Abstract: The purpose of this lab is to determine the ability of an enzyme, which is cellobiased, to increase the conversion rate

of a clear substrate, p-nitrophenyl glucopyranoside, into a colored product, which is p-nitrophenol and glucose under five different scenarios. The scenarios are the presence or absence of an enzyme, the effect of temperature, the effect of pH, the effect of enzyme concentration and the effect of substrate concentration on the reaction rate. By manipulating the spectrophotometer to detect each result of absorbance, the data for amount of p-Nitrophenyl produced was collected in each reaction and used to determine the rate of reaction for each variable. Presence of an enzyme, increase in temperature, lower pH (more acidic), a more concentrated enzyme, and a more concentrated substrate increased the rate of reaction. Introduction: Enzyme is a large protein molecule that made up of amino acid. Enzymes shape is specific; therefore, each enzyme can only react with certain kind of substrate, and the enzymes shape determines which substrate that is. The substrate binds in to the active site of the enzyme, which is where the reaction takes place. (Campbell 8th edition, 2007) An enzymes active site needs to be the right shape so that the substrate molecule can fit into it properly. The chemical groups of the amino acids interact forming hydrogen, ionic, covalent, and Van der Waals interactions to create a specific shape. (Biofuel, 2011) If these amino acids are not in the correct orientation, the active site will not be able to catalyze the reaction. However, if the reactant molecule is forced into the correct orientation, enzyme can speed up the chemical reactions and lowers the energy of activation. The energy of activation is the amount of energy that is required to bring the substrate molecule into the transition state. An enzyme stabilizes the transition state and lowers the amount of energy of the transition state molecule. (Brown, LeMay, Bursten, Murphy, Woodward, 2009) This will result into a faster rate of converting substrate molecule to product due to its less energy requirement. In this lab, the enzyme involved in this laboratory experiment is cellobiase enzyme. Its function is to break down cellulous to glucose. Cellobiase is applied in several industrial applications such as pulp and paper factory. (Vermelho, 2011) It has been used for biomodification of fiber properties with the aim of improving drainage and beatability in the paper mills before or after beating of pulp. The natural product of cellobiase is glucose, which, in natural process is used by many fungi and bacteria to produce glucose as a food source. The natural substrate of cellobiase is cellobiose, a disaccharide composed of two beta glucose molecules. The purpose of the lab is to determine the rate of enzyme activity of cellobiase at each time period under the effect of change in pH, temperature, substrate concentration and enzyme concentration. In order to determine the rate of cellobiase activity, p-nitrophenol was used as a substrate due to its property of producing both glucose and p-nitrophenol. As the p-nitrophenyl glucopyranoside, a substrate to bind with cellobiase, was broken down by cellobiase, it produced glucose and p-nitrophenol, which turned yellow as it mixed with the stop solution. The amount of yellow color is proportional to the amount of p-nitrophenol present. Therefore, the degree of yellowness can be used to compare with the standards, which contain a known amount of colored product, to get an estimation of the amount of product that is produced at each time period.

