2023 Fall BIOL2315 Manual Rate It

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Langara College *

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2315

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Biology

Date

Feb 20, 2024

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7

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Rate It! Submission guide Must be completed Assignment due date (yyyy-mm-dd) 2023-11-18 Last 4 digit of your student ID 3228 Group name Group- G ● Use this page as cover page for your lab report. ● Be sure to meet formatting requirements, and avoid plagiarism. ………………………………………………………………..Refer to important guide page 1 and 2 Please combine the following 1. Cover page: use this page as cover page. 2. Discussion. 3. Figure 1. Graph (Hand-drawn OR typed) 4. Answers to questions (typed) 5. List of references in APA style Sharing data within a group is okay. However, lab components and questions are an individual effort. No plagiarism. No copying data between different groups. No fabrication of data. The group name is assigned in the beginning of lab. Lab report is marked as 0 when students ● Copy and paste any content from the internet (with OR without reference provided). ● Copy and paste any content from other groups (plagiarism) ● Submit previous lab report ● Fabricate data ● Hand in your assignment more than 24 hr. late after the due date Page 1/7
★ Discussion (3 marks) will be marked for sure. Students will need scientific papers to support the discussion. • Must be typed. Do not scan your typed discussion. • Observe the general trend. Explain what your general trend (result) means. • Think carefully about where your bacterial catalase came from and support your reasoning with scientific papers. Use the temperature that you used to perform the titration when you interpret your data. This is a process called critical thinking. Be sure to discuss both types of bacteria. general trend of catalase reaction (rate of hydrogen peroxide decomposition) is decreasing meaning the amount of substrate (hydrogen peroxide) is decreasing over period of time as substate is decreasing with respect to time the number of collisions between catalase with substrate will decrease eventually which will result in decreasing rate of reaction. As the hydrogen peroxide is getting decomposed, it tells us that the catalase is working. The catalase we used came from a Staphylococcus spp. Because optimal temperature it requires to work at full efficiency is 30- 37degrees Celsius (Martin & Chaven, 1987). However, at room temperature 20-25 degree Celsius, a staphylococcus spp. (staphylococcus aureus) shows reduced activity of the catalase in converting hydrogen peroxide to water and oxygen and They work at 10-15% efficiency under room temperature (Martin & Chaven, 1987). On the other hand, catalase of the thermophile bacteria is not able to catalyze the reaction under room temperature because their optimal temperature range in high. (60- 70 degrees Celsius) (Kimoto et al., 2012). Page 2/7
Figure1: graph represents the amount of hydrogen peroxide decomposed over period. Five samples containing bacterial catalase ( Staphylococcus spp) and hydrogen peroxide of different time interval were titrated against potassium permanganate to calculate the amount of hydrogen peroxide decomposed. Orange color represent negative control and blue color represent experimental group. Lab Questions (must be typed – do not scan typed answer) Lab components and questions will be randomly selected for marking. Q1 . Define baseline in your own word for Rate it experiments (1 mark) : Baseline is a control group in this experiment because it gives us a non-enzymatic medium with everything else exactly same as the enzymatic medium. So, when we are measuring the rate of Page 3/7
