Renin experiment

The effect of varying temperature on thee Rennin enzyme

Abstract

The purpose of this experiment was to see what affect different temperate had on the reaction rate of the enzyme rennin. The experiment was performed by placing test tubes filled with renin and milk into water bath which was heated or cooled to one of the temperatures trialed. The hypothesis justified because it found because it though data that the enzyme reacted faster with the 45° temperate because it was closer to the optimum conditions of the enzyme rennin. These results can clearly be seen throughout this report though graphs, calculations and observations

Introduction

The average human body withholds about 100 trillion cells; within each of these cells reactions are always taking place. (Wikipedia The Free Encyclopedia , 2013) In all living organisms the reactions that take place are all vital for survival and make the living organism function. The speed of a reaction such as breaking down foods is performed from a catalyst. A catalyst is a substance that increases the rate of reactions in all living organisms. They speed up the reactions by providing reactants with different pathways so they can react faster.

An enzyme is a biological catalyst it speeds up a chemical reaction by lowering the activation energy required to start the reaction. (Boundless, 2013) An enzyme can be put under different conditions to see whether it will stay active or become denatured. When an enzyme is at its optimum conditions, the rate of reaction is the faster. For example increasing or decreasing the temperature will indicate whether the enzymes are still active or denatured and also when the optimal temperature is for that particular enzyme.

An enzyme works in very complex ways almost like the way a key fits into a lock. When a certain molecule which holds the right chemical compound passes the enzyme it will attach itself. (See figure 1) The area in which the molecule attaches itself is called the active site of the enzyme; this is the spot where the reaction process takes place. The substrate is another name which can be used to describe the molecule which attaches itself to the enzyme. Once the reaction has taken place the substrate leave and get ready to start again. (ABPI, 2013)

(Casiday, 2012)
Figure 1: Enzyme structure, explaining the lock and key process

There are many factors in which effect how well the rennin enzyme reacts with the substrate. Environmental conditions are one of the main factors why the enzymes activity levels change an example of this is changing the temperature. When some of these factors change it will also are changes the reaction rate of the rennin enzyme.

The rennin enzyme is important to the human body because it helps breakdown proteins and/or peptides into certain acids. Rennin’s main job is to assist in the digestion of dairy foods and to coagulate milk the stomach. Newborns reply of the rennin enzyme because it is their only source of protein, once newborns have reached the maximum amount of rennin it is replaced with the digestive enzyme, pepsin. The coagulation of milk in the stomach is very important for within young animals. This is because if the milk was not curdled, it would run straight through the stomach and would not properly digest so no nutrients wouldn’t be absorbed and converted into energy (Bowen, 1996).

In this experiment the enzyme rennin was use. Rennin is a coagulating enzyme and is a mixture of enzymes called Chymosin. Chymosin is a proteolytic that found in the stomach of mammals it role in digestion is to curdle or coagulate milk in the stomach (Jirage, 2013). If mammals were unable to coagulated milk, it would rapidly flow through the stomach and miss the opportunity for initial digestion of its proteins (Wikipedia, the free encyclopedia, 2013)

The variable that will be tested in this experiment is the temperature. The reason for using the variable of temperature is to see if the rennin enzyme will have enough heat and kinetic energy for the enzyme to stay active; it will be observed at what temperate the enzyme will become denatured.

Temperature is a factor of the particle theory because depending on how much temperature is increased will depend on how fast the particles collide. If you heat a substance, the particles move faster and so collide more frequently this will speed up the rate of reaction. Collisions only result in a reaction if the particles collide with enough energy to get the reaction started in this experiment there will need to be enough energy within the collisions to react with the rennin or no reaction will take place and it will either be inactive because there is not enough energy and particle collisions or become denatured because it is too far out of range for the optimum conditions. (Clark, 2002)

(Carnot, 2006)
Figure 2: The collision theory

The aim of the experiment is to see at what temperature the rennin can coagulate with the milk. Also at what temperatures the rennin enzyme will become inactive and what temperature it will become denatured this means no reaction will take place.