Besides qualitative analysis, quantitative analysis can also be executed by using spectrophotometer that measures the amount of light that is absorbed by the sample. The darker the color of yellow of a sample results into more light being absorbed, thus, indicating a more concentrated sample. The absorbance values of a set of standards can first be measured to create a standard curve, a plot of the absorbance values of samples of known concentration of p-nitrophenol. The absorbance values of the reaction samples can then be measured, and the standard curve can be used to convert the absorbance value to a concentration value. The rate of reaction of the amount of p-nitrophenol produced must be measured in order to determine the influence of each factor has on the enzymes ability to break down its substrate. In the experiment, the amount of the product produced in a certain amount of time is calculated by determining the initial slope of the graph plotted with the product produced as a function of time. In order to determine the reaction velocity at any point in time, the difference in amount of product produced between two time points has to be divided by the time interval. Therefore, the rate of reaction is obtained. By calculating the amount of pnitrophenol produced over time, the rate of reaction can be calculated and therefore the effects of pH, temperature, substrate concentration and enzyme concentration have on the initial rate of reaction can be compared. Procedure: In activity 1, the reaction rate under the presence or absence of the enzyme was determined. Four 15ml conical tubes were labeled as, stop solution, 1.5mM Substrate, enzyme, and buffer were located and initialed. Five cuvettes were labeled with E1-E5, and two other cuvettes were labeled separately as start and end. Using a disposable pipet (DPTP), 500 microliters of stop solution was put into each labeled cuvette. Two more 15ml conical tubes were labeled as enzyme reaction and control. A clean DPTP was used to put 2ml of 1.5mM substrate into the 15ml conical tube labeled as enzyme reaction. The same DPTP was used to put 1ml of 1.5mM substrate into the conical tube labeled control. The DPTP was disposed of after this process. A new DPTP was used to put 500 microliters of buffer into the 15ml conical tube of control, which, was then stirred gently. Next, 500 microliters of this control was added to the start cuvette. A clean DPTP was then used to put 1m of enzyme into the 15mlconical tube labeled enzyme reaction. This was then gently stirred and the timer was started. The data were collected at each time interval which was at one minute, two minutes, four minutes, six minutes, and eight minutes by putting 500 microliters solution from the conical tube labeled enzyme reaction, and put into the cuvettes labeled E1-E5, respectively. After all of the samples had been collected, a new DPTP was used to remove 500 microliters of the control solution, and that solution was put into the cuvette labeled end. After all of the data was recorded, the DPTPs were thrown out, and the cuvettes and conical tubes were rinsed with water. In activity two, the reaction rates under the effect of different temperature were determined. Three cuvettes were labeled with 0, 22, and 37. A DPTP was used to put 500 microliters of stop solution into each cuvette. Three 1.5ml microcentrifuge tubes were labeled with 0C Enzyme, 22C Enzyme, and 37C Enzyme. A new DPTP was used to put 250 microliters of enzyme into each microcentrifuge tube. Three other 1.5ml microcentrifuge tubes were labeled with 0C Substrate, 22C Substrate, and 37C Substrate. A new DPTP was used to put 500 microliters of 1.5mM substrate into each microcentrifuge tube. The tubes labeled 0C Enzyme and 0C Substrate were placed in an ice cup. The tubes labeled 22C Enzyme and 22C Substrate were placed on a lab bench. The tubes labeled 37C Enzyme and 37C Substrate were placed in a beaker of warm (37C) water. The tubes were

left for 5 minutes. A new pipet was used to put 250 microliters of enzyme from the tube labeled 0C Enzyme, into the one labeled 0C Substrate. This was then done with the 22C tubes and the 37 tubes. The empty enzyme tubes were discarded and the tubes containing the enzyme and substrate solutions were placed back into their respective locations according to temperature. A timer was then started. After two minutes a new DPTP was used to transfer 500 microliters of 0C solution, 22C solution, and 37C solution into their corresponding cuvettes. The data was then recorded. The DPTPs and the microcentrifuges were then thrown out, and the cuvettes were rinsed with copious amounts of water. In activity three, the rates of reaction under different pH environment were determined. Three cuvettes were labeled with pH 5.0, pH 6.3, and pH 8.6. A new DPTP was used to put 500 microliters of stop solution into each cuvette. Three microcentrifuge tubes labeled pH 5.0, pH 6.3, and pH 8.6. Another new DPTP was used to put 250 microliters of 3.0mM of substrate into each microcentrifuge tube. Another DPTP was used to add 250 microliters of enzyme to each of the labeled microcentrifuge tubes. Timers were then started. After two minutes, another DPTP was used to transfer 500 microliters of each reaction from the microcentrifuges into its corresponding cuvette containing stop solution. The data was then recorded. The DPTPs and the microcentrifuges were then thrown out, and the cuvettes were rinsed with water. In activity four, the effects of different concentrated enzyme on rate of the reaction were determined. One 15ml conical tube was labeled low concentration enzyme and another, high concentration enzyme. A new DPTP was used to put 1ml of buffer into the tube. A new DPTP was used to put 1ml of high concentration enzyme into the tube labeled low concentration enzyme. This mixture was the stirred. Three cuvettes were labeled H1, H2, andH3. Three more cuvettes were labeled L1, L2, and L3. A new DPTP was then used to put 500 ml of stop solution into each cuvette. Another DPTP was used to put 250 microliters of 1.5mM substrate into the 15ml conical tube labeled high concentration enzyme. A new DPTP was used to put 250 microliters of 1.5 mM substrate into a 15ml conical tube labeled low concentration enzyme. A timer was then started. After one minute, 500 microliters of high and low concentration enzymes were taken from each of the 15ml conical tubes. The high concentration enzyme was put into the cuvette labeled H1 and the low concentration enzyme was put into a cuvette labeled L1. After two minutes, 500 microliters of high and low concentration enzymes were again taken from each of the 15ml conical tubes. The high concentration enzyme was put into the cuvette labeled H2 and the low concentration enzyme was put into a cuvette labeled L2. After eight minutes, 500 microliters of high and low concentration enzymes were taken from each of the 15ml conical tubes. The high concentration enzyme was put into the cuvette labeled H3 and the low concentration enzyme was put into a cuvette labeled L3. The data was then recorded. The DPTPs were thrown out, and the cuvettes were rinsed with water. In activity five, the effect of substrate concentration on the rate of reaction was determined. Two conical tubes were labeled with Low Concentration Substrate and high concentration substrate. A DPTP was used to put 1.5 ml of 1.5 mM substrate into conical tube labeled High Concentration Substrate. A new DPTP was used to put 1.25ml of buffer into the 15 ml conical tube labeled low concentration substrate. A new DPTP was used to put 250microliters of 1.5 mM substrate into the conical tube labeled low concentration substrate. This was then mixed. Three cuvettes were labeled H1, H2, andH3. Three more cuvettes were labeled L1, L2, and L3. A new DPTP was then used to put 500 ml of stop solution into each cuvette. Another DPTP was used to put 750 microliters of an enzyme into the conical tube labeled high concentration substrate, and 750 microliters of an enzyme into the conical tube labeled low concentration substrate. A timer was then started. After one

