Experiment 2
ENZYME KINETICS
Beatriz E. Saldana Farias
8797
BIO 4110
Biochemistry 2 Laboratory
Experiment Performed: February 4, 2016
Report Submission Date: March 4, 2016
Instructor: Wade Dauberman
Prepare for your exams
Study with the several resources on Docsity
Earn points to download
Earn points by helping other students or get them with a premium plan
Guidelines and tips
Prepare for your exams
Study with the several resources on Docsity
Earn points to download
Earn points by helping other students or get them with a premium plan
Community
Ask the community
Ask the community for help and clear up your study doubts
University Rankings
Discover the best universities in your country according to Docsity users
Free resources
Our save-the-student-ebooks!
Download our free guides on studying techniques, anxiety management strategies, and thesis advice from Docsity tutors
Biochemistry 2 Laboratory Lab which is proposed by Beatriz E. Saldana Farias
Typology: Lab Reports
2020/2021
1 / 11
This page cannot be seen from the preview
Don't miss anything!
On special offer
Related documents
Partial preview of the text
Download Lab Experiment: Enzyme Kinetics and more Lab Reports Biochemistry in PDF only on Docsity!
Experiment 2
ENZYME KINETICS
Beatriz E. Saldana Farias
BIO 4110
Biochemistry 2 Laboratory
Experiment Performed: February 4, 2016
Report Submission Date: March 4, 2016
Instructor: Wade Dauberman
ABSTRACT
The purpose of this experiment was to test the reaction rate of the decomposition of p- nitrophenyl glucopyranoside into glucose and p-nitrophenol, in the presence of an enzyme called cellobiase. The experiment consisted of six different activities where the enzyme was exposed to a variety of conditions and the difference in reaction rate was analyzed using a spectrophotometer. In conclusion, the enzyme efficiency is increased by about two orders of magnitude in the presence of cellubiase, and the enzyme activity in greately affected by the environment it is exposed to due to the fact that it evolved to function under very specific conditions. INTRODUCTION Enzymes are bundles of proteins with the function of catalyzing biological reactions. Different types of enzyme are designed specifically for the substrate they bind to; the substrate molecules bind to the active site of the enzyme. Enzymes create instability in the molecular bonds of the substrate they are bound to, and therefore speed up the reaction rate [3]. Enzyme activity can be drastically affected by the environment they evolved in, thus external factors such as temperature, pH, and concentration change the efficiency of the enzyme. Enzyme efficiency is examined by analytically measuring the changes in the reaction rate. In this experiment, the reaction rate of cellobiase will be tested in a variety of different conditions. Cellobiase is a class of enzymes produced by fungi and other organisms that are involved in the decomposition of cellulose. The enzyme catalyzes the decomposition of p- nitrophenyl glucopyranoside into glucose and p-nitrophenol. This enzyme is incredibly significant to the process of decomposition on the food chain, due to the fact that most organisms
can affect the rate of reaction by increasing or decreasing the statistical probability of a reaction occurring [4]. The last activity in the series was more intricate and required the extraction of mushroom enzymes. The first part of the activity was to successfully extract the accurate compounds containing the enzyme. After the extraction the enzymes were analyzed using a spectrophotometer. Mushrooms were used for the last activity because they are one of the organisms that produce cellobiase, and thus it was important to learn the extraction process and biochemical location of the enzymes inside an organism. MATERIALS AND METHODS This experiment consisted of six different activities that all consisted of testing the rate of breakdown of p-nitrophenyl glucopyranoside into glucose and p-nitrophenol in the presence of cellobias. The purpose of the first activity was to determine the reaction rate in the presence or absence of cellobias. The first step was to label five corvettes E1 – E5 , and two others “Start” and “End”, which served as the controls. Then 500ul of stop solution was pipetted into all seven curvettes. Then two 15ml conical tubes were labeled “Enzyme Reaction” and “Control”, and 2ml and 1ml of 1.5 mM substrate were pipetted into each respectively. 500ul of the buffer solution were pipetted into the tube labeled “Control”, the solution was mixed, and 500ul of the solution was pipetted into the curvet labeled “Start”. Then 1ml of the enzyme solution was pipetted into the tube labeled “Enzyme Reaction”, the solution was mixed and the timer was started. After 1min 500ul of the “Enzyme Reaction” solution was pipetted into the curvet labeled E1, after 2min the solution was pipetted into the E2 curvet, then 4min into E3, 6min into E4, and 8min into E5. Finally, 500ul from the control solution were pipetted into the curvet labeled “End”. The
samples were then analyzed using a spectrophotometer at 410nm blanked with the curvet labeled “S1”. The results were recorded on a table and the p-nitrophenol produced was determined using a standard curve. The second activity consisted of testing the enzyme reaction rate at different temperatures, 0ºC, 22ºC, and 37ºC. To do that first three curettes were labeled 0, 22, and 37 and 500ul of stop solution was pipetted into each. Then six micro-centrifuge tubes were labeled 0E, 22E, 37E, 0S, 22S, and 37S and 250ul of Enzyme was pipetted into the microcentrifuge tubes containing the letter “E” and 1.5 mM substrate was pipetted into the tubes containing the letter “S”. The tubes with a “0” on it were placed in an ice cup, the ones with a “22” were left on the lab bench, and the ones with a “37” were placed in a beaker with warm water; the tubes were left there for about 5min. After the time went by, 250ul of the tubes containing the enzyme were pipetted into the tubes containing the substrate of their same temperature, and the timer was started. After two minutes 500ul from each tube containing the substrate and enzyme were pipetted into the curvets labeled with their respective temperature. The samples were then analyzed using a spectrophotometer at 410nm blanked with the curvet labeled “S1”. The results were recorded on a table and the p-nitrophenol produced was determined using a standard curve. The purpose of the third activity was to determine the effect of pH on the enzyme. The activity was executed just like the second activity but instead of exposing the solution to three different temperature conditions, it was exposed to different acidities of pH of 5, 6.3, and 8.6. The resulting solutions were analyzed using a spectrophotometer. The fourth and fifth experiments varied the enzyme and substrate concentration respectively. The sixth activity consisted of first extracting enzymes from white mushrooms and then testing the efficiency of the enzyme in a very similar manner than the first activity.
