milk as a substrate. Full fat milk provides the lipid substrate for the enzyme (lipase) to break down and digest.

Possible Variables That Can Affect Enzyme Activity

Temperature
pH level
Substrate concentration
Enzyme concentration

The pH level would not be an interesting variable to alter because as lipase works in the duodenum, the optimum pH is 8/9 (alkaline) so the reactions will be a clear and predictable shape on a graph, so I do not feel it is worth investigating. The graph would be shaped like this:

I would rather investigate the temperature than the substrate concentration or quantity of enzyme because I would expect a fairly basic relationship where the rate is proportional to either one. I know that more enzymes or substrate would increase the rate of reaction.

The higher the temperature, the more collisions occur between enzyme and substrate, which means there is more change of an enzyme binding with the correct substrate to complete the Lock and Key Hypothesis more rapidly. When a certain temperature is reached, enzymes will denature and no longer be able to fit substrate molecules into them. Because of this, I predict a graph in the shape shown below:

Preliminary Method

First, I labeled Test Tube A and added 5cm of full fat milk (I used this type of milk rather than skimmed or semi- skimmed because it has a higher fat percentage so that there is sufficient lipid substrate), 1cm of 3% bile solution which is secreted by the liver to emulsify fat droplets, 1cm phenolphthalein indicator solution which is pink in alkaline solutions but goes colourless at a lower pH, and 1.5cm of 0.1M sodium solution which has an alkaline pH level. I add this because I want to see how long it takes the lipids to break down the fat droplets into fatty acids and glycerol, and I would judge this on the basis that the alkaline solution would be neutralized and the pH indicator would go colourless which would indicate when the fat droplets have gone, and sufficient fatty acids have built up.

I mixed them all together, which turned the liquid in the test tube a bright pink. I then added 2cm of 1% lipase solution to another tube and labeled it Test Tube B, keeping the enzyme and substrate separate until they were of equal temperatures. Then I poured the contents of tube A into tube B and simultaneously started the stop clock.

I then recorded the time taken for the contents of the tube to turn white. This happens when it has a low pH.

Break Down Fats and Oils⇒ Fatty Acids + Glycerol ⇒ Low pH

Prediction

I predict that the closer the temperature of the liquids to human body temperature (37˚C), the faster the reaction will take place. This means the very high and very low temperatures would take a longer time to react because and the closer temperatures would grow gradually faster until they reached 37˚C, where I would expect the reaction time to be quickest as the temperature is optimum.

: I predict that the enzyme will become denatured, and therefore will work at a slower rate after 40 - 45°C. I think the reason for this prediction is because every enzyme has a temperature range of optimum activity. Outside that temperature range the enzyme is rendered inactive. This occurs because as the temperature changes enough energy is supplied to break some of the molecular bonds. When these forces are disturbed and changed the active site becomes altered in its ability to accommodate the substrate molecules it was intended to catalase. Most enzymes in a human body shut down beyond certain temperatures. This can happen if body temperature gets too low (hypothermia), or too high (overheating).
From my background knowledge it is evident that as temperature increases, the rate of reaction also increases. However, the stability of the protein also decreases due to thermal degradation. Holding the enzyme at a high enough temperature for a long period of time may denature the enzyme. Reaction rate is the speed at which the reaction proceeds toward equilibrium. For an enzyme-catalysed reaction, the rate is usually expressed in the amount of product produced per minute. The energy barrier between reactions and products governs reaction rate. In general, energy must be added to the reactants to overcome the energy barrier. This added energy is termed "activation energy", and is recovered as the reactants pass over the barrier and descend to the energy level of the products assuming the reaction is exothermic. Enzymes can accelerate the rate of a reaction. Catalysts accelerate the rates of reactions by lowering the activation energy barrier between reactants and products. In general chemical reactions speed up as the temperature is raised. As the temperature increases, more of the reacting molecules have enough kinetic energy to undergo the reaction. Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster with increasing temperature. However, if the temperature of an enzyme catalyzed reaction is raised still further, an optimum is reached: above this point the kinetic energy of the enzyme and water molecules is so great that the structure of the enzyme molecules starts to be disrupted. The positive effect of speeding up the reaction is now more than offset by the negative effect of denaturing more and more enzyme molecules. Many proteins are denatured by temperatures around 40 – 50°C, but some are still active at 70 – 80°C, and a few withstand being boiled. So, my first prediction is that the enzyme will become denatured at around 40°C, and secondly, that as the temperature increases the reaction rate will increase by 50%, due to the molecules colliding together at a higher speed (kinetic theory) due to their extra energy obtained by the increase in temperature. My prediction is supported by Kinetic Theory in that if I apply twice as much heat there will be twice as much particle vibration therefore the reaction will happen twice as quickly. It is also backed by Collision Theory in that if I apply twice as much heat there will be twice as many collisions and therefore the rate of reaction will double. This will only be so until the enzyme denatures after its optimum temperature: 37°C.

