Enzymes are the large biomolecules which catalyse and speed up chemical reactions which take place in our bodies. They can increase reaction rates by 100 million to 10 billion times faster than normal. (Gurung, 2013) Enzymes are widely used for different commercial applications like refining fruit juice and making detergents which can remove stains more effectively.
Protein molecules are polymers of amino acids bonded together by peptide bonds. (Biology Online, 2018) Proteases are enzymes which catalyse the breakdown these peptide bonds, breaking down the proteins. The idea of this experiment is to view the effects of different enzymes on the milk proteins. The composition of milk includes: water, fats, protein, lactose, and minerals. (Dairy Processing Handbook, 2018) As seen in the diagram to the right 87.5% of milk is water and only 3.4% is actually protein. (Dairy Processing Handbook, 2018) Although there is a very small amount of protein in milk, it gives off a white colour because just over 80% of the protein is casein, which always gives off a white colour, especially when combined with fats. (Mauk, 2013) In addition to the casein, the other 20% is whey protein which takes up the rest of the 3.4g per 100 grams.
Trypsin is a protease which is found in the human body. It is excreted by the pancreas and helps with the digestion of food proteins in the small intestine. It is produced by the inactive proenzyme, trypsinogen being activated by another enzyme called enteropeptidase. (Worthington Biochem, 2018) Trypsin only acts on the peptide bonds in proteins that have the amino acids Arginine and Lysine in their carboxyl groups. (The Columbia Encyclopedia, 2018) The optimal conditions for Trypsin are between pH of 7.5 and 8.5, in the presence of calcium ions and at the temperature of 37°C because it’s the average body temperature. (Rawlings and Salvesen, 2013) Bromelain, also known as bromolase is a protease which is found in pineapple juice and the stems of pineapples. (WebMD, 2018) It is often used in medicine for reducing swelling in the nose and sinuses after surgery or injury. Bromelain is also used for removal of dead tissue, hay fever and many other medical conditions. (WebMD.com, 2018) It does this by breaking down proteins and and muscle fibres, producing substances which seem to cause the body to reduce pain, dead tissue and swelling. (WebMD, 2018) It is also a natural anticoagulant which works by breaking down the blood-clotting protein fibrin. (Freeman, 2005) Laundry detergent is used to remove stains from clothing and clean clothes in general. Laundry detergent consists of either a liquid or powder of condensed enzymes with a mix of Proteases, Amylases and Lipases which make up 0.4 to 0.8% by weight. (Mehic, 2018) They usually only contain one type of enzyme, though some have two or all three. (Science Learning Hub, 2007) When the detergent reacts with H₂O, the enzymes spread out and then break down the stains which are made from proteins, carbohydrates and lipids. (Science Learning Hub, 2007) Amylases are enzymes that convert starch into sugars such as fructose, glucose and sucrose. (Mojsov, 2016) It is in your saliva, which is the beginning of digestion. Amylase is an important component and step in the process in the digestive system as it’s the first stage and is the primary way starch is broken down. (Encyclopedia Britannica, 2018) Amylase was sourced from saliva for this experiment.
Fun fact: foods that contain lots of starch and not much sugar like rice and potatoes develop a sweet in in your mouth as they are chewed because the enzyme amylase turns some of their starch into sugar. (Wikipedia, 2018)
Pectinase breaks down pectin, in the cell walls of fruits like apples and oranges. (Mojsov, 2016) Pectinase is used commercially to help extract juice from fruit by breaking down the pectin in the cell walls, releasing the juice from the cells. (Science Buddies, 2017) The purpose of this experiment is to test the different effects of enzymes on proteins, and support the idea that trypsin & bromelain should have the most effect because these enzymes are proteases and specific to proteins, by testing with other enzymes.
The independent variable is the choice of enzyme used, as when it changes, it affects the reaction between the milk agar and the enzyme. This reaction depends on the different enzymes hence is the dependent variable. The petri dish with the trypsin is the controlled variable, because it\’s already known that trypsin breaks down casein as humans produce it in their small intestines.
If comparing trypsin and other enzymes in a controlled environment to see which enzyme breaks down the greatest amount of protein in milk agar, then Trypsin and Bromelain will have the greatest amount of protein breakdown in the milk agar, because they are proteases which are enzymes which break down protein, and the environment it’s set in is perfect for both of the enzymes (37°C).
5 Petri dishes
Trypsin (3% solution)
Bromelain (also called Bromelase) (pineapple juice)
Pectinase (Apple Juice)
Prepare 5 petri dishes with milk agar
With each dish, make 3 wells in each dish using a straw
Label each petri dish from 1 to 5
Place a drop of each enzyme solution in the wells of each petri dish as follows:
petri dish 1: Trypsin (3% solution)
petri dish 2: Bromelain (Pineapple Juice)
petri dish 3: Laundry Detergent
petri dish 4: Amylase (Saliva)
petri dish 5: Pectinase (Apple Juice)
Leave for 72 hours in 37°C incubator
Petri Dish #
Nearly all the milk agar became transparent.
The agar has a mushy consistency.
A large area of the agar became transparent.
A lot of yellow and blue mold grew.
Smells like detergent. A large area of the agar became transparent.
The agar is a near solid.
A very small area of agar became transparent.
And yellow and white dots formed.
Very small area of agar became transparent.