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disappearance of hydrogen peroxide and compare with baseline, we can say that the change in decomposition rate is due to the enzyme. Q2. 1mL catalase was used for measuring enzyme reaction rate. Why is it necessary to add 1mL of H2O when you were measuring baseline? What will happen to your result if you do not add 1mL of H2O? Explain. (2 marks) When we are adding 1ml of water in baseline, it provides us same medium without enzyme and hence it works as a control, and we can compare this with sample tube containing 1ml of catalase to measure the decomposition rate of hydrogen peroxide and we can say that this effect is only due to enzyme not any other factor and this helps us in ensuring that the change is only due to presence of enzyme. If we do not add 1ml of water in baseline it will lead to improper control group and the result which we will calculate will not be accurate. ★ Q3. This question will be marked. You will need scientific papers as reference(s). (2 marks) Describe the function of catalase with specific mechanisms. Your answer must include: ● Function of catalase. ● Each step of biochemical reaction from catalase. ● Identify and provide function of cofactor and key amino acid residues that are associated with catalase. Your answer must not include: (0 mark) How enzyme work in general . Decomposition of hydrogen peroxide is carried out by enzyme catalase. It converts hydrogen peroxide into water and oxygen. Hydrogen peroxide is a reactive oxygen species, if not processed further, it will damage the cell. The reaction takes place in two steps. Catalase has an iron ion (heme group) as a cofactor (Catalase - Proteopedia, Life in 3D, n.d.). First step results in reduction of hydrogen peroxide forming water and oxidizing iron ion, enzyme with oxidized iron group is the intermediate which in second step is getting reduced and oxidizing another hydrogen peroxide resulting in regeneration of enzyme (Heck DE;Shakarjian M;Kim HD;Laskin JD;Vetrano AM;). Iron ion undergo redox reaction to generate the products. 3 major amino acid residue that are associated with the active site of the catalase are histidine, asparagine, and tyrosine. Histidine and asparagine help to do a proton transfer between the oxygens of hydrogen peroxide and tyrosine helps the iron ion group to get oxidize by gaining that deprotonated oxygen from the hydrogen peroxide in the first step (Catalase - Proteopedia, Life in 3D, n.d.). histidine do proton transfer in the active site by acid-base catalysis. Page 4/7
(Nandi et al., 2019) Q4. Based on facts related to enzyme structure and chemistry, explain the inhibiting effect of sulphuric acid on the function of catalase. (1 mark) As we are adding sulphuric acid to the solution, we are making the medium more acidic meaning the pH will drop below 7 making the medium acidic and catalase only work at neutral or close to 7. Also, catalase will undergo chemical modifications (protonation and deprotonation, change in other electrostatic forces due to the acidic pH) which will result in the denaturation of the protein. Q5. Explain the effect of lowering the temperature on enzyme activity rates. (1 mark) Lowering the temperature will decrease the activity rate of the enzyme because as we are decreasing the temperature after certain point the catalase cannot function because the temperature is not in the optimal range for enzyme to function. Also lowering the temperature results in decreasing the amount of collision between enzyme and substrate. So therefore, it will result in lowering the activity rate of enzyme. Page 5/7
References 1. Martin SE;Chaven. (n.d.). Synthesis of catalase in Staphylococcus aureus MF-31. Applied and environmental microbiology. https://pubmed.ncbi.nlm.nih.gov/3606102/ 2. Kimoto, H., Yoshimune, K., Matsuyma, H., & Yumoto, I. (2012). Characterization of catalase from psychrotolerant Psychrobacter Piscatorii T-3 exhibiting high catalase activity. International journal of molecular sciences. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3291989/#:~:text=On%20the%20other %20hand%2C%20mesophile,and%2070%20°C%2C%20respectively. 3. K;, M. F. (n.d.). Effect of high compost temperature on enzymatic activity and species diversity of culturable bacteria in cattle manure compost. Bioresource technology. https://pubmed.ncbi.nlm.nih.gov/16051089/ 4. Catalase - Proteopedia, life in 3D. (n.d.). https://proteopedia.org/wiki/index.php/Catalase 5. Heck DE;Shakarjian M;Kim HD;Laskin JD;Vetrano AM; (n.d.). Mechanisms of oxidant generation by catalase. Annals of the New York Academy of Sciences. https://pubmed.ncbi.nlm.nih.gov/20716293/ 6. Nandi, A., Yan, L.-J., Jana, C. K., & Das, N. (2019, November 11). Role of catalase in oxidative stress- and age-associated degenerative diseases. Oxidative medicine and cellular longevity. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885225/#:~:text=Catalase%20is %20a%20key%20enzyme,essential%20for%20cellular%20signaling%20processes. Page 6/7
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