minute, 500 microliters of high and low concentration substrates were taken from each of the 15ml conical tubes. The high concentration substrate was put into the cuvette labeled H1 and the low concentration substrate was put into a cuvette labeled L1. After two minutes, 500 microliters of high and low concentration substrates were again taken from each of the 15ml conical tubes. The high concentration substrate was put into the cuvette labeled H2 and the low concentration substrate was put into a cuvette labeled L2. After eight minutes, 500 microliters of high and low concentration substrates were taken from each of the 15ml conical tubes. The high concentration substrate was put into the cuvette labeled H3 and the low concentration substrate was put into a cuvette labeled L3. The data was then recorded. The DPTPs were thrown out, and the cuvettes were rinsed with water. (Biofuel, 2011) Results: a) Activity 1: Determine the Reaction Rate in the Presence or Absence of an Enzyme
Table 1.p-Nitrophenol standards
Standard S1 S2 S3 S4 S5 Amount of p-Nitrophenol (nmol) 0.000 12.5 25.0 50.0 100

Table 2. Comparison of reaction cuvettes to standard cuvettes

Time (minutes) 0 8 1 2 4 6 8 Cuvette Start End E1 E2 E3 E4 E5 Standard That is most similar S1 S5 S2 S3 S3 S4 S5 Amount of pNitrophenol (nmol) 0.000 100 12.5 25.0 25.0 50.0 100

Table 3. Absorbance Values for standards

Standard S1 S2 S3 S4 S5 Amount of p-Nitrophenol (nmol) 0 12.5 25 50 100 Absorbance at 410nm 0.000 0.400 0.640 1.10 1.68

Table 4. Determining p-Nitrophenol produced using a standard curve.

Time (minutes) 0 8 1 2 4 6 8 Cuvette Start End E1 E2 E3 E4 E5 Amount of p-Nitrophenol (nmol) from the standard curve 0.00 58.9 0 14.0 22.4 38.6 58.9 Absorbance at 410nm 0.000 2.13 0.400 0.64 1.10 1.68 2.13

Graph 1: Absorbance versus Amount of p-Nitrophenol

Absorbance versus Amount of p-Nitrophenol 3 2.5
Absorbance

2.66

2 1.5 1 0.5 0 0 0.51 0.88 1.67

y = 0.0285x

Amount of p-Nitrophenol (nmol)

Graph 2: Amount of p-Nitrophenol from the standard curve versus Time

Amount of p-Nitrophenol from the standard curve versus. Time

Amount of p=Nitrophenol (nmol) 80 70 60 50 40 30 20 10 0 0 74.73684211 58.94736842 38.59649123 22.45614035 14.03508772 0 2 4 Time (minutes) 6 8 10

b) Activity 2: Determine the Effect of Temperature on the Reaction Rate Table 5. p-Nitrophenol standards
Standard S1 S2 S3 S4 S5 Amount of p-Nitrophenol (nmol*) 0.000 12.5 25.0 50.0 100

