pH and temperature amylase activity from fungal and mammal
Introduction
The first enzyme that was produced industrially is amylase from a fungal source in 1894, it was used to treat digestive disorders. Amylase are groups of enzyme that breaks down starch into sugar and starts the process of chemical digestion. Its primary function is to digest enzymes and its optimum pH is 7. Amylase is measured by mixing a substrate with a buffer and measuring the change of the mixture. The reason why we measure amylase is to assist in diagnosis of different diseases such as abdominal pain (Reynolds, 2009). Starch breakdown of amylase has received a great deal of attention because of their technological significance and economic benefits, and is also
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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 …show more content…
Two spot plates were placed on a napkin that has pH 3, 4, 5, 6, 7, 8. 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 pH and enzyme source Mammal or Fungal Amylase. Add 1.5ml of starch and 1ml of the pH buffer solution to each test tube. Another six test tubes were labeled with different pH and enzyme source Mammal or Fungal Amylase. Add 0.5ml of amylase into the test tubes, and 0.5ml of pH buffer to each test tube. Allow the tubes to equilibrate for five minutes, then transfer a few drops of the starch solution from each pH treatment add them into the first row of wells on the spot plate. This serves as a control to make sure that the breakdown of starch is not affected by pH. Then add two drops of iodine reagent to each of these wells and wait one minute, then add 0.5ml of amylase to each of the appropriate tubes. After two minutes, add a few drops from each tube, and place in the corresponding wall. After collecting the data use a color coding scheme to convert the qualitative data to quantitative data. The numerical data from each group was used to calculate the mean starch concentration and the standard deviation. The mean and the standard deviation values were calculated after entering all the data into
To begin the study, I first calculated how much of each solution I would need. I knew that the final volume of my reaction solution needed to me 30ml, so I calculated how much of starch, amylase, and tris buffer I would need. I used the formula Concentration (initial stock solution) x Volume (initial stock solution)= Concentration (final solution) x Volume (final solution). Using this formula, I found that I would need an initial concentration of 21 ml of starch, 1 ml of amylase, and 8 ml of the tris buffer. After calculating the amounts of substances I would need, I created two different solutions, one with the Carb Cutter and one without. Carb Cutter claims to block starch, however, to find this I needed to test the absorbance level of the control to compare the effect Carb Cutter had on the solution. Below is a graph showing the concentration of the control reaction over one minute intervals through the
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
For the heat inactivation, two sets of 11 tubes were set up. The indicated amounts of buffer, water, and ONPG listed in table 10 were added to each tube. In addition, the control enzyme (0.1ml) was added to each tube of the control set and the same amount of heated enzyme was added to each tube of the heated set. The absorbance readings were taken and recorded in table 10. Finally, two Lineweaver-Burk plots were created. The plot for the heated set is represented by graph 10 and graph 11 represents the control set. The Km and the Vmax for the heated set and the control set were determined.
Iodine is used to distinguish starch from mono/di/polysaccharides. In the test only two of the six solutions showed any signs of starch being present. The potato juice turned a brownish color with a precipitate indicating a slight presence of starch. The stach solution turned a dark blue/black color indicating a very high presence of starch in the solution. The presence of starch in the potato juice indicates that it does not have as high an amount of carbohydrates as onion juice, but a higher concentration of starch.
However, the decrease varied depending on the temperature. The lowest temperature, 4 degrees Celsius, experienced a very low decrease of amylose percentage. Temperature at 22 degrees Celsius and 37 degrees Celsius, both had a drastic decrease in amylose percentage. While the highest temperature, 70 degrees Celsius, experienced an increase of amylose percentage. In conclusion, as the temperature increases the percentage of amylose decreases; however, if the temperature gets too high the percentage of amylose will begin to increase. The percentage of amylose increases at high temperatures because there is less enzyme activity at high temperatures. However, when the temperature is lower, more enzyme activity will be present, which results in the decrease of amylose percentage. This is why there is a decrease of amylose percentage in 4, 22, and 37 degrees Celsius. In this experiment the optimal temperature is 37 degrees Celsius, this is because this is the average human body temperature. Therefore, amylase works better at temperatures it is familiar
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.
