The Effect of Varying Temperature and pH on enzymatic activity of α-amylase Introduction
An enzyme is a biological molecule which acts as a catalyst for chemical reactions without being altered in the process. Enzymes have a defined three-dimensional globular structure composed of approximately 100-500 amino acids. The first enzyme to be discovered was diastase which catalyses the breakdown of starch into maltose by the French chemist Anselme Payen in 1833.
An enzyme substrate is the material which interacts with an enzyme. An enzymatic reaction occurs if there is a substrate present and if the active site of the enzyme is empty, generally an enzyme will only react and bind to a specific substrate or group of substrates. Enzymes are not
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If an enzymes active site is altered it cannot function and is therefore denatured.
The objective of this experiment is to analyse the effectiveness of alpha-amylase reacting with 1% Starch and NaCl under varying temperatures and pH. The effectiveness of the alpha-amylase is measured by recording the amount of starch present during varying stages of the reaction.
Method
The experiment was carried out in two parts, reaction rate verses temperature and reaction rate verses pH. For reaction rate verses temperature five test tubes were labelled with temperatures, E 0°C, E RT, E 37°C, E 60°C and E 100°C for the enzyme alpha-amylase. Another five test tubes were also labelled with the temperatures S 0ºC, S RT, S 37ºC, S 60ºC and S 100ºC for the substrate 1% Starch and NaCl.
A pipette was used to move 2ml of the substrate into each of the corresponding five test tubes and 2ml of buffer solution, pH 6.8, was added into the five substrate test tubes (S 0ºC, S RT, S 37ºC, S 60ºC and S 100ºC). The same amount of enzyme was transferred into its corresponding test tubes (E 0°C, E RT, E 37°C, E 60°C and E 100°C). The corresponding enzyme and substrate test tubes were equilibrated to their corresponding temperatures. Both the test tubes E 0°c and S 0°c were cooled down to 0°c using a beaker of ice and a thermometer. The room temperature test tubes were already
Enzymes are known as protein catalysts. The name protein catalyst suggests that most enzymes are made of proteins. A catalyst is a substance that speeds up chemical reactions without being consumed in the process. (Giuseppe, M 2002, p.69). After a reaction has been catalyzed, the catalyst can be used again to catalyze the same reaction. Enzymes reduce the activation energy (minimal energy) it takes for a reaction to take place. Enzymes can either catabolize (destroy), or anabolize (build up) a chemical system.
In this lab or experiment, the aim was to determine the following factors of enzymes: (1) the effects of enzymes concentration the catalytic rate or the rate of the reaction, (2) the effects of pH on a particular enzyme, an enzyme known and referred throughout this experiment as ALP (alkaline phosphate enzyme) and lastly (3) the effects of various temperatures on the reaction or catalytic rate. Throughout the experiment 8 separate cuvettes and tubes are mixed with various solutions (labeled as tables 1,3 & 4 in the apparatus/materials sections of the lab) and tested for the effects of the factors mentioned above (concentration, pH and temperature). The tubes labeled 1-4 are tested for pH with pH paper and by spectrophotometer, cuvettes 1a-4a was tested for concentration and cuvettes labeled 1b-4b was tested for temperature in four different atmospheric conditions (4ºC, 23ºC, 32ºC and 60ºC) to see how the enzyme solution was affected by the various conditions. After carrying out the procedures the results showed that the experiment followed the theory for the most part, which is that all the factors work best at its optimum level. So, the optimum pH that the enzymes reacted at was a pH of 7 (neutral), the optimum temperature that the reactions occurs with the enzymes is a temperature of 4ºC or
The use of multiple test tubes and Parafilm was used for each experiment. Catechol, potato juice, pH 7 phosphate buffer, and stock potato extract 1:1 will be used to conduct the following experiments: temperature effect on enzyme activity, the effect of pH on enzyme action, the effect of enzyme concentration, and the effect of substrate concentration on enzyme activity. For the temperature effect on enzyme activity, three test tube were filled with three ml of pH 7 phosphate buffer and each test tube was labels 1.5 degrees Celsius, 20 °C, and 60 °C. The first test tube was placed in an ice-water bath, the second test tube was left at room temperature, and the third test tube was placed in approximately 60°C of warm water. After filling the test tubes with three ml of the
Amylase experiment # 2 was done to see how the pH affected the efficacy of the enzyme. First we collected all of the materials that were necessary to make this experiment. We needed five clean test tubes, the following standard solutions, 1% Starch Solution pH 3,1% Starch Solution pH 5,1% Starch Solution pH 7,1% Starch Solution pH 9,1% Starch Solution pH 11
Enzymes are biological catalysts that speed up chemical reactions, without being used up or changed. Catalase is a globular protein molecule that is found in all living cells. A globular protein is a protein with its molecules curled up into a 'ball' shape. All enzymes have an active site. This is where another molecule(s) can bind with the enzyme. This molecule is known as the substrate. When the substrate binds with the enzyme, a product is produced. Enzymes are specific to their substrate, because the shape of their active site will only fit the shape of their substrate. It is said that the substrate is complimentary to their substrate.
