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Biology lab enzyme activity
Biology lab enzyme activity
Biology lab enzyme activity
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Because it’s very difficult to accurately measure lactose or glucose in solution, we use a false substrate (known as analog) for the enzyme known as ONPG (ortho-nitro-phenyl-galactoside). ONPG has a structure similar to lactose, so it can bind to the enzyme and be cleaved. The products are galactose and ortho-nitrophenolate. ortho-nitrophenolate is yellow in color and can be measured by spectrophotometry. the rate of an enzyme can be measured. The reaction is usually expressed like this E is the enzyme, S is the substrate, ES is the complex of the two and P is the product. Each step 1) formation of ES and 2) formation of product goes forward, as well as backwards. The backwards direction increases over time (as the reaction approaches equilibrium). …show more content…
In order to make that correlation, you would need to measure a series of known dilutions of ortho-nitrophenolate, measure them with the spectrophotometer. the standard curve plotted Absorbance versus concentration for known amounts of ortho-nitrophenolate will be drawn. This will be useful in determining the rate of reaction for the Lactase enzyme in different conditions (amount of product/time = rate). D ifferent concentrations of different concentrations of ONPG is needed. At this point the amount of lactase activity in your stock is unknown, so it would be good to determine a proper concentration of lactase to, therefore a series of dilutions of this first stock will be made and tested. Make a set of 4 serial 1:10 dilutions and label them as 1:10; 1:100; 1:1,000 and 1:10,000. This will give you a set of stock enzyme solutions of different concentrations. After the working concentration of Lactase has been found, the two main measurements of enzyme kinetics need to be calculated: Vmax (maximum rate of reaction for this enzyme and this substrate under these conditions) and Km (the Michaelis constant, the concentration of substrate that gives one half Vmax – it’s an indirect measure of the affinity of the enzyme for the
Data from Table 1. confirms the theory that as the concentration of glucose increases so will the absorbance of the solution when examined with the glucose oxidase/horseradish peroxidase assay. Glucose within the context of this assay is determined by the amount of ferricyanide, determined by absornace, which is produced in a one to one ratio.1 Furthermore when examining the glucose standards, a linear calibration curve was able to be produced (shown as Figure 1). Noted the R2 value of the y = 1.808x - 0.0125 trend line is 0.9958, which is statistically considered linear. From this calibration curve the absorbance values of unknowns samples can be compared, and the correlated glucose concentration can then be approximated.
The purpose of this study is to analyze the activity of the enzyme, catalase, through our understanding
For example, incubating the samples at different temperatures would create more data points to establish an optimal temperature. From the results in the experiment in this study, it is known as temperature increases, enzymatic activity increase, and vise versa. However, what can not be observed is at what point does the increase in temperature begin to denature the enzyme, above 60°C. Furthermore, assays can be preformed to determine optimal pH, as well. From Dutta’s, and his partners, experiment it shows that there is a range where the Heliodiaptomus viduus’s lactase shows the most activity, which is between 5.0 and 6.0
These labels indicated the lactose solution that was be placed into the mini-microfuge tubes. The varying lactose ph solutions were obtained. The four miniature pipets were then used, (one per solution,) to add 1mL of the solution to the corresponding mini-microfuge tubes. When this step is completed there were two mini-microfuge tubes that matched the paper towel. Then, once all of the solutions contained their respective lactose solutions, 0.5mL of the lactase enzyme suspension was added to the first mini-microfuge tube labeled LPH4 on the paper towel, and 4 on the microfuge tube. As soon as the lactase enzyme suspension was added to the mini-microfuge tube, the timer was started in stopwatch mode (increasing.) When the timer reached 7 minutes and 30 seconds, the glucose test strip was dipped into the created solution in the mini-microfuge tube for 2 seconds (keep timer going, as the timer is also needed for the glucose strip. Once the two seconds had elapsed, the test strip was immediately removed, and the excess solution was wiped gently on the side of the mini-microfuge tube. The timer was continued for 30 addition seconds. Once the timer reached 7:32 (the extra two seconds accounting for the glucose dip), the test strip was then compared the glucose test strip color chart that is found on the side of the glucose test strip
Mader, S. S. (2010). Metabolism: Energy and Enzymes. In K. G. Lyle-Ippolito, & A. T. Storfer (Ed.), Inquiry into life (13th ed., pp. 105-107). Princeton, N.J: McGraw Hill.
The independent variable for this experiment is the enzyme concentration, and the range chosen is from 1% to 5% with the measurements of 1, 2, 4, and 5%. The dependant variable to be measured is the absorbance of the absorbance of the solution within a colorimeter, Equipments: Iodine solution: used to test for present of starch - Amylase solution - 1% starch solution - 1 pipette - 3 syringes - 8 test tubes – Stop clock - Water bath at 37oc - Distilled water- colorimeter Method: = == ==
Investigating the Effect of Substrate Concentration on Catalase Reaction. Planning -Aim : The aim of the experiment is to examine how the concentration of the substrate (Hydrogen Peroxide, H2O2) affects the rate of reaction. the enzyme (catalase).
