Alkaline phosphatase, or AP, is an enzyme that is responsible for dephosphorylation, or removing phosphate groups, of various types of molecules such as proteins and nucleotides. It has a molecular weight of 140-160 kilo-Daltons and as said in the name, alkaline phosphatase works in an environment with pH values ranging from 7.5-9.5, which is an alkaline, or basic, environment. And according to Sigma-Aldrich, alkaline phosphatase can be used to “to dephosphorylate the 5'-termini of DNA or RNA to prevent self-ligation” (sigmaaldrich.com). Alkaline phosphatase in gram-negative bacteria can be found in the periplasmic space, which is found outside of the cell membrane. Because of this, it is subject to various types of environmental activity, making alkaline phosphatase more resistant to being denatured, inactivated, and degraded, while also giving AP a higher rate of activity.
Every cell has a plasma membrane, which is a protein-lipid bilayer. It forms a barrier that separates the cell contents from the outside environment. Amphipathic lipids make up the plasma membrane, meaning they have hydrophilic and hydrophobic parts. The hydrophilic heads and hydrophobic tails form a “sheet” containing lipids whose hydrophobic tails face each other. In order to obtain something from inside the cell, or in this case alkaline phosphatase, one could use the process of cell lysis. Cell lysis is a process that breaks down or destroys a cell wall due to an outside force. Cell lysis can occur both naturally and artificially. Natural lysis can occur through viral infections whereas artificial lysis can occur by adding a substance that can break down a cell wall.
One such substance that can cause artificial cell lysis is lysozyme. Lysozyme, or N-acetyl...
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...tions collected, a spot test can be used. The spot test is a qualitative determination to see if a molecule exists in solution. For this spot test, para-nitrophenylphosphate, PNPP, in solution was used to determine fractions from IEC had AP or not. PNPP was used specifically because when PNPP comes into contact with AP in basic conditions, a phosphate from PNPP is cleaved off, becoming para-nitrophenol, PNP. PNP turns yellow in basic conditions. If AP was in a fragment, the yellow tint shown would indicate such.
The experiment was performed using mutated E. coli, cell lysis, two centrifugations, two dialysis processes, heat denaturation, salting out via ammonium sulfate, anion exchange chromatography, and spot testing. By following these procedures, one should be able to obtain a lot of purified alkaline phosphatase and should see a yellow tint during the spot test.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
The isolate possesses some enzymes required for hydrolytic reactions. Hydrolytic enzymes found to be secreted from the bacterium, are -amylase, casein, and PYRase. In the starch hydrolysis and casein tests, there was a zone of clearing around the bacterium, which was indicative of the secreted enzymes necessary to break down starch and casein. In the PYR test, the presence of PYRase was detected by a color change to red on the PYR disc after the addition of the PYR reagent (p-dimethylaminocinnamaldehyde). Hydrolytic enzymes for which the EI tested negative were urease, gelatinase, and DNAse. In the Urea Hydrolysis test, it was observed that the urea broth did not have a color change, indicating that there was no urease secreted to break down urea in the broth. Similarly, there was no gelatinase present to break down gelatin in the Gelatin Hydrolysis test, so the nutrient gelatin remained solid. It was concluded that the EI does not possess DNase because there was no clearing zone around the bacteria, indicating that DNA had not been
The control for both curves was the beaker with 0% concentration of substrate, which produced no enzyme activity, as there were no substrate molecules for...