Enzymes react differently under different conditions and concentrations, being the most productive at the enzymes specific optimum condition and concentration. The enzyme sucrase, extracted from yeast, breaks down the complex sugar sucrose into the simple sugar glucose. Testing for sucrase’s optimum environment, multiple reactions were ran using varying amounts and concentrations of sucrose and sucrase at different pHs and temperatures. The product was then treated with Benedicts solution to visually observe what amount of glucose was present after the reaction was ran; negative results being little to no glucose present and positive results being glucose present. The varying levels of colors created by the Benedicts test were recorded as 1 (negative) blue; 2 green; 3 yellow; 4 (positive) orange; and 5 (positive) red. After running the reactions to determine the optimum temperature and pH, 37 degrees Celsius was determined to be the optimum temperature while pH 2.0 was determined to be the optimum pH. This means that under these conditions the enzyme will help produce the most product from the substrate. After finding the optimum conditions the two different concentrations of sucrose were ran for ten minuets taking an initial sample and then subsequent samples every minuet, under those conditions, one concentration at 2.5% sucrose and again at 10% sucrose. The 10% sucrose had the most positive results because there were sufficient active sites to bind with most of the sucrose present producing glucose more efficiently than the 2.5% sucrose. As with the 2.5% sucrose all of the sucrose was bond to the active sites of sucrase and there were ...
Affect of the Rate of Reaction of Amylase on Starch and How Its Affected by the Concentration of the Substrate
...the lactase concerning the pH level. We hypothesized that enzyme functions are under the influence of environmental factors, in this particular experiment it is pH. In the results, the test tubes with the pH of 4 and 7, yielded color changes with the pH of 7 having the most mg/dL. The results proved our hypothesis, we were right to conclude that because the pH was in between the pH in which the lactase is found, that it would have yielded a reaction. The reason why the pH of 7 yielded the most is because that is the the closets to the pH of the small intestine where the enzyme lactase is found. The significance of the results is that the optimal pH for the biochemical reaction to occur is at the pH of 7 and it would yield the best breakdown of lactose. This fits the bigger picture because it shows that everything must be in balance in order for a successful reaction.
Test-tube C had the best concentration according to the results. Three test-tubes were labelled A-C. Test-tube A had 1ml enzyme solution which was added to test-tube B which had 4ml buffer (pH 5 was used). 1ml of the solution from test-tube B was then added to the test-tube C which also had 4ml buffer (pH 5). Test-tube C was used as the enzyme in all the reactions. Nine test-tubes were taken out of them one was used as the the blank, labelled as test-tube 9. The blank had 5ml buffer (pH 5), 2ml hydrogen peroxide, 1ml guaiacol and no enzyme. Then, 3ml of buffer (pH 3) and 2ml of enzyme were added to test-tube 1. Test-tube 2 had 2ml hydrogen peroxide and 1ml guaiacol. Test-tube 1 and 2 were mixed. The same procedure was used for test-tube 3 as test-tube 1, but this time the buffer was pH 5. Test-tube 4 was prepared the same way as test-tube 2. Then, Test-tube 3 and 4 were mixed. Test-tube 5 was prepared as test-tube 1 but with buffer of pH 7 and test-tube 6 was prepared as test-tube 2. Next, test-tube 5 and 6 were mixed. Last but not the least, test-tube 7 was prepared as test-tube 1 but with buffer of pH 9 and test-tube 8 was prepared as test-tube 2. Then, test-tube 7 and 8 were mixed. The spectrophotometer was set to 470nm and using the blank it was set to zero. The four test-tubes with different pH’s (pH 3, pH 5, pH 7, pH 9) were read
The hypothesis is supported by the experiment in that with increased starch concentration, the amylase activity increased each time and the salivary amylase functioned best at higher concentrations of starch. Also, for the most part, the reaction followed the general trend of how at lower concentrations, the increase in reaction rate is greater, while at higher concentrations, the increase in reaction rate is less. Despite some discrepancies in trend, specifically at the 40g/L concentration, figure 1 still displays how the amylase activity eventually reaches a plateau, as mentioned in the hypothesis. From the results it appears that the point of saturation for this reaction is at the concentration 50g/L as the amylase activity rates for 50g/L