It is hypothesized that the that the optimum condition for the rennin enzyme will be 37 degrees Celsius because it works in the body of a mammal, the typical mammalian body temperature is around 37°C. It is predicted that when the temperature is lower than thirty two degrees, the rate will be slow, gradually increasing as the temperature because the rennin enzyme will not have enough kinetic energy for the reaction to take place and the rate of reaction will be very slow due to the very low diffusion of enzyme and substrate molecules (123helpme.com, 2013). It is predicted that if the temperature of the rennin enzyme exceeds 40°C the enzyme will start to denatured If the temperature to of the rennin enzyme becomes too hot then the reaction will give a slower rate, decreasing the further from the optimum as it increases. At a certain point the reaction will stop altogether, this is when the enzyme is now denatured and to reaction process of the reaction will stop (123helpme.com, 2013).

When the enzyme is at its optimum temperature, the substrate molecules gain more kinetic energy, so they move around more. This means more of the molecules will be able to enter the active enzyme (Freeman, 2002). When they collide more energy is created so the bonds will be broken so the reaction can occur.

The structure of an enzyme is made up of proteins which are divided into sub- units called amino acids these are called peptides bonds (123helpme.com, 2013). When the temperature goes beyond the optimum temperature more of the bonds are formed. The thermal energy breaks the hydrogen bonds and the enzyme and loses its shape to become a random coil (123helpme.com, 2013). Therefore the substrate can no longer bind and the reaction is no longer present at very high temperatures this is irreversible

Methodology

A table was drawn for each of the variables that were tested 15,30,45,60 degrees. These tables were used after the experiment to record data that was collected. Then 4 15ml test tubes were used for each temperate and labeled from A to D. They were then placed in the test tube rack. Rennin tablets were then broken in half. Next 30 mL of full cream milk was poured into a 100 mL beaker, and then using a pipette, 3mL of milk was added to each individual test tube. Next water was added to tee 100ml and ice was added until reached 15 degrees Celsius, it was monitored closed and checked using a thermometer. When the temperature was 15°C all 4 test tubes. The temperature of the milk was closely monitored when the milk had reached 15 degrees half of the rennin tablet was placed into each of the test tubes expect “A” because test tube was used as a controlled variable. As this was done a timer was started using a stop watch to see at what time the milk would start to change or coagulate, when the test tube showed a jelly- like consistency the timer was stopped and all data and notes was recorded in the table shown above.

temperature the different temperatures will be 15,30,45 and 60 degrees Celsius each temperature will have 3 different trials all data will be recorded and the results in a table to give a clear view of the results

Photo: Showing the set up for the experiment

This process was repeated four times at each different temperature, all with test tube “A” only containing milk as this was used as the controlled data. The water was held in a kettle before being placed in the beaker for the water bath. The temperature was altered using either ice cubes or more heated water and stirred with a thermometer until reaching the temperature that was being tested at that time the rest of the experiment was repeated identically.

Results

Qualitative

The reaction of the rennin solution to the milk at different water temperatures has reacted at different times this depended on the temperature, during some of the trials a reaction did not occur. At the temperatures of 30 and 45 degrees Celsius a reaction occurred and it was visible that a jelly substance formed on the top of the solution and also some bubbles formed on the top. At 45°C a gelatinous mass formed at the top and there were air bubbles visible within the coagulation. The rate of reaction occurred faster at 45°C because the kinetic energy within the reaction moves faster with a hotter temperature. Although when the temperature is trialed at 60°C the gel strength from the rennin is reduced. This is because heat precipitates the lactoglobulins onto the kappa casein which interferes with gel formation (food.oregonstate.edu, 2012)

Table 1: Averages from each temperature trialed

Temperature
Time to react (minutes)
15°C
10 minutes +
30°C
1.40
45°C
0.88
60°C
10 minutes +

Graph 1: Showing the rate of reaction

Discussion

The hypothesized that the optimum temperature for the enzyme rennin to react would at 37°C the hypothesis was justified. This can be seen in graph 1 , in the results section because the graph line and the best fit line this collide which shows that 37°C is the optimum conditions for the rennin enzyme. The enzyme is most active because of when rennin’s molecules are near optimum conditions the molecules start to collide it forms kinetic energy (123helpme.com, 2013).

