Enzyme Activity Lab Report PDF

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Full report (abstract, intro, materials and methods, results, and discussion) for enzyme activity lab...

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Lab # 11: Enzyme Activity I.

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Abstract Enzymes are multi-structured proteins that behave and function as biological catalysts. As most other biological components, enzymes require specific physiological conditions in order to maximize their functional performance as catalysts. Apart from enzymes catalyzing biological processes in one’s own body, enzymes prove to be greatly useful in large scale productions of different products as well. In order to maximize the rate at which enzymes catalyze substrate to a wanted product, it is crucial to study and understand the optimum conditions at which the enzyme of interest requires. The optimal physiological conditions for which the enzyme cellobiase, which is utilized in large-scale ethanol production, are unknown. Here it is shown that the optimum temperature required for cellobiase to maximize rate of reaction of p-nitrophenyl glucopyranoside to p-nitrophenol and glucose is about 57°C without the addition of glucose solution. Due to enzymes comprising of proteins in tertiary and quaternary structure, it was also found that over-heating the solution containing cellobiase would denature the enzyme and cause its activity to reduce significantly. Also, it was found that the longer the enzyme was left in the water bath closest to its optimal temperature, the more product was produced as a result. Considering the data recovered from the experiments presented, it is hypothesized that further study into the physiological properties of cellobiase should be pursued. A possible area of study would include looking for a strongly effective cofactor to pair with cellobiase that would work at 57°C. These areas of study could lead to cheaper, more effective methods of mass producing ethanol. Introduction Cellobiase, an enzyme common to mushrooms, is an enzyme responsible for the cleaving of glucose-glucose bonds in cellulose. Cellulose is a molecule comprised of bundled up long chains of glucose, found in plant cell walls. Cellulose and cellobiase alike have become crucially important to the biofuel and ethanol production industry. In the production of ethanol, as well as biofuels, massive quantities of biomass are treated due to their rich abundance in cellulose. The biomass is cut down into shreds and pretreated with heat and chemicals so that cellulose is easily accessible to the cellobiase once it is introduced. After the biomass has been pretreated, cellobiase, along with endocellulases and exocellulases, is introduced to the biomass so that they can easily cleave glucose-glucose bonds holding cellulose together. Once the solution is mostly glucose, microbes can then be introduced for the fermentation of glucose into ethanol. The assays performed in this lab relates to the use of cellobiase in the production of ethanol. Cellobiase will be extracted from mushroom samples and used to catalyze the production of p-nitrophenol and glucose from p-nitrophenyl glucopyranoside. The optimum physiological surroundings of cellobiase will be tested for and analyzed. By doing this, information extracted from these experiments can be applied to the usage of cellobiase for ethanol and biofuel production as a means for a more cheap, effective production process. Materials and Methods

