Eznymez Lab Rpt

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Isabele Dimino Partial Lab Report Results Effect of Enzyme Concentration Our data suggests that the rate of a reaction increases as the enzyme concentration increases. The data shows a consistent upward trend and directly relates to the amount of enzyme concentration. With an increase in enzyme concentration would cause for a higher reaction rate due to a higher chance of the enzyme reacting with the substrate. Figure 2 below shows the different dilutions of the enzymes, one to one thousand, one to one hundred, and one to ten. The one to ten dilutions provided the highest reaction rate due to a higher concentration of the enzyme. Figure 2 is the graph made from figure 1 results. The graph shows an upward trend of reaction rate and enzyme concentration. Figure 1: Effect of enzyme concentration on enzyme activity 0.5 1 1.5 2 2.5 3 3.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Reaction Rate vs. Enzyme Concentration Enzyme Concentration Reaction rate Figure 2: Graph represents Reaction Rate vs. Enzyme Concentration. Effect of Substrate Concentration

Figure 3: Effect of substrate concentration on enzyme activity 0.5 1 1.5 2 2.5 3 3.5 0 0.01 0.02 Reaction Rate vs. Substrate Concentration Substrate Concentration Reaction Rate Figure 4: Graph represents Substrate Concentration vs. Reaction Rate Effect of Temperature on Enzyme Activity Enzyme activity highly depends on temperature conditions. Enzymes are known to have highest function at room temperature, this is because 37 degrees Celsius is most similar to the reactions occurring within the human body. Our results showed otherwise, this may be due to a human error or incorrect incubation temperatures. The data suggested that 65 degrees Celsius was the optimal temperature for enzyme activity just by a .04 difference. Figure 5 depicts the table in which we collected the data, and we can see that the 37 degrees Celsius slot should have the highest value but does not. Figure 6 shows the graph acquired from the results in figure 5, it is expected to look like a mountain, with the highest point being at 37 degrees Celsius. Figure 5: Effect of temperature on enzyme activity.

4 14 24 34 44 54 64 0 0.5 1 1.5 2 2.5 3 Reaction rate vs. Incubation Temperature Incubation Temperature (C) Reaction Rate Figure 6: Graph represents Reaction rate vs. Incubation temperature Effect of pH on enzyme activity Enzymatic activity is affected by a change in pH, a very low or very high pH may result in a loss of activity in many enzymes. The optimal pH value for enzyme activity is 7.0. With an increase or decrease will alter the enzymes conformation, thus altering its functionality. Our data suggested that the optimum pH was valued at 11.0, but this is too basic. This may have been because we were required to use three different buffers at three different pH values, there may have been a switch up or complete loss of a buffer. Figure 7 depicts the table in which we gathered our data in, and figure 8 is the graph generated from the absorbance values in figure 7. Figure 7: Effect of pH on enzyme activity

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2 3 4 5 6 7 8 9 10 11 12 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Reaction rate vs. pH Buffer pH Reaction rate Figure 8: Graph represents Reaction Rate vs. pH Discussion Lactase is an enzyme required to break lactose into its constituents, galactose and glucose. This is done through the process of lactase binding to the disaccharide lactose, and the active sites cleave lactose into its two constituents. The first portion of the experiment was to determine enzymatic activity or catalytic activity based on enzyme concentration. This was done through serial dilutions with one to ten being the most concentrated form. Increasing enzyme concentration will result in an increased reaction rate, as long as there is enough substrate for the enzyme to bind to, the reaction rate will continue to increase. Denaturation of an enzyme is when the structure or conformation of the enzyme is broken down to high temperatures which results in the weak hydrogen bonds breaking down within the enzyme. Denaturation essentially results in an ineffectual enzyme because it is no longer able to bind to a substrate because its structure is altered. Enzyme activity is affected by many factors like pH, temperature, and substrate concentration. Our data did not suggest that any denaturation of the enzyme occurred within the experiment and it also showed that when an enzyme is at a higher concentration, there is a higher reactive rate. Substrate concentration is a factor that also affects catalytic activity. The reaction rate for an enzyme increases as the substrate concentration increases until the enzyme is at its point of saturation. Our data did not suggest any denaturation occurred but out data was not consistent with expected readings. This may have occurred by human errors like wrong dilution factor or wrong measurements. Another factor that has a great affect on catalytic activity is temperature. The optimal temperature for enzymatic activity is 37 degrees Celsius, and generally with an increase in temperature causes for a decrease in the reaction rate due to the denaturation of the enzyme. Our data did not show any significant results to make a definitive statement on the affect of temperature. Rather than showing the highest value of the reaction rate at 37 degrees Celsius, it showed it at 65 degrees Celsius which is not consistent with previous studies.

Lastly, an important activity factor is pH. Most enzymes are proteins; therefore, they are impacted by changes to pH. The optimum pH for catalytic activity is 7, being neutral. A small change in pH may alter the enzymes’ structure and deforming the active site. Our data suggested that a pH of 11 would allow for maximum function but to our knowledge, this does not correlate. This may have been due to a use of wrong buffers or a spectrophotometer error. An enzyme may be denatured if the pH has reached beyond its optimum range of pH for maximum activity. This experiment specifically provided me with more knowledge about how an enzyme works, and what factors impacts an enzymes activity. Enzymes are essential for many reactions in the human body, by understanding the chemical basis of them has allowed us to create medicine, like lactase tablets which helps people who lack the enzyme to digest lactose. Throughout the experiment, there may have been a few errors, more specifically within the pH and temperature portion. This could have been caused by inconsistent techniques or improper handling of buffers. With more research, our data is slightly similar to others. We know what the expected graph are meant to depict, our data points are less consistent but going the same direction. Upon doing more research about factors, it was shown that a pH of 5.5 and a temperature range of 40-50 degrees Celsius were shown to have optimum function. [1] I believe with more specific experiments, like a more diverse range of pH, temperature, and different enzymes we will be able to generate a definitive statement about how these factors affect catalytic activity. [1] Da Silva LCA, Honorato TL, Cavalcante RS, Franco TT, Rodrigues S. 2011. Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced Under Solid Stated Fermentation by Trichoderma spp. Indian Journal of Microbiology. 52(1):60–65. doi:10.1007/s12088-011- 0196-0. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298583/.

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