Alkaline Phosphatase (APase) is an important enzyme in pre-diagnostic treatments making it an intensely studied enzyme. In order to fully understand the biochemical properties of enzymes, a kinetic explanation is essential. The kinetic assessment allows for a mechanism on how the enzyme functions. The experiment performed outlines the kinetic assessment for the purification of APase, which was purified in latter experiments through the lysis of E.coli’s bacterial cell wall. This kinetic experiment exploits the catalytic process of APase; APase catalyzes a hydrolysis reaction to produce an inorganic phosphate and alcohol via an intermediate complex.1 Using the Michaelis-Menton model for kinetic characteristics, the kinetic values of APase were found by evaluating the enzymatic rate using a paranitrophenyl phosphate (PNPP) substrate. This model uses an equation to describe enzymatic rates, by relating the
at a volume of 4cm3. The preliminary work also proved to me that my basic method worked without any setbacks that may affect my results. Variables:.. The variables involved in the rate of reaction between amylase and starch are. The volume of amylase The volume of starch
Investigating the Effect of Enzyme Concentration on the Hydrolysis of Starch with Amylase Aim: Investigate the effect of enzyme concentration on the rate of an enzyme-controlled reaction. Using amylase and starch as my example. Introduction: I am investigating the effect of the concentration of the enzyme, amylase on the time taken for the enzyme to fully breakdown the substrate, starch to a sugar solution. The varied variable will be the concentration and all other variables are going to be fixed. The different concentrations will be: 0.5% 0.75% 1.0% 1.5% 2% An enzyme is a class of protein, which acts as a biological catalyst to speed up the rate of reaction with its substrates.
Therefore, it is expected that the methyl meta-nitrobenzoate would be the product formed faster and in greater quantities because it has the more stable intermediate. Thin layer chromatography uses a solvent (in this case 85% hexane–15% ethyl acetate) to separate different products based on differences in polarity of the molecules. Typically more polar compounds will have more interaction with the stationary phase, and will not move as from the solvent front. This means that the less polar a substance is, the farther it will move. Using the mechanism of electrophilic benzylic substitution, it can be determined at where each step of the mechanism is occurring, and at what procedure it is occurring at.
I have a prediction for the investigation concerning the concentration of Rennin, another reason I chose this factor. Prediction I predict that as the concentration of rennin increases the rate of reaction will increase. I believe this to be true because if there are an increased number of enzymes, more milk particles will be broken up at any one time into the substance, which coagulates the milk,
Abstract: Enzymes are catalysts therefore we can state that they work to start a reaction or speed it up. The chemical transformed due to the enzyme (catalase) is known as the substrate. In this lab the chemical used was hydrogen peroxide because it can be broken down by catalase. The substrate in this lab would be hydrogen peroxide and the enzymes used will be catalase which is found in both potatoes and liver. This substrate will fill the active sites on the enzyme and the reaction will vary based on the concentration of both and the different factors in the experiment. Students placed either liver or potatoes in test tubes with the substrate and observed them at different temperatures as well as with different concentrations of the substrate. Upon reviewing observations, it can be concluded that liver contains the greater amount of catalase as its rates of reaction were greater than that of the potato.
an enzyme is used to speed up the process in the equation above. In my
Two spot plates were placed on a napkin that has Temperature 0˚, 25˚, 35˚, 50˚, 65˚, 90˚ Celsius. Three groups tested fungal amylase which is Alpha-amylase Aspergillus Oryzae and two groups tested mammal amylase which is Alpha-amylase from Porcine pancreas. Six test tubes were labeled with different temperature and enzyme source Mammal or Fungal Amylase. Add 2.5mL of 1% starch was added to each test tube. Afterwards, each test tube was placed into its respective temperatures. Another Six test tubes were obtained and were labeled with different temperatures and enzyme source Mammal or Fungal Amylase. Add 1ml of amylase Mammal or Fungal in each test tube and placed into its respective temperature. The test tubes were allowed to equilibrate for five minutes in their temperatures. After the calibration process, few drops of each solution were transferred from each test tube without removing the test tubes from their temperatures. These drops were added into the first row of wells on the spot plate, then add two drops of iodine to the wells on the spot plate and wait 1 minute. Use a color- coding scheme to convert the data to qualitative data into quantitative data this will serve as the control. Add 0.5 ml of amylase to the appropriate test tube containing starch and wait two minutes. Then add two drops of the starch-amylase mixture from each tube to the third row of