Catechol Oxidase Lab Report

The preparation for the experiment started by gathering the solutions of enzyme Peroxidase, substrate hydrogen peroxide, the indicator guaiacol and distilled water. Two small spectrometer tubes and three large test tubes with numbered labels. In addition, one test tube rack, one pipet pump and a box of kimwipes were also gathered. Before the experiment, the spectrometer must be set up to use by flipping the power switch to on. Following, the machine was warmed up for 10 minutes and the filter lever was moved to the left. In addition, I set the wavelength to 500 nm with the wavelength control knob. Before the experiment, I had to create the blank solution by pipetting 0.1 ml of guaiacol, 1.0 ml of turnip extract and 8.9 ml water into tube #1. Following the creation of the blank, a control 2% solution was created.

In this experiment, the 5mM catechol (substrate) reacted with catechol oxidase in the presence of 5 different pH buffers mentioned above. This experiment was used to measure the buildup of the colored product, benzoquinone, to observe the change in the absorbance of the mixture in a spectrophotometer at a wavelength of 486 nm. Ithypothesized that since each enzyme has an optimal pH and that the enzymes are proteins, the enzyme activity will increase with pH level and will be at its highest at pH 7, which is water. As seen in figure 1, absorbance is low pH 2 because catechol oxidase activity was minimal at low pH concentration due to the catechol oxidase denaturing in the acidic solution. The catechol oxidases also denatured at high pH concentration such as pH 11, which is a basic solution, lowering catechol oxidase activity and absorption. Catechol oxidase activity was highest at pH 8 making it an optimal pH for catechol oxidase to catalyze the reaction and create more product which in turn increased the absorption of blue

During this experiment, the activity of catechol oxidase and the absorbance of benzoquinone was measured in five solutions with different pH concentration. Five cuvettes provided by the instructor were used during the experiment. The transmittance on the spectrophotometer was zeroed on an empty chamber and it was set at a wavelength of 486 nm. Because the shape of an enzyme changes with different pH concentration and each pH has different chemicals, so five cuvettes containing 0.5 mL of enzyme solution and 4.5 mL of pH 2, pH 6, pH 7, pH 8, pH 11 were used to make five different blank solutions (Scott, S.M. et al 2016). (Each blank was made when needed, not all blanks were made at the same time.)

The human body is an incredible system that is capable of working a multitude of diverse functions. Without the help of the many different protein molecules, the human body would not be able to function properly. One major group of proteins called enzymes are mandatory for essential life. These proteins are constantly at work assembling molecules, metabolizing energy, and fighting off infections. An enzyme is a macromolecule that acts as a catalyst that speeds up a chemical reaction without being consumed by the reaction. Without these proteins, these reactions would take place too slowly to keep us alive. Essential parts in your body like vitamins and minerals cannot do any work without

The role of an enzyme is to catalyse reactions within a cell. The enzyme present in a potato (Solanum Tuberosum) is catechol oxidase. In this experiment, the enzyme activity was tested under different temperature and pH conditions. The objective of this experiment was to determine the ideal conditions under which catechol oxidase catalyses reactions. In order to do this, catechol was catalyzed by catechol oxidase into benzoquinone at diverse temperatures and pH values. The enzyme was exposed to its new environment for 5 minutes before the absorbance of the catechol oxidase was measured at 420 nm using a spectrophotometer. The use of a spectrophotometer was crucial for the collection of data in this experiment. When exposed to hot and cold temperatures, some enzymes were found to denature causing the activity to decrease. Similarly, when the pH was too high or low, then the catechol oxidase enzyme experienced a significant decrease in activity. It can be concluded after completing this experiment that the optimal pH for catechol oxidase is 7 and that the prime temperature is 20º C. Due to the fact that the catechol oxidase was only tested under several different temperatures and pH values, it is always possible to get a more precise result by decreasing the increments between the test values. However, our experiment was able to produce accurate results as to the

The purpose of the experiment is to measure the level of browning, due to the effects of the enzyme called polyphenol oxidase (phenolase), on apple slices exposed to different Ph. levels.

The enzyme Polyphenol Oxidase (PPO) belongs to a family of about 10 closely related genes that are mostly responsible for the browning of apples. The genetically modified (GM) apple is nonbrowning because it expresses less PPO by preventing the enzymatic/oxidative browning reaction from occurring (Arctic® Apple, 2016). This reaction is initiated when there is superficial damage to the apple’s cells, such as when the apple is sliced, bitten, or bruised. Colorless polyphenols are oxidized as they come into contact with the PPO and result in colored polymer compounds consisting of quinones (Xu, 2013); therefore, this reaction is the core of why apples brown. By increasing the amount of PPO being suppressed, researchers were able to conclude that high percentage reductions (90-91%) in PPO activities of mature fruit

Within the experiment, pure catechol was mixed with different concentrations of catechol oxidase and the rate at which each solution produced benzoquinone was measured. The amount of benzoquinone made throughout the trials was measured by using a colorimeter to measure the level of “brownness” of the liquid. The colorimeter worked by shining a light through the liquid and then measuring that light on the other side to see how much of it was absorbed. In this experiment, absorbance of blue light was measured because blue light is absorbed by the color brown. The amount of blue light absorbance was measured every 15 seconds for five minutes. Because enzymes speed up reactions, more enzymes would cause the reaction to be even faster.1