Table 6: Determination of p-nitrophenol produced at three different temperatures based on p-nitrophenol standards
Temperature (C) 0 22 37 Standard That is Most Similar S2 S3 S4 Amount of p-Nitrophenol produced (nmol) 12.5 25.0 50.0

Table 7. Determination of p-nitrophenol produced at three different temperatures

Temperature (C) 0 22 37 Absorbance at 410nm 0.270 0.350 0.636 Amount of p-Nitrophenol Produced (nmol) 9.47 12.3 22.3

Graph 3: Amount of p-nitrophenol Produced versus Temperature for 2mins

Amount of p-Nitrophenol Produced versus Temperature for 2 mins in C
25 20 15 10 5 0 0 5 10 15 20 25 Temperature (t) in C 30 35 40 9.473684211 12.28070175 22.31578947

c) Activity 3: Determine the Effect of pH on Reaction Rate

Table 8. p-Nitrophenol standards
Standard S1 S2 S3 S4 S5 Amount of p-Nitrophenol (nmol*) 0.000 12.5 25.0 50.0 100.0

Amount of p-Nitrophenol Produced (nmol)

Table 9. Determination of p-nitrophenol produced at three different pH values based on pNitrophenol standards
pH pH 5.0 pH 6.3 pH 8.6 Standard that is most similar S4 S3 S3 Amount of p-Nitrophenol produced (nmol) 50.0 25.0 25.0

Table 10. Determination of p-Nitrophenol produced at three different pH values based on standard curve
pH pH 5.0 pH 6.3 pH 8.6 Absorbance at 410nm 0.436 0.356 0.352 Amount of p-Nitrophenol Produced (nmol) 15.3 12.5 12.4

Graph 4: Amount of p-Nitrophenol versus pH for 2mins

Amount of p-Nitrophenol versus. pH for 2 mins

Amount of p-nitrophenol (nmol)

18 16 14 12 10 8 6 4 2 0 0 1 2 3 4 5
pH

15.29824561 12.35087719 12.49122807

10

d) Activity 4: Determine the Effect of pH on the Reaction Rate Standard

S1 S2 S3 S4 S5

Table 11. p-Nitrophenol standards Amount of p-Nitrophenol (nmol)

0.000 12.5 25.0 50.0 100

Table 12: Determination of p-nitrophenol produced using a high and a low enzyme concentration based on p-nitrophenol standards. Cruvette Standard That is Most Amount of p-Nitrophenol Similar (nmol)
H1 H2 H3 L1 L2 L3 S3 S4 S5 S1 S2 S4 25 50 100 0 12.5 50

Table 13. Determination of p-nitrophenol produced using a high and low enzyme concentration based on a standard curve Cuvette Absorbance at 410nm Amount of p-Nitrophenol Produced (nmol)
H1 H2 H3 L1 L2 L3 0.98 1.28 1.90 0.34 0.53 1.32 34.4 44.9 66.7 11.9 18.6 46.3

Graph 5: Amount of p-Nitrophenol versus Time under High and Low Substrate Concentration
Amount of p-Nitropheonl Produced (nmol)

Amount of p-nitrophenol versus Time under High and Low Substrate Concentration

90 80 70 60 50 40 30 20 10 0 0

66.66666667 44.9122807 34.38596491 18.59649123 11.92982456 0 2 4

Time (min)

46.31578947

High Concentration Low Concentration

10

e) Activity 5: Determine the Effect of Substrate Concentration on Reaction Rate Standard

S1 S2 S3 S4 S5

Table 14. p-Nitrophenol standards Amount of p-Nitrophenol (nmol)

0.000 12.5 25.0 50.0 100

Table 15. Determination of p-nitrophenol produced using a high and a low substrate concentration based on p-nitrophenol standards Cuvette Standard That Is Most Amount of p-Nitrophenol Similar Produced (nmol)
H1 H2 H3 L1 L2 L3 S3 S4 S5 S1 S2 S4 25.0 50.0 100 0.000 12.5 50.0

