Beta Amylase Lab Report

The experiment calls for the extraction and purification of a protein. The protein chosen for the experiment was an amylase, specifically beta-amylase. Amylase protein can be typically found in plants and animals. The role of this protein is to hydrolyze organic materials, such as starch and dextrin, in order to form glucose, maltose, and limit dextrin. One distinction between beta amylase and alpha amylase is that beta amylase is not found in animal. It is found primarily in plants, bacteria, and fungi. In these types of organisms, beta amylase plays a huge role in its survival. It contributes to the hydrolysis of glycosidic bonds. Also, in plants like sweet potatoes (source of the experiment), the protein performs the hydrolysis of starch molecules and converts them into the products of beta maltose and limit dextrin [1]. These organic materials are essential in energy production in an organism. In order to obtain an optimal form of the protein in this experiment, it had to be purified. There were several methods used in order to purify beta amylase. The first purification step performed was the peeling, cutting, and blending of sweet potatoes. The blending of potatoes was done with 0.020M sodium phosphate buffer in order to make the homogenized mixture that can be used to easily extract the protein. Then the excess solids were removed through the use of cheesecloth, centrifugation, and the addition of ammonium sulfate “cuts”. After another session of centrifugation, the solution is undergoes dialysis for a couple of days with the buffer being changed a few times. The protein solution then undergoes through an increase in pH known as an acid treatment. It is followed by ultracentrifugation, which is longer than a session of centrifugation and involves creating a smaller volume for the high concentration of protein present in the solution. Afterwards, the protein was thoroughly purified through the use of Ion-Exchange Chromatography, based on its negative charge, and gel filtration, based on its molecular weight [2]. The methods used in this experiment were used to due to their impact on the protein. When finding a source of beta amylase, sweet potatoes were used because they were the more convenient and inexpensive option compared to sugar cane. It made it easier to make a homogeneous mixture and to centrifuge it. Then, when it came to fractioning the solubility of the solution after the first session of centrifugation, ammonium sulfate was used as a form of partial purification and it lead to the precipitation of some parts of the solution that are then separated through another session of centrifugation. The process of dialysis was used afterwards in order to normalize the amount of solution by regulating the level of salt. It was followed by the use of acid treatment, which is very important, because the enzyme would prefer an acidic environment. When the columns were performed, the types of chromatography chosen were Ion Exchange and gel-filtration. The ion exchange allowed for the proteins present in the solution to …show more content…
Like the sodium phosphate buffer, the solid was dissolved in 300 milliliters of distilled in a 500-mL beaker using a stirrer and a magnetic stirring bar. The pH of the solution was then checked using the pH meter and it was brought down to the desired pH of 4.8 using a couple of drops of glacial acetic acid. Then it was poured into a 500-mL graduated cylinder and brought up to a volume of 1000 milliliters using distilled water. The buffer was poured into a labeled glass bottle and stored in the fridge. Another sodium acetate buffer was made using the same …show more content…
Each fraction was prepared in an eppendorf tube and the solutions were added using a 100uL-1000uL micropipette and micropipette tips. The first was the blank solution that only contained 800 microliters of distilled water and 200 microliters of the BioRad protein assay reagent. The second fraction was made with 200 microliters of distilled water, 600 microliters of the BioRad protein assay reagent, and 200 microliters of Bovine serum albumin standard solution (BSA). The following fractions had the amount of water used decrease by 200 microliters and the amount of BSA solution used increase by 200 microliters. The amount of BioRad reagent used in each fraction was kept constant. Each fraction had a total volume of 1000 microliters or 1