Diameter of Reaction (cm)
Optimum pH of Enzyme
7.5 to 8.5
5.0 to 8.0
8.0 to 10.5
4.0 to 5.0
2.5 to 3.5
Optimum temp of Enzyme
up to 60ºC
40 to 55°C
Protein, Carb & Lipid Breakdown
Cell Wall Breakdown
The results from the experiment align with the hypothesis and previous research and statements mentioned in the introduction. The Trypsin and pineapple juice (Bromelain) broke down the greatest the amount of protein. This was primarily due to the fact both bromelain and trypsin are proteases, which are enzymes which are specific in the catalyse of hydrolysing protein.
Another reason for trypsin and Bromelain breaking down the greatest amount casein is because of the temperature the milk agar was left in (37 Degrees), this increased the rate at which enzymes can catalyze the breakdown of the proteins.
The detergent was the third most effect. Again the detergent was already known to work as enzymes are used in detergents to breakdown stains in clothing. There was 0.4-0.8% of enzymes in the detergent and of that an unknown proportion protease enzymes. Thus the detergent was also effect but not quite effect as trypsin and bromelain, generally because it didn’t have as many enzymes as trypsin and bromelain. Saliva (amylase) and apple juice (pectinase) were the least effect because both of these enzymes don’t specifically break down protein, instead break down others substances such as carbohydrates.
- But a small breakdown still occurred, a possible the reason for this is that the amylase and the pectinase could be an induced fit model and change shape slightly to react, but this is really slow hence for milnalistic reaction.
- There we multiple problems with the experiment, firstly there was no choice with the type of washing detergent, this meant another detergent which could be cold active could’ve been used to have a more drastic to reaction.
- Another problem was that mould had grown on the top of the agar of the bromelase and there were yellow and white dots on top of the saliva agar.
- This showed there was either a small amount of bacteria or a mystery substance in the bromelain and amylase. This was a clear issue as it meant there were other substances in the bromelain and amylase. This obviously didn’t affect bromelain because it broke down a lot of the protein.
- But it could’ve possibly made a massive effect on the amylase especially because the saliva came from a person and the person could’ve consumed food with non competitive enzymes in it earlier and there may still have been traces of it when giving saliva for the experiment.
- In consideration of these issues the general practice of the experiment was efficient and smooth. Overall the experiment performed has reinforced previous research and helps encourage for more in depth investigation in the similar areas in future research.
JACOB\’S DISCUSSION (JASON JAKIE JAQOS)
The results of the experiment support the hypothesis and previous research. The Trypsin and pineapple juice (Bromelain) had the most effect on the milk agar, this was expected because they are both proteases, and are specific to proteins. The third most effective enzymes were in the laundry detergent, this was expected as well because detergent contains a mix which includes proteases as well. The saliva had the lowest reaction rate, which was expected because it contains amylase which primarily breaks down starch in the mouth, and does not break down proteins. Surprisingly the Pectinase in apple juice had more of an effect than the saliva, which was unexpected because Pectinase is an enzyme which breaks down cell walls in plants, not proteins. Apple juice must have other enzymes present which can break down the casein. From research, apple juice does contain some Amylase which is interesting as this is the same enzyme found in saliva. The Amylase must be more concentrated in the apple juice than saliva or there are other enzymes present that hydrolysed the milk proteins.
A factor on the rate of reaction was the concentration of the enzymes, as this was not a controlled variable. The trypsin used was a 3% solution and the concentration of enzymes in pineapple juice, saliva, apple juice and detergent were arbitrary and uncontrolled. This makes replicating the experiment difficult as Trypsin was the only known concentration. The accuracy of results could be improved by making all the enzymes the same concentration. The saliva was sourced from two people into a beaker, its enzyme content may have varied greatly: some areas of the mouth might contain more enzymes than others, and food consumed prior to the experiment may have introduced non-competitive enzyme inhibitors. Also the bubbles in the saliva may have reduced the surface contact between the Amylase in the saliva and milk agar substrate, affecting the results.
Mould grew on top of the agar in the pineapple juice petri dish and yellow and white dots formed on top of the saliva agar. This may have been caused by the bi-products of the enzymes or from contaminants in the pineapple juice or bacteria in the saliva. This was an issue as there may have been other substances interacting with the Bromelain and Amylase. Clearly, this didn\’t affect the Bromelain but may have been a factor for the rate of reaction for the Amylase. The experient design could be improved by recording the enzyme action over time, which will give insight to any inhibitors acting on the enzymes or other unexpected behaviour. Another problem with the experiment was the choice of laundry detergent used, as the enzyme content varies greatly across the brands and types of detergent. This means that if a different detergent was used like cold power, it would have a greater reaction as it contains more enzymes than the detergent used in this experiment.
- the results support the hypothesis
- critically analyses, interprets and explains data to draw a valid conclusion that relates to a hypothesis
The conclusion summarises key results and interpretations of the experiment. The conclusion should be concise and brief. Importantly, the conclusion should not introduce any new information.
- Rawlings, N. and Salvesen, G. (2013). Handbook of proteolytic enzymes. 3rd ed. Amsterdam: Elsevier Academic Press.
- Science Buddies. (2017). A Juicy Project: Extracting Apple Juice with Pectinase.
- Mojsov, K. (2016). New and Future Developments in Microbial Biotechnology and Bioengineering.
- WebMD. (2018). Bromelain: Uses, Side Effects, Interactions, Dosage, and Warning.
- Science Learning Hub. (2007). Enzymes in washing powders.
- Freeman, B. (2005). Bromelain (Pineapple enzyme).
- The Columbia Encyclopedia. (2018). 6th ed. The Columbia University Press.