Biology

Experiments investigating the effect of a particular factor on an enzyme-catalysed reaction are commonly used as assessed practicals in Biology. The reason for this is there are a number of factors that can be investigated, and therefore, a number of factors that can also be controlled.

From our lecture, we are aware that the following can affect the rate of reaction: 1) Temperature: a. Independent variable: A range of temperatures should be investigated, with particular attention to the area around 35-45oC. b. Control variable: You will need to use a water bath or other method to control the temperature; you cannot just say you did the experiments at the same time in the same room etc. You will need to monitor this using a thermometer. 2) pH: c. Independent: A range of pH should be used to establish the bell-shaped curve. Buffers should be used to do this. d. Control: You will need to use the same buffer (appropriate to the optimum pH of the enzyme used) for every sample. 3) Substrate concentration: e. Independent: Serial dilutions, or % concentrations (unless you are doing Chemistry, wherein you could use molar concentrations) could be used to find the optimum, and a few samples above the optimum should be done to observe the plateau. f. Control: Just use the same concentration throughout your samples, and make note of it in your Design. 4) Enzyme concentration: g. Independent: Depending on the form of the enzyme, you could do serial dilutions of digestive enzymes, or increase the surface area of your ‘vector’ (ie. yeast, potato, liver). h. Control: Just use the same concentration throughout your samples, or the same surface area, or the same ‘vector’ throughout. Make note of it in your Design. 5) Inhibitors: i. Independent: It is generally agreed that these are extremely difficult experiments to do, due to the fact that usually, it is an ‘all or nothing’ scenario. It is very difficult to get the levels of inhibitors correct. See your teacher if you wish to do an experiment with inhibitors. j. Control: You will need to make sure your equipment is clean, dry etc. before using it because unknown substances could potentially be inhibitors of your enzyme.
Experiments investigating the effect of a particular factor on an enzyme-catalysed reaction are commonly used as assessed practicals in Biology. The reason for this is there are a number of factors that can be investigated, and therefore, a number of factors that can also be controlled.

From our lecture, we are aware that the following can affect the rate of reaction: 6) Temperature: k. Independent variable: A range of temperatures should be investigated, with particular attention to the area around 35-45oC. l. Control variable: You will need to use a water bath or other method to control the temperature; you cannot just say you did the experiments at the same time in the same room etc. You will need to monitor this using a thermometer. 7) pH: m. Independent: A range of pH should be used to establish the bell-shaped curve. Buffers should be used to do this. n. Control: You will need to use the same buffer (appropriate to the optimum pH of the enzyme used) for every sample. 8) Substrate concentration: o. Independent: Serial dilutions, or % concentrations (unless you are doing Chemistry, wherein you could use molar concentrations) could be used to find the optimum, and a few samples above the optimum should be done to observe the plateau. p. Control: Just use the same concentration throughout your samples, and make note of it in your Design. 9) Enzyme concentration: q. Independent: Depending on the form of the enzyme, you could do serial dilutions of digestive enzymes, or increase the surface area of your ‘vector’ (ie. yeast, potato, liver). r. Control: Just use the same concentration throughout your samples, or the same surface area, or the same ‘vector’ throughout. Make note of it in your Design. 10) Inhibitors: s. Independent: It is generally agreed that these are extremely difficult experiments to do, due to the fact that usually, it is an ‘all or nothing’ scenario. It is very difficult to get the levels of inhibitors correct. See your teacher if you wish to do an experiment with inhibitors. t. Control: You will need to make sure your equipment is clean, dry etc. before using it because unknown substances could potentially be inhibitors of your enzyme.

For this practical, you will be assessed on D, (for this one just Design) DCP, CE and MS.

Abstract: Enzymes are found in all living organisms. They catalyse the metabolic reactions necessary for life to exist. However, enzymes are also sensitive to many environmental factors. Many can be easily denatured by changing conditions, which makes them ineffective. All are affected by the concentrations of enzymes and substrates.

Task: You are going to plan an experiment to investigate the effect of one of these factors. You may choose from the reactions listed below. Be sure to consider how you will change and control all your variables.

You will have one lesson to design your method wherein you will be able to run trials and decide which equipment you will need - you will make your orders from the science technician based on the available equipment in the department. Any other planning work will be assigned as homework. The following double-lesson will involve carrying out your experiment and collecting results. During this time you will be assessed on MS.
The write-up of the report (including DCP, CE) will be due one week after your results are collected.

Background Information on various Enzyme-Catalysed Reactions

Amylase is an enzyme that catalyses the breakdown of starch into sugars. Amylase is present in human saliva, where it begins the chemical process of digestion. Foods that contain much starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed because amylase turns some of their starch into sugar in the mouth. The pancreas also makes amylase (alpha amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylase. All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds. Starch can be detected using iodine solution, and simple reducing sugars can be detected using heated Benedict’s Solution.

Catalase: Catalase is a common enzyme found in nearly all living cells. It catalyzes the decomposition of hydrogen peroxide to water and oxygen. Likewise, catalase has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of molecules of hydrogen peroxide to water and oxygen each second. The optimum pH for human catalase is approximately 7, and has a fairly broad maximum. The pH optimum for other catalases varies between 4 and 11 depending on the species. The optimum temperature also varies by species.

Pectinase: The enzyme pectinase breaks down pectin present in the peel of fruits - a polysaccharide substrate that is found in the cell walls of plants, the jelly-like matrix which helps cement plant cells together and in which other cell wall components, such as cellulose fibrils, are embedded. Therefore pectinase enzymes are commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of fruit juice from fruit, including apples. This is a reason why it is used to increase the volume of juice extracted from fruits. They can be extracted from fungi such as Aspergillus niger. The fungus produces these enzymes to break down the middle lamella in plants so that it can extract nutrients from the plant tissues and insert fungal hyphae. If pectinase is boiled it is denatured (unfolded) making it harder to connect with the pectin at the active site, and produce as much juice.

Lipase is an enzyme that catalyzes the hydrolysis of fats (lipids) into fatty acids and glycerol. The formation of acids can be detected by changes in pH using the indicator phenolphthalein. Lipases perform essential roles in the digestion, transport and processing of dietary lipids (e.g. triglycerides, fats, oils) in most, if not all, living organisms. Genes encoding lipases are even present in certain viruses. For example, human pancreatic lipase (HPL), which is the main enzyme that breaks down dietary fats in the human digestive system, converts triglyceride substrates found in ingested oils to monoglycerides and two fatty acids.

Pepsin and Trypsin are enzymes released by cells of the stomach and small intestine, respectively, and that degrade food proteins into peptides. They were the first animal enzymes to be discovered. They are digestive proteases. During the process of digestion, these enzymes, each of which is specialized in severing links between particular types of amino acids, collaborate to break down dietary proteins into their components, i.e., peptides and amino acids, which can be readily absorbed by the intestinal lining.