While the tube for specimen Cb turned a tannish white in the lower half of the tube while the top stayed the lavender inoculated tube color. Do to this evidence I determined that both specimens Ca and Cb cannot use the process Casein hydrolysis or Casein coagulation due to lack of soft or firm curds in both tubes. Since there was no casein curds formed, I concluded that specimens Ca and Cb also cannot perform the process of proteolysis. My conclusion is supported by the fact that there was no clearing of the medium. I have also determine that neither of my organisms can make the enzymes rennin, proteolytic or even proteases. I know my specimens cannot produce proteases due to the fact that there was no blue coloring in the tubes which means that the byproduct Ammonia was not produced to increase the pH. Since neither of my specimens can make these enzymes, I concluded that my specimens cannot break down lactose or casein. Although I did learn that specimen Cb can reduce litmus due to the evidence that the lower part of the tube turned a tannish white color with a purple ring at the top. This color change from a purple to a white means that the litmus was reduced turning it clear and leaving the white of the milk to show. Finally I know that specimen Ca cannot reduce litmus due to the fact that the tube had no change in
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
The beet Lab experiment was tested to examine bio-membranes and the amount of betacyanin extracted from the beets. The betacyanin is a reddish color because it transmits wavelengths in red color and absorbs most other colors. The membrane is composed of a phospholipid bilayer with proteins embedded in it. The phospholipid bilayer forms a barrier that is impermeable to many substances like large hydrophilic molecules. The cells of beets are red and have large vacuoles that play a big role for the reddish pigment. This experiment aimed to answer the question, “How do cell membranes work?” The hypothesis we aim to test is: Cell membranes work as a fluid mosaic bilayer of phospholipids with many embedded proteins. We predicted that the 50% Acetone will break down the most betacyanin. Our hypothesis was proven wrong by our data collected. We could test our predictions by doing the experiment multiple times and compare the
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
...on dioxide, within the body, affecting the pH balance of the blood. This will then affect proteins within the body, being known as enzymes, which can only function if their surrounding environment is in balance. Any alteration to this environment, will prevent the enzymes from functioning effectively.
In life, it is critical to understand what substances can permeate the cell membrane. This is important because the substances that are able to permeate the cell membrane can be necessary for the cell to function. Likewise, it is important to have a semi-permeable membrane in the cell due to the fact that it can help guard against harmful items that want to enter the cell. In addition, it is critical to understand how water moves through the cell through osmosis because if solute concentration is unregulated, net osmosis can occur outside or inside the cell, causing issues such as plasmolysis and cytolysis. The plasma membrane of a cell can be modeled various ways, but dialysis tubing is especially helpful to model what substances will diffuse or be transported out of a cell membrane. The experiment seeks to expose what substances would be permeable to the cell membrane through the use of dialysis tubing, starch, glucose, salt, and various solute indicators. However, before analyzing which of the solutes (starch, glucose, and salt) is likely to pass through the membrane, it is critical to understand how the dialysis tubing compares to the cell membrane.
With combined testing, APHIS can precisely track the quantity of specimens tried by State of cause. This guarantees satisfactory testing to meet individual state observation prerequisites under present PRV program gauges. To minimize expenses, tests in overabundance of State observation prerequisites are not tried.
From looking at the results I can conclude that when the pH was 3 and 5. No oxygen was produced, therefore no reactions were taking place. This was because the pH had a high hydrogen ion content, which caused the breaking of the ionic bonds that hold the tertiary structure of the enzyme in place of the syringe. The enzyme lost its functional shape.
Purpose: The purpose of this lab is to explore the different factors which effect enzyme activity and the rates of reaction, such as particle size and temperature.
The Effect of pH on the Activity of Catalase Planning Experimental Work Secondary Resources Catalase is a type of enzyme found in different types of foods such as potatoes, apples and livers. It speeds up the disintegration of hydrogen peroxide into water because of the molecule of hydrogen peroxide (H2O2) but it remains unchanged at the end of the reaction.
Investigate the Effect of pH on Immobilised Yeast Cells on the Breakdown of Hydrogen Peroxide
Introduction: Purifying proteins is an important part of biology because it can help identify the function of that protein. Once a protein’s function has been identified, it can be manipulated to see how the function would change if the protein was changed. A common way to purify a protein is through Ion Exchange Chromatography, which is where charged proteins will bind to the beads in the column to purify it from the solution (Berg JM, 2002). The purpose of this experiment is to use Ion Exchange Chromatography to purify cellulase.