Hypothesis

I predict that the closer the temperature of the liquids to human body temperature (37˚C), the faster the reaction will take place because the lipid substrate would be accustomed to working at body temperature. This would result in a faster rate of a chemical reaction and so the substrate keys would connect significantly more rapidly with their enzyme locks than if they were at a much lower or excessively high temperature.

My prediction is supported by Kinetic Theory in that if I apply twice as much heat there will be twice as much particle vibration therefore the reaction will happen twice as quickly. It is also backed by Collision Theory in that if I apply twice as much heat there will be twice as many collisions and therefore the rate of reaction will double. This will only be so until the enzyme denatures after its optimum temperature.

Chosen Variable

10˚C
20˚C
30˚C
37˚C
40˚C
50˚C
60˚C

Fair Test

I will keep this experiment a fair test by:

Using fresh pipettes for each substance to avoid contamination
Altering only the stated variable
Keeping all other variables the same
Using the same stop clock for each experiment
Having the same person timing and deciding when it is the correct time to stop the clock so the judgments will not alter
Stirring the liquids continuously and equally
Including the same quantity of phenolphthalein (1cm) in each concentration of milk, because the indicator is slightly acidic so varied measures may sway the results
I will take three readings of each temperature to ensure a fair average and no anomalous results.

Safety

I will wear safety goggles for the duration of the experiment because sodium is a caustic alkali and the enzyme lipase is an irritant.
I will try not to touch any substance directly with my hands because sodium is a caustic alkali and the enzyme lipase is an irritant.
I will use tongs when lifting beakers filled with hot water in case I burn myself

Results of preliminary work:

The results did not show much of a correlation or any type of relationship that I had expected. I think I may have made an error in the recording because I only took one reading. I am still going to investigate the same variable (temperature) although it was difficult and time consuming.

Equipment

I have decided to use the following equipment in order to carry out my experiment:

Water bath
Hot water
Water at room temperature
Ice
Phenolphthalein
Sodium carbonate
Lipase
3% Bile salt
Undiluted full cream milk
Test tubes
Thermometer
Stirring rods
Syringes
Pipettes

Method

First, I set up a water bath at the lowest temperature which is 10˚C and used a thermometer to keep track of the temperature to check it does not become too hot or too cold.

Then I labeled two of the test tubes Tube A and Tube B. To Tube A, I added 5cm of undiluted full cream milk, 1cm 3% bile solution, 1cm phenolphthalein indicator solution and 1.5cm 0.1M sodium carbonate solution. In Tube B, I measured 2cm 1% lipase solution.

I then placed both tubes into a water bath at the required temperature and left them both together for 10 minutes (it doesn’t matter how long you leave them in there for as long as it is over 2 minutes and they are taken out at the same time so they are the same temperature.

I then poured the contents of Tube B into Tube A, started the clock and stirred constantly for 10 minutes.

I repeated this three times to find a mean for this temperature. The procedure was repeated for the following temperatures: 10°C, 20°C, 30°C, 37˚C, 40°C, 50°C and 60°C. I have evolved on this plan as a result of preliminary work on the topic in which a number of procedures and variables were demonstrated.

Results:

Analysis of results

From the graph, I am able to back up my theory. I can see when the enzyme is most active and when it starts to denature. From the graph, I have found out that, as the temperature increases, so does the catalase activity, as it does not take as long to move the same distance, up to a certain point (40°C) where the activity has past its optimum. I found that the optimum for a catalase is at 37°C because it is body temperature. This is where the greatest number of collisions takes place between the enzyme and the substrate and therefore the highest rate of reaction is.

