Post LAB Experiment 2 Biochem (2)

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Jagjyot Kaur (301286821) 1

Jagjyot Kaur (301286821) 2 RESULT Table 1. Absorbance Data for the p-nitrophenol standard Standard Absorbance Amount of p -Nitrophenol ( nmol) S1 0 0 S2 0.100 12.5 S3 0.196 25 S4 0.380 50 S5 0.777 100 0 20 40 60 80 100 120 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 0.1 0.2 0.38 0.78 f(x) = 0.01 x + 0 R² = 1 Standard Curve of concetration of p Nitrophenol vs Absorbance at 410nm Concentration of pNitrophenol in nmol Absorbance at 410nm Equation to calculate concentration of p-Nitrophenol. x = (y-0.0004) / 0.0077 Initial rate of product formation = slope of the line = change in y/change in x

Jagjyot Kaur (301286821) 3 ACTIVITY 1 Table 2: Determination of p Nitrophenol produced using a high and a low enzyme concentration based on p nitrophenol standards. Cuvette Standard That is most similar Amount of p Nitrophenol Produced (nmol) H1 S4 50 H2 S5 100 H3 S5 100 L1 S3 25 L2 S4 50 L3 S5 100 Table 3: Determination of p Nitrophenol produced using a high and a low enzyme concentration based on spectrophotometer. Cuvette Absorbance 10 Amount of p -Nitrophenol Produced (nmol) H1 0.273 35.66 H2 0.321 41.12 H3 0.489 63.07 L1 0.167 21.64 L2 0.209 27.10 L3 0.380 49.30 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 0 21.64 27.1 49.3 0 35.66 41.12 63.07 Effect of concentration of Enzyme Cellobiase on Reaction Rate High Enzyme Concentration Low Enzyme Concentration Time in minutes Concetration of pNitrophenol in nmol Using the linear portion of the curve to determine initial rate of reaction via the slope method Initial Rate of reaction for high enzyme concentration = slope of the line = (63.07-41.12)/ 8-2 = 3.66 nmol/min

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Jagjyot Kaur (301286821) 4 Initial Rate of reaction for low enzyme concentration = slope of the line= (49.3- 21.64 )/ 8-1 = 3.95 nmol/min POST-LAB QUESTIONS 1. Does the amount of enzyme change the initial rate of reaction? Explain why based on your data. Answer: The reaction rate is directly proportional to the enzyme concentration (Robinson, 2015). Increasing the amount of enzyme increases the rate of reaction, as there are more active sites available to bind substrate molecules. As seen in the graph, for the same amount of time more product is obtained when high concentration enzyme is introduced in comparison to low concentration enzyme. 2. Does the amount of enzyme change the final amount of product, assuming that you start with the same amount of substrate and that you let each reaction proceed for a really long time? Answer: The amount of enzyme present affects the rate or speed of the reaction, but not the final equilibrium amount of product. For a given amount of substrate, the maximum amount of product that can be formed is determined by the stoichiometry of the reaction, not the enzyme concentration. Therefore, while a higher enzyme concentration speeds up the initial rate, allowing the equilibrium to be reached faster, it does not affect the final maximal amount of product formed from a fixed amount of starting substrate. 3. If you were a scientist who is responsible for determining the concentration of enzyme to use in the hydrolysis process of producing sugar from cellulose, what advantage would there be. to using a high concentration of enzyme? What disadvantage would there be to using a high? concentration of enzyme? Advantages of High Enzyme Concentration Disadvantages of High Enzyme Concentration Faster reaction rate due to more catalytic sites Increased enzyme cost as enzymes is expensive Higher initial hydrolysis yields and glucose concentrations Enzyme inactivation at very high solid loadings due to high viscosity and poor

Jagjyot Kaur (301286821) 5 mass transfer (Chen & Liu, 2017). Helps overcome product inhibition effects caused by accumulation of sugars like glucose and cellobiose Extremely high enzyme concentrations can cause substrate inhibition, where substrate molecules vie for active sites, or enzyme inhibition, where enzymes disrupt each other's activity. In some cases, enzymes at higher concentrations can exhibit increased stability, possibly due to the protective effect of enzyme-enzyme interactions or the crowding effect that reduces the rate of denaturation. Enzymes are proteins and have limited solubility in aqueous solutions. At very high concentrations, enzymes may precipitate out of solution, leading to a loss of catalytic activity.

