Horseradish Peroxidase: A Case Study

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.

The type of peroxidase is used is called turnip peroxidase. Turnip peroxidase is made up of Guaiacol and hydrogen peroxide. The reactants to the product are turnip peroxidase or called tertraguaiacol and water. The color of the react is brown. In the experiment was conducted there were baseline experiment, temperature, pH, 10X substrate, Inhibitor, and half the amount of enzyme.

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

Hydrate the yeast packets in a beaker with 400 mL of distilled water at a 10% concentration. In a 50 mL

The data in proves that our hypothesis was correct. When we increased the temperature to 35°C, the the enzyme activity increased because kinetic energy increased, increasing the collisions between the substrate and the enzyme, and thus creating a higher chance of reaction. When we increased the temperature to 45°C, the enzyme activity decreased as the enzyme became denatured,because the atoms in the enzyme had enough energy to overcome the hydrogen bonds between the R groups that give the enzyme its shape From our data, we could conclude that the optimal temperature of turnip peroxidase is around 35°C and around 45°C, it will start to denature.

We hypothesized that a medium pH buffer added to the hydrogen peroxide an peroxidase reaction would be the best condition for the enzyme activity due to it being the more neutral than the high, being basic, and low, being acidic, pH.

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.

will be working at the pH 7 the majority of the time and our bodies

HRP has been around for a long time and used in a wide variety of products such as food taste enhancer or spices to pharmaceutical products. Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century but the first ever recorded observation of a reaction catalysed HRP is found in a note published by Louis Antoine Planche (Veitch, Nigel C 2004). HRP was first detected by Theorell in horseradish roots.Horseradish peroxidase has been known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols, but for the first time it is shown that HRP is effective in degrading and precipitating industrially important azo dyes(Bhuniaet al. 2001).

Pyranose 2-oxidase catalyzes the oxidation of aldopyranoses by using molecular oxygen as an electron acceptor to yield the corresponding keto-aldoses and hydrogen peroxide. This enzyme belongs to the Glucose-Methanol-Choline (GMC) oxidoreductase superfamily and contains flavin adenine dinucleotide (FAD) as a cofactor. P2O catalyzes regio-specific oxidation at the C2 position. Here, the hydride transfer reaction from glucose to oxidized flavin catalyzed by pyranose 2-oxidase (P2O) was investigated by density functional theory calculations and transient kinetics. Our findings suggest that the first step of the P2O reaction is a hydride transfer from C2 position of glucose to N5 of the flavin. Then, the proton abstraction occurs by the conserved

With the desire of wanting to know how to increase the enzyme’s activity rate, the experimenters formulate a question that is which temperature will cause the potato enzyme to decompose the most hydrogen peroxide? In order to find the answer to this inquiry, experimenters started the procedure by preparing four different potato samples, where there were, hot, cold, room temperature, and baked potato samples. Each sample was half of two Idaho potato, which was cut into fourths. Then, each group of potatoes were examined when each section was submerged in 150 mL of hydrogen peroxide in individual beakers. The results were recorded based on the physical appearances of each reaction.

In this lab we used catalase (enzyme), and this enzyme was a catalyst used to breakdown the hydrogen peroxide. An enzyme will bind with a substrate, then form an enzyme substrate complex, and then the new products then leave the enzyme. The enzyme binds with the substrate in the active site. The active site is where enzymes act. Enzymes do not undergo permeant changes during the reaction, (ch302). The catalyst is not part of the reactant, instead it is added to the reactant to speed up the reaction. The catalyst overall will lower the activation energy for the reaction. This is because the enzyme changes the way the reaction happens, (Enzyme and…). In our lab the enzyme sped up the breakdown of the hydrogen peroxide. Instead of the

LiP (EC 1.11.1.14) catalyzes the H2O2-dependent oxidative depolymeriza¬tion of lignin. It was first discovered in the the white-rot fungus, P. chrysosporium. Multiple LiP isoforms exist in P. chrysosporium and other white-rot fungi such as Phanerochaete sordida, T. versicolor, Phlebia radiata, and Phlebia tremellosa (Abdel-Hamid et al., 2013). LiPs are stronger oxidants because the iron in the porphyrin ring in theses enzymes is more electron deficient than in classical peroxidases.

The use of Bacillus and Pseudomonas spp. as bioagents increase the activity of some enzymes such as chitinase, peroxidase and polyphenol oxidase, which play an important role in plant defense mechanisms against plant pathogens (Li et al., 2009 and 2015). 2. Materials and Methods 2.1. Isolation, Purification and Identification of the Associated Bacteria