Table 16. Determination of p-nitrophenol produced using a high and low substrate concentration based on a standard curve Cuvette Absorbance at 410nm Amount of p-Nitrophenol Produced (nmol)
H1 H2 H3 L1 L2 L3 0.17 0.31 0.99 0.07 0.13 0.49 5.96 10.9 34.7 2.46 4.56 17.2

Graph 6: Amount of p-Nitrophenol Produced versus Time under High and Low Enzyme Concentration
Amount of p-Nitrophenol Produced versus Time under High and Low Enzyme Concentration

Amount of p-Nitrophenol Produced (nmol)

40 35 30 25 20 15 10 5 0 0
0 10.87719298 5.964912281 4.561403509 2.456140351 17.19298246 34.73684211

High Concentration Low Concentration

6 Time (min)

10

Analysis: In activity 1, the reaction rate in the presence or absence of an enzyme is determined. Initial rate of product formation with enzyme present is 8.97nmol/min Sample Calculation Initial rate of product formation = slope of the line = change in y/change in x = (72.4 nmol 9.59 nmol)/(8 min 1 min) = 8.97 nmol/min Rate of product formation with no enzyme present is 0 nmol/min During the time that reaction was occurring, both solutions remained clear with no visible changes in the enzyme reaction and control reaction conical tubes. When enzyme and substrate mixture is added to the solution in each cuvette, the solution turned yellow. The solution gradually turned into a darker yellow after a period of time. However, the control cuvette without enzyme did not change color. The change in color indicated that there was chemical reaction occurring, which, was the enzyme breaking down the artificial substrate, p-nitrophenol glucopyranoside, into glucose and p-nitrophenol. In the control cuvette, which is the reaction without enzyme, there was no detectable product produced after 8 minutes. However, the reaction with enzyme produced about 72.4 nmol of product after 8 minutes or an absorbance at 410nm of 2.66. Sample Calculation Where y equals absorbance and x equals the amount of p-nitrophenol
y = 0.0285x x=72.4nmol y= 2.66

The amount of product produced is increasing at each time interval according to Graph 1. Therefore, if the reaction goes up to the time point at 15 minutes, the product will be produced more than at 8 minutes because there would be more time allow the enzymecatalyst activity. However, the rate of product formation will eventually drop to zero when there is no more substrate available for reaction. In qualitative analysis, the amount of product produced by the enzyme was estimated by visually compared the amount of yellow in our samples with a standard that contained a known concentration of product. The amount of light absorbed by the sample is proportional to the amount of product produced is

due to the fact that product produced turns yellow in the presence of the stop solution. The more yellow the color, the more products presents. The spectrophotometer measures the amount of light, at the wavelength of 410nm, which is absorbed by the sample. The more profound in color indicates a larger amount of light that is absorbed by the sample and therefore more products is presented in the sample. The initial rate of product production for the absorbance measurements is 9.59nmol/min or 0.40 AU/min. The rate of product production appears to be constant over time for earlier time points, however, the slope got steeper for the later time point and therefore the overall rate of product production is not constant over time. After completing the standard curve for amount of p-Nitropheonal produced, the effect of temperature on the reaction rate was determined in activity two. After 2 minutes, the 37C sample gave an absorbance reading at 410 nm of 0.636, which looked most similar to standard S4. Qualitatively (using the standards), indicating that there was ~50 nmol of pnitrophenol. Quantitatively, which is determined by the standard curve generated in Activity 1, the amount of p-nitrophenol with an absorbance of 0.636corresponds to 22.3nmol. Using the same process for the 0C sample, the qualitative analysis indicates that there was about 12.5 nm because it resembles the S2 color; the quantitative analysis indicates that there was 9.47nmol of p-nitrophonal produced with an absorbance of 0.270 at 410 nm. As for and 22C sample, the qualitative analysis indicates that there was about 25nmol of p-nitrophenol produced because it resembles the S3 color; the quantitative analysis indicates that with an absorbance of 0.350 corresponds to 12.28070175 nmol. The initial rate of reaction for quantitative data at 0C is 4.74nmol/min, at 22C is 6.14nmol/min, at 37C is 11.2nmol/min. Sample Calculation The initial rate for the qualitative data for 0 = (9.473684211nmol 0 nmol)/(2 min 0 min) = 4.736842106nmol/min. =4.74nmol/min (significant digit) The initial rate is determined by dividing the amount of absorbance between the 0 and 2 minute time point with the time interval, which is 2 minutes. The maximum optimum temperature activity seems to be at 37C in this experiment. The way to figure out initial rate is using the amount of absorbance between 0 and 2 minute time point divided by 2 minutes. This is also the initial slope of the line of product produced versus time. Chemical reactions occur faster at higher temperatures because it increases the kinetic energy in molecule, which, results into faster movement in molecules and therefore increases the number of collisions between molecules. The average kinetic energy of the substrates is higher and therefore more substrate molecules have the required activation energy in achieve the transition state. However, chemical reactions occur tend to occur more slowly at low temperatures because molecules are moving slower at lower temperature, therefore, fewer collisions occur. This leads to lack of energy to reach the transition state and therefore the chemical reaction is slower at low temperatures. ( Although high temperature does stimulate more molecular collisions, enzymatic reactions slow down at extremely high temperatures because enzymes are made of proteins. The structure of proteins denature at high temperatures due disconnection of the weak bonds between amino acids. According to the data from the experiment, a scientist would want the enzyme to react at 37C in order to achieve the fastest reaction rate. Besides the change in temperature would affect on the rate of the reaction, the scale of pH would also make an impact on the rate of reaction, which, was determined in activity three. After 2 minutes, the pH 5.0 sample gave an absorbance reading at 410 nm of 0.436,