Jagjyot Kaur (301286821) 6 ACTIVITY 2 Table 4: Determination of p Nitrophenol produced using a high and a low enzyme concentration based on p nitrophenol standards. Cuvette Standard That is Most Similar Amount of p -Nitrophenol Produced (nmol) H1 S3 25 H2 S4 50 H3 S5 100 L1 S1 0 L2 S2 12.5 L3 S3 25 Table 5: Determination of p Nitrophenol produced using a high and a low enzyme concentration based on standard curve. Cuvette Absorbance at 410 Amount of p Nitrophenol Produced (nmol) H1 0.196 25.40 H2 0.668 86.70 H3 0.628 81.03 L1 0.016 2.03 L2 0.037 4.75 L3 0.118 15.28 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 0 2.03 4.75 15.28 0 25.4 86.7 81.03 Effect of concentration of Substrate p-nitrophenol glucopy- ranoside on Reaction Rate High Substarte Concentration Low Subtrate Concentration Time in minutes Concentration of pNitrophenol in nmol Using the linear portion of the curve to determine initial rate of reaction via the slope method

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Jagjyot Kaur (301286821) 7 Initial Rate of reaction for high substrate concentration = (86.7-25.4)/ -1=61.3 nmol/min Initial Rate of reaction for low substrate concentration = (15.28 – 4.75) / 8-2= 1.76 nmol/min

Jagjyot Kaur (301286821) 8 Post-Lab Questions 1. How does the amount of substrate present change the initial rate of reaction? Answer: The rate of reaction for high subtrate concentration is higher than the low subtrate concentration reaction mixture. Since more substrate molecules are available to bind to the enzyme’s active sites, leading to a higher reaction rate. With the same amount of enzyme, the reaction mixture with high concentration subtrate gave a product concentration of 25.4 nmol in 1 minute whereas for low substrate concentration it was2.03 nmol in 1 minute. 2. Explain how changing the concentration of substrate affects the rate of product formation. Answer: Since at a given point of time more subtrate – enzyme complex will be formed in a reaction mixture with higher substrate concentration it will lead to high product concentration indicating high initial rate. For a comparatively low substrate concentration less number of complex will be formed at a given time leading to less product. A similar trend is observed in the data recorded. With initial rate of reaction of higher substrate concentration mixture being 61.3 nmol/min and low substrate concentration being 1.76 nmol/min 3. Does the amount of substrate change the final amount of product, assuming that you let each reaction proceed for a really long time? Answer: No this isn’t a possibility, since it is the substrate that is converted to the product. If a reaction mixture contains x amount of substrate it will produce a maximum x amount of product. If the reaction mixture contains 2 x substrate it will produce a maximum 2x amount of product. Even if the reaction mixture is left for longer period of time we cannot accept the former mixture to form 2x product since the substrate is like a precursor of product. If the precursor is exhausted and has given x amount of product no amount of time or enzyme will lead to formation of more product.

Jagjyot Kaur (301286821) 9 Activity 3 Table 6: Determination of p Nitrophenol produced by mushroom extract breaking down the substrate based on standard curve Cuvette Absorbance at 410 Amount of p Nitrophenol Produced (nmol) 1 0.261 33.84 2 0.363 47.10 3 0.727 94.36 4 0.746 96.83 5 1.110 144.10 6 0.00 0 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 0 33.84 47.1 94.36 96.83 144.1 f(x) = 18.51 x R² = 0.98 Effect of Extract of Shiitake Mushroom on Reaction Rate Time in minutes Concnetration of pNitrophenol in nmol Initial Rate of reaction for Mushroom Extract = (94.36 – 33.84)/ 4-1= 20.17 nmol/min

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Jagjyot Kaur (301286821) 10 Post Lab Did your mushroom extract break down the substrate (i.e., was there any formation of a yellow product)? Answer: Yes, there was a formation of yellow product indicating product formation. 2. Why did we use a blank for this activity that was different from the one used in earlier activities? Answer: This is because the reaction mixture was different than the previous 2 activities as activity 3 had extraction mixture as part of the reaction mixture which had to be considered while auto zeroing. Hint: What would be the effect on the absorbance readings if a mushroom naturally had some yellow color to it? Answer: If the mushroom extract itself had a yellow color it would have led to higher absorbance value however, we would still have observed the same trend since the yellow color added by the extract would have been constant in all cuvettes. However, the amount of p nitrophenol could not have been reported with confidence

Jagjyot Kaur (301286821) 11 References 1. Robinson, P. K. (2015). Enzymes: principles and biotechnological applications. Essays in Biochemistry, 59, 1–41. https://doi.org/10.1042/bse0590001 2. Chen, H., & Liu, Z. (2017). Enzymatic hydrolysis of lignocellulosic biomass from low to high solids loading. Engineering in Life Sciences, 17 (5), 489– 499. https://doi.org/10.1002/elsc.201600102

Post LAB Experiment 2 Biochem (2)

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