Lab Experiment: Enzyme Kinetics, Lab Reports of Biochemistry

Download Lab Experiment: Enzyme Kinetics and more Lab Reports Biochemistry in PDF only on Docsity! 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 Beatriz E. Saldana Farias 8797 3 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 Beatriz E. Saldana Farias 8797 6 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. Beatriz E. Saldana Farias 8797 7 RESULTS Activity 1: Determine the Reaction Rate in the Presence or Absence of an Enzyme Table 1: Absorbance values for standards Standards Amount of p-Nitrophenol (nmol) Absorbence at 410 nm S1 0 0.00 S2 12.5 0.111 S3 25 0.208 S4 50 0.427 S5 100 0.804 Table 2: Determining p-nitrophenol produced using a standard curve Time (min) Cuvette Amount of p-nitrophenol (nmol) from the Standard Curve Absorbance at 410 nm 0 Start 0.75 0.006 8 End -0.012 1 E1 12.5 0.100 2 E2 16.75 0.134 4 E3 29.25 0.234 6 E4 43.376 0.347 8 E5 54.875 0.439 Activity 2: Determine the Effects of Temperature on the Reaction Rate Table 3: P-nitrophenol produced at three different temperatures Beatriz E. Saldana Farias 8797 8 Temperature Absorbance at 410nm Amount of p-Nitrophenol Produced (nmol) 0ºC 0.081 10.125 22ºC 0.093 11.88 37ºC 0.230 28.75 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 47.50 6.3 0.477 59.63 8.6 0.072 9.00 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 Beatriz E. Saldana Farias 8797 11 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 118.22 (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 19.7 (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 Beatriz E. Saldana Farias 8797 12 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.