The purpose of this experiment was to determine (1) the reaction rate of an amylase enzyme in starch and (2) the environmental factors that can affect the enzymatic activity. The hypothesis, in relation to the enzymatic activity by variables such as the substrate concentrations, temperature, PH and chemical interactions on the rate of reaction, stated
The purpose of this experiment was to record catalase enzyme activity with different temperatures and substrate concentrations. It was hypothesized that, until all active sites were bound, as the substrate concentration increased, the reaction rate would increase. The first experiment consisted of five different substrate concentrations, 0.8%, 0.4%, 0.2%, 0.1%, and 0% H2O2. The second experiment was completed using 0.8% substrate concentration and four different temperatures of enzymes ranging from cold to boiled. It was hypothesized that as the temperature increased, the reaction rate would increase. This would occur until the enzyme was denatured. The results from the two experiments show that the more substrate concentration,
An experiment was performed to test how temperature variations affect enzymatic activity of the enzyme amylase. The results of the experiment will also determine the optimal temperature of the amylase enzyme. The results of the experiment provide evidence for determining the environments that the enzyme amylase would most likely be present. By determining the possible environments, one can predict what and how environmental factors will affect the enzyme amylase. Two forms of amylase (Bacterial - Bacillus licheniformis and Fungal - Aspergyllus oryzae) were combined with starch molecules at four different temperatures (0⁰, 25⁰, 65⁰, 85⁰ Celsius). The combination of starch and the amylase enzyme resulted in a visual chemical reaction that was recorded. The enzyme activity was recorded every two minutes, starting at 0 and ending at 10. The start time 0 served as the control group of the experiment. The results concluded that both bacterial and fungal amylase has an optimal temperature around 65⁰C. This was possible to determine by recording the color change of the spot plate wells. Amylase catalyzes efficiently at its optimal temperature which resulted in yellow spot plate wells. Enzymatic activity decreased when the temperature was less than 65⁰C, resulting in a green-brown well. The green wells indicated that starch wasn’t broken down completely and was still present. Temperatures greater than 65⁰C resulted dark-green wells which resembled the denaturing of
Enzyme = any one of many specialized organic substances, composed of polymers of amino acids, that act as catalysts to regulate the speed of the many chemical reactions involved in the metabolism of living organisms. (Microsoft® Encarta® Encyclopedia '99.)
Enzymes are a key aspect in our everyday life and are a key to sustaining life. They are biological catalysts that help speed up the rate of reactions. They do this by lowering the activation energy of chemical reactions (Biology Department, 2011).
During these experimental procedures, the implication of multiple different temperatures on fungal and bacterial amylase was studied. In order to conduct this experiment, there were four different temperatures used. The four temperatures used were the following: 0 degrees Celsius, 25 degrees Celsius, 55 degrees Celsius, and 80 degrees Celsius - Each temperature for one fungal and one bacterial amylase. Drops of iodine were then placed in order to measure the effectiveness of the enzyme. This method is produced as the starch test. The enzyme was tested over the course of ten minutes to determine if starch hydrolysis stemmed. An effective enzyme would indicate a color variation between blue/black to a more yellowish color towards the end of the time intervals, whereas a not so effective enzyme would produce little to no change in color variation. According to the experiment, both the fungal amylase and bacterial amylase exhibited a optimal temperature. This was discovered by observing during which temperature and time period produced a yellow-like color the quickest. Amylase shared a similar optimal temperature of 55 degrees Celsius. Most of the amylases underwent changes at different points, but some enzymes displayed no effectiveness at all. Both amylases displayed this inactivity at 0 degrees Celsius. At 80 Celsius both the enzymes became denatured due to the high temperatures. In culmination, both fungal and bacterial amylase presented a array of change during it’s
This experiment consisted of setting up a control group of starch in various temperature and then placing both fungal amylases and bacterial amylases in a mixture of starch and placing the solution of amylase and starch in various temperatures of water. After a certain amount of time- different amount of time needs to be used in order to have reliable results- iodine is added in a well on spot plates, then two drops of the mixture of amylase-starch is added from each temperature used, by adding iodine into the plates the mixture will show how much starch was hydrolyzed, this is used to calculate the amount of
An enzyme is a protein that makes a chemical reaction happen without being structurally changed depending on its environment. Yeast was used because it has an enzyme in it called catalase that breaks down the hydrogen peroxide with a product of water and oxygen gas.(1) This happens when the enzyme connects with a substrate molecule. A Substrate molecule has what is called an active
There are thousands of chemical reactions that occur in an organism that make life possible. Most of these chemical reactions occur too slowly on their own. Enzymes are protein catalysts that speed up chemical reactions in a cell. Catalysts are not changed by the reactions they control, and are not used up during the reaction. Enzymes therefore, can be used over and over again. Enzymes are large complex proteins made by the cell and allow chemical reactions to take place at the temperature of the cell. These catalysts are needed in only very small amounts because a single enzyme molecule can complete the same reaction thousands of times in one minute.
In this lab our group observed the role of pancreatic amylase in the digestion of starch and the optimum temperature and pH that affects this enzyme. Enzymes are located inside of cells that increase the rate of a chemical reaction (Cooper, 2000). Most enzymes function in a narrow range of pH between 5 through 9 (Won-Park, Zipp, 2000). The temperature for which enzymes can function is limited as well ranging from 0 degrees Celsius (melting point) to 100 degrees Celsius (boiling point)(Won-Park, Zipp, 2000). When the temperature varies in range it can affect the enzyme either by affecting the constant of the reaction rate or by thermal denturization of the particular enzyme (Won-Park, Zipp, 2000). In this lab in particular the enzyme, which was of concern, was pancreatic amylase. This type of amylase comes from and is secreted from the pancreas to digest starch to break it down into a more simple form called maltose. Maltose is a disaccharide composed of two monosaccharides of glucose. The presence of glucose in our experiment can be identified by Benedicts solution, which shows that the reducing of sugars has taken place. If positive the solution will turn into a murky reddish color, where if it is negative it will stay clear in our reaction. We can also test if no reduction of sugars takes place by an iodine test. If starch is present the test will show a dark black color (Ophardt, 2003).