From this Experiment we demonstrated the process of proteins purification by isolating lysomes from the egg white. Both Enzyme assays and spectrophotometry were used to determine purity of the lysozyme of the samples ( A,B and C) at each stage. In order to conduct this experiment, The experiment was divided into two session; The First session was to isolate and partially purify egg white lysozyme while the second session involved the assay of lysozyme activity and degree of purification.
“mucopeptide N-acetylmuramylhydrolyase.” is the systematic name for lysozyme. Lysozyme of egg white, was chosen to hydrolyze bacterial cell walls of Micrococcus lysodeikticus
Lysozyme catalyzes the hydrolysis of ? -1,4-linkages between N-acetylmuramic acid and 2-acetamide-2-deoxy-D-glucose residues in mucopolysaccharides or mucopeptides of a variety of microorganisms (“BC 367 Experiment 3 Purification And Characterization Of The Enzyme Lysozyme”).
Generally, Lysozyme has a lower molecular mass of about 14.6 kDa, at neutral pH. Lysozyme surface is dominated by its basic side chain therefore it bears a net positive charge therefore; considerable purification can be achieved by simple gel- filtration, which separates proteins on the basis of size.
The first filtrate known as EWF(egg white filtrate) was obtained and labeled as “A” 5mL of the filtrate was kept at 40C to maintain active enzyme. Further determination of enzyme activity and protein estimation were carried out. After calculating the protein sample A was found to be 40.39mg
Sample B was prepared by mixing 4.0mL of EWF, buffer solution and Amberlite together and swirled gently on ice. Amberlite is a cation exchanger. It was then homogenate and filtrated out the sample and labelled as “Sample B”. protein of higher PI(PI>8.0) was removed by rinsing the amberlite with a NaCL of PH 8.0.
Ater we calculated the protein of sample B it was found to be 28.208 mg. That shwed a significance difference relative to Sample A, sample B was much lower than sample A which makes sense.
Sample C Sample C was prepared by filtration with Amberlite(Ion-exchange chromatography) from previous sample. Proteins with lower pI (pI>4.5) were removed by rinsing the sample with citrate buffer at pH 4.5. The total protein of sample C was found to be 1.62 mg. This sample was stored at 4 ? to be used in second session
This Second session was carried out to determine the assay of lysozyme activity and degree of purification. The assay method is based on observing changes in turbidity of a cell suspension. This is because active lysozymes hydrolyze cell walls of
Micrococcus lysodeikticus. An appropriate rate is a decrease in (lamda symbol)=450nm at room temperature.
This is a spectrophotometric assay. A continuous assay was carried out to measure the enzyme activity in which the absorbance of the cell wall suspension was measured after every 0.5 minutes up to 6.0 minutes. From the results obtained, it was observed that absorbance decreased per time. This shows that the Lysozyme was present in active form and was able to hydrolyze the cell walls of Micrococcus lysodeikticus.
The total enzyme activity of proteins after each purification steps was calculated. The total activity of crude egg white lysosyme “A” was 390 U and after the first and the second purification steps, the total enzyme activity of “B” and “C” significantly reduced to 306 U and 180 U respectively.
In additional to, protein assay was employed too to figure out the total proteins, specific enzyme activity, fold purification and overall yield of desired protein from enzyme concentration. A protein standard curve was plotted to determine the concentration of enzyme in sample A, B and C.
The final protein concentrations for the samples A, B, and C were 3.226 mg/ml, 0.656 mg/ml and 0.538 mg/ml respectively. The total protein decreased after each step of purification. Furthermore, the specific activity and fold purification of enzyme increased. In contrast to the expected results, our sample B enzyme activity was slightly lower 17, than Sample C 18; that was as a result of the buffer we used in the suction part did not discard all, causing the volume to be quite high which significantly contributed to the low concentration obtained. Some proteins lost in the procedure, during the suction pump not all proteins could pass through and collected as filtrate.
The overall yield of protein was was found to be 46.15% which is quite acceptable.