The rate was higher at the higher temperatures (up to 40°C) because as the temperature is raised, so is the energy level of the enzymes and substrate molecules. This means that they have more kinetic energy so they collide more often and therefore more reactions take place between them. This, in turn, means that the rate increases as more oxygen (O) is produced.

The enzyme started to denature at about 40°C because the weak bonds, which hold the molecule into the specific shape for one substrate, are broken. The increase in molecular collisions and vibrations at higher temperatures is great enough to permanently change the shape of the active site. The enzyme is said to be denatured because it can no longer form an enzyme-substrate complex as its active site has been unalterably changed.

At lower temperatures, the enzymes simply did not have enough energy to catalyze, which is why they were slower than at the higher temperatures where they were beginning to denature but still had enough energy to catalyze. Although 40˚C was just 3˚C higher than the optimum of 37˚C, the hydrogen bonds are weakening and breaking. This explains why the rate of reaction dropped suddenly and the time taken slowed down at 40˚C.

The rate of reaction graph also shows a good trend which backs up my prediction because they react faster and faster until 37˚C when they reach their peak and then they significantly slow down.
My prediction was correct in that there was very little activity in the ice bath because the speed at which the enzymes and substrate molecules were moving was very slowly so there were not many collisions between them. The optimum temperature was the same as I predicted at body temperature.

Evaluation:

Although I conducted the experiment as accurately as I could there were sources of error in the method that I used. Firstly, some help from a friend was needed to start the experiment and this led to a small delay in starting the stopwatch. I needed to pour the contents of Tube A into Tube B, remove the empty tubes from the water bath so as not to obscure the view and start the stopwatch all at the same time.

I also think it would have been better if I had used the same milk from the whole experiment but was unable to due to the time and equipment restrictions. This meant I probably had a mixture of a variety of milks at different ages, heats and bacteria count. I needed the tubes to all have the some constant pink colour before I should start. This meant I had to modify the rate of sodium carbonate to neutralize the solution. I had to compete with sour milk with a lower, more acidic pH as well as differing bio salts in souring milk. This is a source of error because the concentration of catalase in the milk may have been different which may have produced an inconsistent rate of reaction.

This might be why the values I obtained for the 20°C and 50°C do not quite fit the pattern of the graph that I expected. To remove this problem, I could repeat the experiment not only with three readings at each temperature, but also with three different milk samples, which would provide an even more accurate reading, as I could calculate an even wider average.

Another problem was that I had to leave the milk solution and lipase solution to acclimatize for longer than I expected would be necessary which consumed a lot more time which may have been used more constructively to carry out more averages.

I think that any anomalous results where mostly due to a longer acclimatization or the fact that I did not allow the lipase to acclimatize for exactly the same amount of time, which I would definitely do if I repeated the experiment.

I also found it difficult to measure all three tubes at the same time because it meant I had to keep moving them so I could see the colour as the beakers were not transparent. This meant I had to use a large amount of human judgment, which is not always entirely reliable in scientific conditions.

If I repeated the experiment I would also take more readings for example at every 5°C and 70˚C because if I did this I would be able to plot a more accurate graph and it would be easier and more accurate to tell when the enzyme got to the optimum and denaturing temperatures. If I had measured 70˚C, I might have discovered the temperature where most of the enzymes denatured so that they had even less force to continue reacting. I found it difficult to draw my line of bestfit on both graphs because of the large jumps in rate and time of reaction. The fact that the optimum results were significantly lower on the temperature/time graph and significantly higher on the temperature/rate of reaction graph meat I had to try to accommodate them as well as the more regular readings leading up to and after the optimum temperature.

The evidence that I obtained is sufficient enough to support the conclusions I have come to about the values for the optimum and denaturing temperatures because I conducted my experiment as accurately as I could with the method I used and did quite a large range and number of repetitions to the results reliable.

If I was to conduct a further experiment, I would measure the relationship of relationship of temperature against time again, but I would use these values:

31˚C
33˚C
35˚C
37˚C
39˚C
41˚C

This is because I would like to investigate closer if the enzymes begin to denature immediately after they have reached the optimum temperature and if they in fact reach a similar rate before hand. If I did this, I would be able to find out if 37˚C really is the optimum temperature for enzymes and if 40˚C is really a potentially dangerous body temperature.

Investigating The Activity Of The Enzyme Lipase On Milk

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