Determining Optimum Temperature and Ph for Enzymatic Reactions of Alpha Amylase

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

Used to see if the temperature of the water is at 37oc – 40oc and if

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

Using the yellow tube, which included everything but starch, as the blank, each group zeroed their spectrophotometer. This was done so that any absorbance observed depends only on the amount of starch present, not on any other reagents (buffer, IKI). To zero the spectrophotometer, the wavelength was first set at 580nm, using knob 3 (45). Next, the groups made sure that the light next to “transmittance” was lit, and the chamber to be tightly closed. Having the chamber empty & closed tightly provides reference for the darkest condition possible. Using knob 1, the transmittance was turned until it read 0.0 (45). Before the groups used their blank test tube to zero the spectrophotometer, each needed to wipe the tube with kimwipes to ensure a clean reading. Turning knob 2, each group was then instructed to zero the absorbance, 0.000. Upon removing the blank, each trial was inserted into the chamber (46). The

amylase enzyme and the optimal temperature for fungal and bacterial amylase. In order to make

In part II of the lab six small glass tubes were obtained in a test tube rack. Ten drops of distilled water were then added to test tube 1, five drops to tubes 2-4, and no drops in tubes 5 and 6. Five drops of 0.1M HCl were added to test tube 5 and five drops of 0.1M NaOH to test tube 6. Five drops of enzyme were then added to all tubes except tube 1. Tube 3 was then placed in the ice bucket and tube 4 was placed in the hot bucket at 80-900C for five minutes, the remaining tubes were left in the test tube rack. After the five minutes five drops of 1% starch was added to every tube and left to sit for ten minutes. After ten minutes five drops of DNSA were then added to all the tubes. All the tubes were then taken and placed in the

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

3. The spectrophotometer was set at 420nm. Distilled water was also used as the ‘blank’.

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

For this experiment, we have to prepare our phosphorylase which extracted from a potato. We prepared by weighed about 250 grams of peeled potato and cut it into cubes. The extracts then blended with 100mL of 0.1M NaF. After filtered the contents into a clean 250mL centrifuge bottle, we centrifuged it for 3 minutes. Then, separated the supernatant into a centrifuge bottle, which is our phosphorylase preparation. The enzyme assay used in this experiment today is the iodine test. As the iodine reacts with starch, it will form a brown, blue or black precipitate due to the iodine ions forcing into a linear arrangement. The endpoint of the enzyme reaction indicates the presence of starch by using the iodine test to determine. The faster the endpoint is reached, the less active the phosphorylase is.

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

The effects of temperature on fungal amylase Aspergillus oryzae, and bacterial amylase, Bacillus licheniformis ability to break down starch into maltose was studied. The study determined the optimal temperature the Aspergillus oryzae and Bacillus licheniformis was able to break down the fastest. The starch catalysis was monitored by an Iodine test, a substance that turns blue-black in the presence of starch. Amylase catabolizes starch polymers into smaller subunits. Most organisms use the saccharide as a food source and to store energy (Lab Manual, 51). The test tubes were labeled with a different temperature (0°C, 25°C, 55°C, 85°C). Each test tube was placed in its respective water baths for five minutes. After the equilibration process, starch was placed in the first row of the first row of the spot plate. Iodine was then added to the row revealing a blue black color. The starch was then added to the amylase. After every two minute section a pipette was used to transfer the starch-amylase solution to place three drops of the solution into the spot plate row under the corresponding temperature. Iodine drops was placed in the row. Color changes were noted and recorded. The results showed Aspergillus oryzae was found to have an optimal temperature between 25°C and 55°C and Bacillus licheniformis was found to have an

This experiment was performed to demonstrate how varying temperatures affects the activity of the enzyme, amylase. Also, it was conducted to determine the optimal temperature for the fungal, Aspergillus oryzae, and bacterial amylases by placing them both into different test tubes with differing temperature conditions. At varying time intervals, both enzymes were removed from their assigned temperatures. A drop of each enzyme will then be placed on two different spot plates with a pipette. All the wells on the spot plates contain three drops of iodine to clearly show whether a reaction has taken place. The pipette was used to mix the iodine with the starch enzyme to organize the levels of starch catabolism based on temperature. At the end of

All biological processes require a constant supply of energy. This energy is produced through multiple chemical reactions that occur in cells and are controlled by biological catalysts called enzymes. Enzymes increase the rate of reactions by reducing the activation energy required to start a reaction. What effects an enzyme whether it is in a positive or negative way? How does temperature effect an enzyme? This leads to the question: Is there a correlation between temperature and enzyme efficiency? In order to answer this

Enzymes are proteins that act as catalysts and help reactions take place. In short, enzymes reduce the energy needed for a reaction to take place, permitting a reaction to take place more easily. Some enzymes are shape specific and reduce the energy for certain reactions. Enzymes have unique folds of the amino acid chain which result in specifically shaped active sites (Frankova Fry 2013). When substrates fit in the active site of an enzyme, then it is able to catalyze the reaction. Enzyme activity is affected by the concentrations of the enzymes and substrate present (Worthington 2010). As the incidence of enzyme increases, the rate of reaction increases. Additionally, as the incidence of substrate increases so does the rate of reaction.