In order to carry out the experiment, the enzyme cellobiase was first extracted from, in this case, shitake mushrooms. Approximately 2 grams of shitake mushroom were weighed out and placed into a mortar with 4 mL of extraction buffer (10 mM Tris). Then, using a pestle, the mushroom and buffer were grinded into a slurry. The semi-homogenized mushroom/buffer solution was then transferred into four 1.5 mL micro-centrifuge tubes. These tubes were placed into a micro-centrifuge to pellet the solid mushroom particles by spinning at top speed for 2 minutes. The separated supernatant, containing cellobiase, was then extracted from each tube and transferred to a clean micro-centrifuge tube labeled as extract. After the enzyme had been isolated, the effect of temperature and time on the enzyme’s activity could then be tested. Three test tubes were labeled with the appropriate temperatures: 40°C, 60°C, and 80°C. 1.2 mL of 0.1 M citrate buffer, 1.2 mL of substrate (1.5 mM p-nitrophenyl glucopyranoside or pNPGP), and 0.3 mL of dH2O were added to each tube and briefly vortexed to mix. Twelve remaining test tubes were labeled with the appropriate temperature and time (2min, 5min, 10min, 15min) and 4.5 mL of 0.1 M NaOH were added to each. These twelve tubes were the stop reaction tubes. The three test tubes containing the buffer/substrate solutions were pre-warmed for approximately 2 minutes in their assigned temperature water baths. After 2 minutes had passed, 0.3 mL of mushroom extract containing cellobiase were added to each tube and vortexed then returned to their assigned water baths. A timer was then started to monitor reaction times. At the times indicated on the 12 labeled stop reaction tubes containing NaOH, 0.5 mL of reaction solution were removed from each test tube in the water baths and transferred to the appropriately labeled ‘stop’ tubes and then vortexed. Great care was taken to transfer the reaction solutions to the each ‘stop’ tube at the indicated temperature and reaction time. The ‘stop’ tubes were then left to cool to room temperature as to assure of the complete stoppage of the reactions. Once cooled, enzyme activity could then be measured with a Spec-20. Before recording the absorbance of each ‘stop’ reaction tube, the Spec-20 was first blanked using 4.5 mL of 0.1 M NaOH and appropriate volumes of citrate buffer, substrate (pNPGP), and enzyme. 405nm wavelength was used as that is the wavelength which p-nitrophenol absorbs. The absorbance of each ‘stop’ tube was then measured and recorded. Similar to the above procedures, the experiment was then repeated with the addition of a cofactor or inhibitor of differing dilution factors (1, 1/10, 1/100). A 10 mM solution of glucose was chosen to be studied. As the above procedures, 1.2 mL of citrate buffer and 1.2 mL of substrate (pNPGP) were added to three test tubes labeled with the appropriate dilution factor of 10 mM glucose solution. In addition, 0 mL, 0.27 mL, and 0.297 mL of dH2O along with 0.3 mL, 0.03 mL, and 0.003 mL of glucose solution were added to tubes labeled with the dilution factors (1, 1/10, 1/100), respectively. The reactions were then carried out exactly as above, however, only at one temperature as the variable here is the concentration of cofactor or inhibitor. The absorbance of each tube was then measured and recorded at each indicated time, excluding the 15 min reaction time. IV.

Results

The purpose of these experiments was to explore the behavior of enzymes and how their activity is affected by changes in temperature and the presence of inhibitors or cofactors. In regards to how cellobiase’s activity was affected by changes in temperature, data suggests that the enzyme’s activity was optimal around 60°C. Figure 1 reinforces this conclusion by showing how as time progressed, the amount of p-nitrophenol produced by cellobiase at 60°C was significantly greater than that of product produced at either 40°C or 80°C. Although the experiment showed that 60°C was the most optimal temperature for enzyme activity of the 3 temperatures tested, Figure 2 shows that 60°C is not the ideal temperature for cellobiase’s activity. In fact, Figure 2 shows that the optimal temperature for the enzyme’s function is about 57°C. In regards to how cellobiase’s activity was affected by the presence of differing concentrations of glucose, data suggests that the enzyme’s activity was optimal at the 1/10 dilution factor (1 mM) of glucose. Figures 3 and 4 reinforce this conclusion. Since enzyme activity was found to be optimal around 60°C in the effect of temperature on enzyme function experiment, this experiment was carried out solely at 60°C. This being said, comparing the amount of product produced over time at 60°C with glucose present to that of the amount of product produced in the absence of glucose suggests that glucose exhibits a slight inhibitory quality to this specific enzyme. V.

Discussion The first experiment in this lab pertained to studying the influence of temperature on enzyme activity. As shown in the data collected, temperature has a large impact on how an enzyme functions. Every enzyme has a temperature at which its activity is at a maximum. In this case, cellobiase’s optimal temperature proved to be about 57°C. That being said, once temperatures start to deviate from this optimal temperature, enzyme activity is affected significantly. At 40°C and lower, enzyme activity is depressed. This phenomenon is most likely due to the fact that molecules at lower temperatures have less kinetic energy, therefore, significantly decreasing the enzyme’s ability to operate to its fullest capacity due to energy loss. At the other end of the spectrum, once temperatures exceed that of the optimal temperature, enzyme activity plummets as well. This phenomenon is most likely due to the fact that as temperature increases, molecules gain more and more kinetic energy and begin to vibrate violently. Enzymes are comprised of proteins which are heavily influenced by temperature. As temperature continues to increase, quaternary and tertiary structures of the enzyme begin to denature, thus, completely neutralizing its ability to function, as structure is vital to protein function. The second experiment in this lab pertained to studying the effect of a cofactor (or inhibitor) on the rate of reaction. In this experiment, a 10 mM glucose solution was selected to be studied. Comparing the amount of p-nitrophenol produced by the enzyme at 60°C from times 2 min – 10 min (the 15 min time was excluded due to shortage of time) with glucose present to that of when glucose was absent, glucose was found to exhibit a slight inhibitory quality. It is hypothesized that this phenomenon occurs because the added free floating glucose acts as a sort of imposter. The free floating glucose could trick the cellobiase into ‘thinking’ that it is binding to a p-nitrophenyl glucopyranoside molecule to cleave the p-nitrophenol/glucose bond when in reality, it is only recognizing