The purpose of this experiment was to record catalase enzyme activity with different temperatures and substrate concentrations. It was hypothesized that, until all active sites were bound, as the substrate concentration increased, the reaction rate would increase. The first experiment consisted of five different substrate concentrations, 0.8%, 0.4%, 0.2%, 0.1%, and 0% H2O2. The second experiment was completed using 0.8% substrate concentration and four different temperatures of enzymes ranging from cold to boiled. It was hypothesized that as the temperature increased, the reaction rate would increase. This would occur until the enzyme was denatured. The results from the two experiments show that the more substrate concentration,

Catechol, in the presence of oxygen is oxidized by catechol oxidase to form benzoquinone (Harel et al., 1964). Bananas and potatoes contain catechol oxidase that acts on catechol which is initially colorless and converts it to brown (Harel et al., 1964). In this experiment, the effect of pH on the activity of catechol oxidase was conducted using buffers ranging from pH2 to pH10. Two trials were conducted due to the first trial results being altered by an external factor. The results were acquired by taking readings every 2 minutes for 20 minutes from a spectrophotometer and then recorded on to the table. The data collected in the table were then made into graphs to illustrate the influence of pH on the catechol oxidase catalyzed reaction. After analysis, the data revealed that pH did have a significant influence on the enzyme as recorded by absorbance per minute. However, the data was collected was not accurate due to external factors, thus the results are debatable and should be experimented again for validation.

The purpose of this experiment is to learn the effects of a certain enzyme (Peroxidase) concentration, to figure out the temperature and pH effects on Peroxidase activity and the effect of an inhibitor. The procedure includes using pH5, H202, Enzyme Extract, and Guaiacol and calibrating a spectrophotometer to determine the effect of enzyme concentration. As the experiment continues, the same reagents are used with the spectrophotometer to determine the temperature and pH effects on Peroxidase activity. Lastly, to determine the effect of an inhibitor on Peroxidase, an inhibitor is added to the extract. It was found that an increase in enzyme concentration also caused an increase in the reaction rate. The reaction rate of peroxidase increases at 40oC. Peroxidase performed the best under pH5 and declined as it became more basic. The inhibitor (Hydroxy-lamine) caused a decline in the reaction rate. The significance of this experiment is to find the optimal living conditions for Peroxidase. This enzyme is vital because it gets rid of hydrogen peroxide, which is toxic to living environments.

The data in experiment one showed the darkest color in the tube containing the substrate catechol, which was scaled at a 10, and showed no change in any of the other tubes containing the other substrate, which were scaled at 0, showing that the enzyme only reacted with the catechol substrate. This supported the hypothesis that the enzyme would be specific to only one type of substrate, in this case the substrate was catechol, this is known as enzyme specificity. The active site of the catechol oxidase enzyme has as structure that is able to remove the hydrogen of the catechol to create benzoquinone. This is because the oxygen atoms in the catechol oxidase are electronegative enough to pull the hydrogen atoms away from the oxygen atoms present in the catechol.

In this lab it will be determined how different exposures of environments will affect the browning of an apple. In order for this experiment to proceed the knowledge of why an apple turns brown must be researched and understood. In this research it was determined that polyphenol oxidase is the enzyme that distorts the colouring of the apple through an oxidation reaction. (Mellidou, I., et. Al, 2014, p.1) the discolouration varies depending on the exposure in different conditions. the ideal condition for an apple is an environment that maintains a low temperature as well as high levels of carbon dioxide and lower levels of oxygen. (Mellidou, I., et. Al, 2014, p.1) the colour change of the apple after effected

reaction rate increases. If the temperature of an enzyme gets to high the reaction rate will slow

The purpose of the “Apple Lab Experiment” was to identify the reasons for the browning of the apple in four different scenarios. Upon exposure to air and when the cortex of the apple is cut, the colour changes, which is also known as the browning of the apple. (Di Guardo et al., 2013) Apples contain an enzyme called polyphenol oxidase (PPO), which is responsible for the browning of the apple. (Di Guardo et al., 2013) When in contact with O2 (Oxygen), the polyphenol oxidase catalyzes quinones which in turn undergo self-polymerization resulting in the brown pigment. (Di Guardo et al., 2013) The degradation of the compartments in the internal cellular structure can also be attributed to the gene known as Polygalacturonase gene. (Di Guardo et al., 2013) In addition to the gene, the degradation of the compartments within the cellular structure causes the browning in apples to appear. (Di Guardo et al., 2013) Temperature plays a vital role in enzyme action as well because enzymes work in certain temperatures. They cannot function properly in very high or low temperatures as they start to denature and therefore their activity is inhibited. (Di Guardo et al., 2013) Ascorbic Acid can be used to temporarily inhibit the activity of the polyphenoloxidase (PPO), which is the enzyme responsible for the browning on the slices of the apple. (Di Guardo et al., 2013) This temporary inhibition of the enzyme occurs due to a series of oxidation, reduction reactions between the ascorbic acid and