The reason that the reaction rate increased at 30°C and 45°C is because it started to become the optimum conditions. The enzymes are most active at 37 degrees Celsius this is because of kinetic energy formed with the rennin molecules (Panse, 2011). Although the kinetic energy formed within the rennin enzyme worked reacted with a higher better with a higher temperature, when it started to exceed 50°C the activity levels started to decrease. This therefore made the enzyme harder to attach to the substrate. The reason the 15°C trial did not have a reaction is because the molecules did not have enough kinetic energy to form a reaction this shows that the trial of 15°C is inactive.

It is shown on graph 1 that the 15 and 60 degree trial is predicted as continuous data. The arrows pointing upwards on the graph show that the data is infinite and had no reaction. It is predicted that the trials would have no outcome and the data is infinite. This happened because it did not react after 10 minutes which made the trials of 15 and 60 degrees Celsius either inactive or denatured. This means that the rennin enzyme could not react within the conditions.

At 15°C it shows that there is no reaction in the rennin enzyme meaning it had become inactive and the active site had changed shape and because it was not in the not let the substrate attach this is because there was not enough kinetic energy and therefore had no reaction (Boundless). The 30°C trial shows that the substrate was able to attach to the substrate and a reaction formed. The 45°C trial was the quickest to react because the rennin enzyme was in closer to the optimum temperature and the substrate attached very quickly. At 60°C the enzyme rennin was denatured and the active site had changed shape and could not let the substrate attach and therefore had no reaction because the trials of 15°C and 60°C had no reaction after 10 minutes and was predicted as infinite data.

There doesn’t seem to be any anomalous data present in the results. Potential errors that could have occurred during this experiment were. Firstly, measuring the rennin solution into the individual test tubes this is could have altered the results because it would have faster or shorter to react to the milk. This could have occurred because when using the pipette air bubbles formed so some of the measurements are incorrect. Secondly, when using the pipette the milk and rennin were used with the same pipette. This could have occurred because the pipette was not cleaned though out each trial which may have caused cross contamination.

Two errors that occurred in the experiment was keeping the temperature at the allocated time when the trial of 60°C it was difficult to keep it at 60°C as the temperature continually varied from 55-60, meaning the rate of reaction may have been a few degrees hotter or cooler, also the rennin temperature could have increased or decreased while it was at room temperature which may have had an effect on the outcome of the results. This could be avoided by maintaining the temperature allocated and taking more care to get it as close as possible if this was done it would ensure the collection of more accurate data.

The second error conducted in the experiment was when the rennin solution was measured into each individual test tube, if was difficult to get it accurate because of the air bubbles that formed within the pipette, this could be avoided by not having the solution measure in milliliters and possibly drops this would ensure that the measurements of the rennin solution would be more accurate, if both of these changes would ensure the collection of more accurate data in the future.

Recommendations

If this experiment was conducted again, the range of temperatures would be decreased, possibly into 5°C intervals. This would enable more specific and accurate results in which the rennin works and its optimum temperature. In future it will be beneficial to note the temperature of the rennin solution and weather distilled or normal water has any effect on the reaction rate. One thermometer was used in the experiment in a water bath of 3 test tubes so the range of the temperature results would have been varied by a few degrees.
It would also be interesting to vary the types of milk, for example soy milk the purpose of this experiment would be to see if rennin enzyme would still react even though the milk produced did not come from a mammal. This could benefit people who could not tolerate cow’s milk or animal related milk products.

Conclusion

In conclusion changing the temperature affects the substrate activity levels of rennin. It is shown in graph 1 that the trials 30°C and 45°C reacted faster because it is closer to 37°C which is the optimum temperature for the rennin enzyme. Very high temperatures such as 60°C caused the enzyme to denature, and very low temperatures didn’t provide the molecules with enough energy to collide and react. When no reaction was taken place it was because the substrate was unable to attract and therefore no reaction was seen. Overall it is clear the temperature is very important with the rennin enzyme and if it is not suited to the enzyme no reaction will take place.

Bibliography
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