Temperature Absorbance at 410nm Amount of p-Nitrophenol Produced (nmol) 0ºC 0.081 10. 22ºC 0.093 11. 37ºC 0.230 28. Initial rate of product formation at 0ºC= 5.0625 nmol/min Initial rate of product formation at 22ºC= 5.94 nmol/min Initial rate of product formation at 37ºC= 14.375 nmol/min Activity 3: Determine the Effects of pH on Reaction Rate Table 4: p-nitrophenol produced at three different pH levels pH Absorbance at 410 nm Amount of p-nitrophenol produced 5.0 0.380 47. 6.3 0.477 59. 8.6 0.072 9. Initial rate of product formation at pH 5.0= 23.75 nmol/min Initial rate of product formation at pH 6.3= 29.80 nmol/min Initial rate of product formation at pH 8.6= 4.50 nmol/min
Activity 4: Determine the Effect of Enzyme Concentration on Reaction Rate Table 5: P-nitrophenol produced using a high and low enzyme concentration based on a standard curve Cuvette Absorbence at 410 nm Amount of p-Nitrophenol Produced (nmol) H1 0.719 89. H2 0.927 115. H3 1.372 171. L1 0.215 26. L2 0.320 40 L3 0.775 96. Activity 5: Determine the Effect of Substrate Concentration on Reaction Rate Table 6: Determination of p-nitrophenol produced using a high and low substrate concentration based on a standard curve Cuvette Absorbance at 410 nm Amount of p-Nitrophenol Produced (nmol) Hi 0.057 7. H2 0.154 19. H3 0.261 32. L1 0.008 1 L2 0.000 0 L3 0.097 12. Activity 6: Test Ability of Mushroom Extracts to Increase Reaction Rate
are specialized for more basic or acidic environments. According to the results of the third activity, cellobiase is also one of the enzymes that preforms better in neutral conditions. Most of the organisms that are capable of decomposing cellulose do not contain complex organs or specialized acidic digestion, thus most of their enzymes work better in a neutral environment. The fifth and sixth activities provided very expected results, the higher the concentration the more p-Nitrophenol produced. That is due to the fact that the more substrate or enzymes present the higher the binding probability. The last activity was very similar to the first one, but required an extraction at first. The extraction was successful and thus the results were the same as the first activity, and for the same reason. In conclusion, enzymes are vulnerable to the environment and the rate of reaction can be drastically influenced by the conditions an enzyme is exposed to. LITERATURE CITED [1] Baars, Johan JP, et al. "Nitrogen assimilating enzymes in the white button mushroom Agaricus bisporus." Microbiology 140.5 (1994): 1161-1168. [2] Craig, Douglas B., et al. "Studies on Single Alkaline Phosphatase Molecules: Reaction Rate and Activation Energy of a Reaction Catalyzed by a Single Molecule and the Effect of Thermal Denaturation The Death of an Enzyme." Journal of the American Chemical Society 118. (1996): 5245-5253. [3] Garcia-Viloca, Mireia, et al. "How enzymes work: analysis by modern rate theory and computer simulations." Science 303.5655 (2004): 186-195. Gong, Cheng‐Shung, Michael R. Ladisch, and George T. Tsao. "Cellobiase from Trichoderma viride: purification, properties, kinetics, and mechanism." Biotechnology and Bioengineering 19. (1977): 959-981. [4] Maguire, R. James. "Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-β-d-glucoside by cellobiase of Trichoderma viride." Canadian journal of biochemistry 55.1 (1977): 19-26. [5] Segel, Irwin H. Enzyme kinetics. Vol. 957. Wiley, New York, 1975. [6] Sengupta, Saswati, Anil K. Ghosh, and Subhabrata Sengupta. "Purification and characterisation of a β-glucosidase (cellobiase) from a mushroom Termitomyces
clypeatus." Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 1076.2 (1991): 215-220. [7] Wolfenden, Richard, et al. "The temperature dependence of enzyme rate enhancements." Journal of the American Chemical Society 121.32 (1999): 7419-7420.