Introduction to Chemical and Biological Engineering Laboratory (CBE 101B) Enzymatic Reaction Rates Experiment Last Revision: 09/08/2023 Background Enzymes for production of biofuels from biomass Current biofuel production in the USA

relies on the conversion of carbohydrates, mainly from corn kernels, to ethanol via fermentation. This technology was developed to reduce dependence on imported oil for transportation needs and to reduce the climate impacts of combusting non- renewable fuels. However, using only the grain from corn neglects the rest of the plant (as is the case when we use it for feeding ourselves or animals). A possible alternative to using only the corn kernels lies in the use of the rest of the plant, including as the leaves, stalks, and corncobs. In the same way, other plants can be used, including grasses that can grow rapidly with less fertilizer. A significant portion of these plant tissues are made up of lignocellulose, an important structural material in cell walls. Lignocellulose has three primary components: cellulose, which is a polymer of glucose; hemicellulose, a polymer of xylose and other sugars; and lignin, a complex polymer that also helps protect plants from predation. Following pretreatment of cellulosic materials (such as mechanical grinding and exposure to dilute acid), enzymes called cellulases can hydrolyze cellulose to produce glucose. This glucose can then be used to grow bacteria or yeast to produce ethanol (and other fuels and chemicals). This experiment is focused on the reaction of cellulose to glucose. Enzyme kinetics Enzymes are biological catalysts - compounds that accelerate the rate of a reaction. In the terminology of enzymes, a chemical that is reacted in an enzyme-catalyzed reaction is called "substrate". Most enzymes act on substrates that are dissolved in water. Cellulases (there are many types) are unusual in that their substrate (cellulose) is a solid and does not dissolve in water. This results in a different form of equation describing the reaction kinetics. Rather than the first-order reaction rate equation you may have seen in a chemistry course, the rate of cellulase-catalyzed reactions is represented by Equation 1: reaction rate = r = KCSCE Kd+CE Eqn. 1 با 10 OH OH OH cellulose O + 2n H₂O cellulases OH LO PO OH OH glucose C6H12O6 Equation 1 relates the rate of the cellulase-catalyzed reaction to the concentration of cellulase (CE). The equation also involves the parameters k (a rate constant), and Kd (a measure of binding affinity of the cellulase to the cellulose), as well as Cs (the initial concentration of enzyme binding sites on the solid substrate). A standard method of determining the rate parameters, k and Kd, for a cellulase is to measure the rate of glucose production at three different enzyme concentrations using the same type and amount of substrate. The glucose meter used in this lab uses an enzyme in a test strip to generate an electrical current proportional to the concentration of glucose. It is designed to measure glucose in human blood samples. In blood, the monitor can measure glucose in concentrations between 20 and 600 mg/dL (What are the equivalent concentrations in g/L?). Research questions 1. Can a blood glucose monitor be used to accurately and precisely measure glucose that isn't in blood? If it isn't accurate, what is the quantitative correlation between the measurement with a known standard concentration? 2. What experimental conditions are required for the cellulase to generate a glucose concentration within the limits of detection for the blood glucose meter? 3. What are the rates of glucose generation for this enzyme? 4. What other variables impact the reaction rate? For example, how would the rate change if you doubled the enzyme concentration or doubled the mass or surface area of the substrate? 5. If you were tasked with designing an industrial process that used these cellulases to generate glucose for biofuel production, qualitatively describe the important process variables you would need to control in order to optimize the amount of glucose generated. Tasks 1. Determine the accuracy of the glucose meter. This will allow us to better account for error in readings during the experiments you will be conducting. To accomplish this you will take several readings with the glucose meter between the range provided earlier (20- CARACA control positive compare Actual glucose concentration 0.42 9/L negative enzyme + ice 600 mg/dL). To make a measurement, place a test strip in the glucose meter, take a small sample (10 μL) and put it on a clean surface. Touch the tip of the strip to the solution sample, when the meter asks how to mark the sample, select "No mark". Please record your readings below: Native Hay 0.29 0.49 0.69 991L 2.79/L 5g/L Glucose meter-reported concentration 68 mg/dL over 600 mg/dL 257 mg/dL 580mg/dL 2. Design an experimental plan to answer as many of the 'Research Questions' as possible. Please determine what factors you plan to vary, how you expect these factors will affect the reaction rate (proportionally or inversely proportionally) what you plan to hold constant, what your dependent variable is, how many levels you will test, what positive and negative controls you will use, how many replicates you will use, and how many total experiments you will need. ● In this experiment you will have the following information from previous studies involving glucose meter: dependent: rate of change Time: 5 minutes ● Substrate: 1 cm x 1 cm squares of Whatman filter paper substrate or blades of Temperature: Room temperature., approx. 25 °C independent varies of amounts grass hay 1:1000 → 10ml: 1000ml • Enzyme dilution: 1:500 to 1:2000 in 1 mL of 25 mM NaHCO3, pH 6 Use these parameters above to ensure glucose readings can be gathered. In this glucose experiment there will be a major limitation, each team will only have access to 20 test strips, and therefore must limit their total experiments. 10 PL enzyme 1000μl buffer When your team has a rough outline of the kind of experiments you want to perform ask an instructor or TA to review your plan and provide feedback and approval. 3. Create an experimental outline in which you will use hay as the source of cellulose, attempt to change the surface area, the independent variable, of the hay for this experimental plan. 4. Once approval is received, begin your experiments and carefully record all data observations. art 32 51 417 87g|RL \/ BL₁=8.5m BL2=10,5mm BL3=6.5MM Data Analysis 1. 2. Part 1 (plotting and curve fitting): a. Use Matlab to plot the glucose standards measurements and determine the correlation describing the relationship between the actual glucose concentration and the glucose meter reading. What is the accuracy and precision of the meter at the low and at the high glucose concentration? b. What is the precision of the measurements? Precision can be described by the x 100%. Is the precision different range as a percent of the average, or at the two different concentrations? Part 2 (use the actual glucose concentration as determined using your correlation): 3. Part 3: a. Assuming zero glucose at t = 0 min., calculate the rate of the reaction in all of your enzyme experiments. What are the units of the rate you have calculated? What is the relationship between the reaction rate and enzyme concentration? b. The rate parameters for the solid substrate enzyme reaction can be found using the rates measured at three different enzyme concentrations. Assume the starting enzyme concentration was 100 g/L and that the cellulase molecular weight is 50,000 g/mol. Use Matlab to plot 1/CE (in units of liters/mol) on the x-axis against 1/rate on the y-axis. Find the correlation between these transformed variables. 1 r Take the inverse of the rate equation, r = 1 ŕ kd CE + KCSCE KCSCE kd KCSCE (max- 1 + average kCs KCSCE ka+CE to obtain: Eqn. 2 Eqn. 3 Compare the form of Eqn. 3 to y = mx + b to see that 1/(k*Cs) is the y-intercept of the correlation and Ka/(k*Cs) is the slope of the correlation. What are the values and units for k*Cs and Ka? a. Estimate by what factor you increased the surface area of the cellulosic material. How does this compare to the change in the reaction rate? [1] Mathematically, the reaction rate is dCs/dt but in experiments, we approximate it as ACs/At 1