As a group the slope of the glucose line was recorded as 59.001 ppm/min. The class average for glucose was conducted from numbers that were varied in size. The lowest number being 3.0136 ppm/min and the highest being 1026.2 ppm/min. The class average was calculated as 471.201 ppm/min for the respiration rate of yeast when metabolizing glucose. Using class averages from multiple periods the result of 679.48 ppm /min was collected as the overall average. Moreover, the collection of data for sucrose was also compressed into averages. The slope of the line for sucrose was recorded as 280.3 ppm/min for a group collection. The data of sucrose included numbers starting at -873.51 ppm/min and ended with 1522.8 ppm/min being the highest. The class average recorded for the respiration rate of yeast when metabolizing sucrose was 649.246 ppm/min. The overall average of sucrose was 575.686 ppm/min. Also, the data for starch and lactose were also calculated into class averages and overall averages. As a group the slope of the lactose line was recorded 155.69 ppm/min, and the slope of the starch line was recorded as 367.34 ppm/min. The class average calculated for lactose was 214.183 ppm/min while the average for starch was 389.439 (units). Lastly, the overall averages were collected for lactose and starch. The overall average of lactose
The purpose of the experiment conducted is to understand the role of enzymes in maintaining life and to be able to identify and explain various factors that affect enzyme functions. Make sure to be wearing lab appropriate clothing, a lab coat, and safety goggles at all times since this experiment involves you handling dangerous chemicals like hydroxylamine. For this experiment one of the main materials needed is a spectrophotometer. The use of the spectrophotometer is very important in this experiment. You will test three concentrations of enzyme (0.5 ml, 1.0 ml, and 2.0 ml of turnip extract) and three concentrations of substrate (0.1 ml, 0.2 ml, and 0.4 ml hydrogen peroxide). You always need to make sure you have a control, the control in this experiment is the turnip extract and the color reagent guaiacol. Increasing the temperature increases the rate of an enzyme reaction, decreasing the temperature decreases the rate of an enzyme reaction. Denaturation is a process in which proteins or nucleic acids lose the quaternary structure, tertiary structure and secondary structure. Hydroxylamine is a colorless inorganic compound and an odorless white crystalline solid.
Without enzymes, reactions wouldn’t occur and living organisms would die. For instance, the enzyme in the stomach breaks down large molecules to smaller molecules to absorb nutrition faster. Researchers experimented with enzyme activity with a potato extract. Researchers will test enzyme activity by increasing and decreasing pH levels, lowering and increasing temperature, and substrate concentration effects. In the first experiment, researchers hypothesized whether different pH levels would change how much Benzoquinone are created and how will the enzymes function in neutral pH levels than higher and lower levels. Researchers used potato extract and different levels of pH to test their hypothesis. In addition, researchers questioned at what temperature does the greatest amount of potato extract enzyme activity take place in. Researchers then hypothesized that the results would indicate the greatest amount of potato enzyme activity level will take place in room temperature. In this experiment, researchers used potato extract and different temperature levels to test the hypothesis. Moreover, researchers wanted to test the color intensity scale and how specific catechol oxidase is for catechol. In this experiment, researchers used dH2O, catechol solution, hydroquinone, and potato extract. Lastly, researchers tested the substrate concentration and how it has an effect on enzyme activity. In this experiment researchers used different measurements of catechol and 1cm of potato extract. Researchers hypothesized that the increase o substrate would level out the enzyme activity
Purpose: This lab gives the idea about the enzyme. We will do two different experiments. Enzyme is a protein that made of strings of amino acids and it is helping to produce chemical reactions in the quickest way. In the first experiment, we are testing water, sucrose solution, salt solution, and hydrogen peroxide to see which can increase the bubbles. So we can understand that enzyme producing chemical reactions in the speed. In the second experiment, we are using temperature of room, boiling water, refrigerator, and freezer to see what will effect the enzyme.