which looked most similar to standard S4. Qualitatively (using the standards), this means that you have ~50 nmol of p-nitrophenol. Quantitatively, you would use the standard curve you generated in Activity 1 to determine the amount of p-nitrophenol with an absorbance of 0.436 corresponds to 15.3nmol. Using the same method for pH 6.3 with an absorbance at 410nm of 0.356, the qualitative measurement is about 25nmol because it resembles to the color of S2; the quantitative measurement is 12.5nmol of p-nitrophenol produced. As for pH 8.6 with an absorbance of 0.352, the qualitative measurement is about 25nmol; the quantitative measurement is about 12.4nmol. The initial reaction rate is the amount of product produced during the first time interval divided by the amount of time of the first time interval. It can also be determined by graphing a graph of product of the reaction versus time. By using the same calculation, each initial rate of reaction can be found. The initial rate of product formation for the quantitative data is 7.65nmol/min for pH of 5, 6.24nmol/min for pH of 6.3, and 6.18nmol/min for pH of 8.6. Sample Calculation The initial rate of product formation for the qualitative data for pH of 5 =(15.29824561nmol nmol 0 nmol)/(2 min 0 min) = 7.649122805nmol/min =7.65nmol/min (significance digit) The data from the enzyme pH showed the highest product production at a pH of 5. Therefore it can be concluded that the best enzyme pH is about 5. If the enzyme had a much higher or much lower pH than 5, the reaction would slow down. Drastically high or low pH can cause unwanted reactions because extreme change in temperature can break the bonds that hold the enzyme in its critical three-dimensional structure. The active site will not be the same shape; therefore, the produce would not be catalyzed. pH of the environment is a crucial factor in terms of the function of the enzyme. The environment must be acidic, but not to a drastic level. An acidic environment such the soil should be a good environment for bacteria or fungi that produce cellobiase enzymes. The pH is not the only thing that affects the rate of reaction of enzymes and substrates. The amount of the enzyme present also affects it, which, its effect is determined in activity 4. Assume that the amount of product at 0 minutes is 0 nmol. The amount of product at 1 minute was used to obtain the initial rate. The initial rate of reaction for high enzyme concentration is 34.4nmol /min. Initial rate of reaction for low enzyme is 11.9nmol /min. Sample Calculation The initial rate of product formation for high enzyme reaction =(34.38596491nmol 0 nmol)/(1min 0 min) = 34.38596491nmol /min =34.4nmol/min (significance digit) According to the data collected, the initial rate of reaction will increase if there is a larger amount of enzymes. The initial rate increases as the amount of enzyme is increased until there is excess enzyme. This occurs because there is many more enzymes present to aid break down the substrate. However, a state of equilibrium will be reached that is no matter how much enzyme is present, the reaction will not occur any quicker. This happens because all the substrate is being broken down by the exact same amount of enzyme, so enzymes will be present which have no substrate to break down. On the other hand, the amount of enzyme presented does not affect the amount of final product being produced because it is determined on the amount of substrate. If there is a low concentration of enzyme, it will take