a single glucose molecule. This, in turn, would slow the rate of the reaction as data shows. Data also showed that enzyme activity in the presence of added glucose solution was (barely) optimal at the 1/10 dilution factor. This directly conflicts with the conclusion that added glucose acts as an inhibitor as the 1/100 dilution factor should be the optimal concentration for enzyme activity in the presence of added glucose due to it being least concentrated. A possible source of error could be a mistiming in the removal of reaction solution to be analyzed, giving the solution a slight increase in time to react, accounting for the slim (0.01) difference in the values of the 1/10 and 1/100 dilution factor solutions. VI.

Figures

Figure 1.

Amount of Product vs. Time at Different Temperatures

Amount of p-nitrophenol (µmol)

0.25

f(x) = 0.02 x R² = 0.93

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f(x) = 0.01 x R² = 0.96

0.15

0.1

0.05 f(x) = 0 x R² = 0.9 0

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Time (min) 40 C Linear (60 C)

Linear (40 C) 80 C

60 C Linear (80 C)

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Figure 1. µmols of Product Produced vs. Time at Different Temperatures Depicted above is the graph of the amount of product (µmol p-nitrophenol) produced over time when cellobiase, an enzyme, is introduced into a solution containing p-nitrophenyl glucopyranoside (pNPGP) for three different temperatures (40°C, 60°C, 80°C). A best fit line, along with its respective equation and R2 value, is also given for all three temperatures to show the approximate constant amount of product produced over time for that given temperature. Figure 2.

Enzyme Activity vs. Temperature 0.02

Activity (µmol/min)

0.01 0.01 0.01 0.01 0.01 0 0 0 35

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Temperature (°C)

70

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Figure 2. Enzyme Activity (µmol/min) vs. Temperature Depicted above is the approximate curve of the enzyme cellobiase’s activity, given in µmol of product produced/min, versus temperature. The graph was made by taking the total amount (µmol) of p-nitrophenol produced by cellobiase at 15min for all three temperatures and dividing that value by 15 min to give µmol of p-nitrophenol produced/min at that given temperature.

Amount of Product vs. Time at Different Concentrations of 10 mM Glucose

Amount of p-nitrophenol (µmol)

0.25

0.2

f(x) f(x) == 0.02 0.02 xx R² R² == 0.98 0.98

0.15

f(x) = 0.02 x R² = 0.98

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0.05

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Time (min) 1 Dilution Factor Linear (1/10 Dilution Factor)

Figure 3.

Linear (1 Dilution Factor) 1/100 Dilution Factor

1/10 Dilution Factor Linear (1/100 Dilution Factor)

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Figure 3. µmol of Product vs. Time at Different Concentrations of 10 mM Glucose Shown above is the graph of the amount (µmol) of p-nitrophenol produced when cellobiase is introduced to solutions containing pNPGP and differing concentrations of glucose. The three best fit lines on the graph account for three differing dilution factors (1, 1/10, 1/100) of glucose added to the reaction tubes.

Figure 4.

Enzyme Activity vs. Glucose Concentration 0.03

Activity (µmol/min)

0.02 0.02 0.01 0.01 0

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Concentration of Glucose (mM)

Figure 4. Enzyme Activity (µmol/min) vs. Concentration of Glucose Depicted above is the approximate curve of the activity of cellobiose, given in µmol of pnitrophenol produced/min, versus the concentration (mM) of glucose present. The graph was made by taking the total amount (µmol) of p-nitrophenol produced at 10 min for all three differing concentrations of glucose present and dividing that value by 10 min to give l f it h l d d/ i t th t i t ti f l...