longer to produce the product, but results into the same amount of product. If a scientist is responsible for determining the concentration of enzyme to use in the hydrolysis process of producing sugar from cellulose, by using high concentration of enzyme can achieve a more efficient reaction. However, enzymes are rather expensive. If a scientist wanted to know whether or not to use high concentration enzyme, the scientist should find a medium amount of enzymes that would effectively catalyze the substrate without being wasted. Enzymes are not the only things that can be manipulated to increase the rate of reaction. The concentration of the substrates present can also make an impact of the reaction rate; this was tested in activity five. Assume that the concentration of product at 0 minutes is 0 nmol. The amount of product at 1 minute was used to find the initial rate of reaction. Initial rate of reaction for high substrate concentration is 5.96nmol/min. Initial rate of reaction for low substrate concentration is 2.46nmol/min. Sample Calculation Initial rate of reaction for high substrate concentration =(Amount of product at 1min 0 nmol)/(1min 0 min) = (Amount of product at 1 min)/1 min = 5.964912281nmol/1min = 5.96nmol/min (significant digit) According to the data collected, increasing the amount of substrate will increase the rate of product production until there is excess substrate present. Once there is excess substrate present, then additional increases to the concentration of substrate will no longer make a difference in the rate of product production. An analogy for this would be a factory that slices hams in package. The hams are equivalent to substrate and the packages are equivalent to product. If there were less hams and excessive amount of slicing machines, the speed of package production would not be fast. If the amount of hams were increased, the rate of production would increase. However, there is a point in which every slicing machine is busy slicing ham. Adding more hams would not increase the rate any further. As oppose to the enzyme, the amount of substrate have an direct impact on the amount of product being produced. In the case of cellobiase breaking down cellobiose into glucose, the more cellobiose added the more glucose would be obtained. Although the empirical relationships in the experience between enzymatic actions under all scenarios of varied in temperature, pH, change in the concentration of enzyme and substrate confirm with the theoretical relationships, the data were not exactly accurate due to several limitations in the study. Examples are such as timing, measuring, and spectrophotometer use. Not all of the timing could have been exactly precise. It is highly possible that the measuring was a few seconds off. A few seconds might not have drastically changed the experiment; however, it could have resulted into inconsistencies and making some result hard to interpret. Measuring the amount of substrate and enzymes were particularly difficult when using the DPTPs. It was hard to get an exact measurement; therefore, estimations were made. The last things that could have possibly altered the results were the use of the spectrophotometer. The spectrophotometer was a very accurate and sensitive machine. However, if it was not set back to zero every time using the standard, the results could not been as precise as it could be.

Conclusion: The ability of cellobiase to increase the conversion rate of p-nitrophenly glucopyranoside into p-nitrophenol and glucose under the presence or absence of an enzyme, the effect of temperature, the effect of pH, the effect of enzyme concentration and the effect of substrate concentration on the reaction rates were determined in the experiment. By manipulating the data collected, the relationship in each scenario was concluded. The presence of enzyme stimulates a faster reaction rate; however, no significant reaction occurred under the absent of enzyme during the same time interval given. High temperature allows cellobiase to catalyze the break down of p-nitrophenly glucopyranoside at a faster rate; 37 degree Celsius is the optimal reacting temperature for cellobiase in the experiment. A pH of 5 allows cellobiase to catalyze the break down of p-nitrophenly glucopyranoside at a faster rate. High concentrated cellobiased would have a faster reaction rate than low concentrated cellobiased. A high concentrated substrate would create a faster reaction rate than low concentrated enzyme, as well. However, excessive amount of enzyme and substrate would not have any further impact on the reaction rate. Bibliography 1. Vermelho, 2011: Microbial Cellulases and Their Industrial Application: http://www.hindawi.com/journals/er/2011/280696/ 2. Campbell AP Biology Textbook 8th edition (Campbell, 2007) 3. Biofuel Enzyme Emanuel (Biofuel, 2011) 4. AP Chemistry: The Central Science 11th AP* Edition (Brown, LeMay, Bursten, Murphy, Woodward, 2009)

Biofuel Enzyme Lab Linda Huang Wednesday, November 23, 2011 Mr. Bowen AP Biology (SBI4U-AP)