Week 4 Lysozyme Extraction

Egg Lysosome in DNA Extraction

The topic is significant because of Lysozyme’s ability to break chemical bonds on outer walls of bacteria. It is important to learn more about lysozymes to understand why and how they break down chemical bonds on outer walls of bacteria. It is important to conduct research in this area so we can find ways to apply lysozymes ability to break down chemical bonds in other scientific fields.

·        Experiment- I will run a procedure of partial purification of lysozyme.

1.     For the partial purification of lysozyme, egg whites, carefully separated from the egg yolks, were diluted 3- or 3.3-fold with 0.05M NaCl solution.

2.     To precipitate the egg white proteins other than lysozyme, the pH of this mixture was set to 4.0 by carefully adding several drops of 1 N acetic acid and it was diluted with an equal volume of 40%(v/v) ethanol. After 30-minute incubation at room temperature in the presence of ethanol, the mixture is centrifuged at 15,000xg for 15 minutes at 4 degrees C: then the precipitates were discarded.

3.     The supernatants were analyzed for their lysozyme activity and protein content to determine if lysozyme extraction was successful.

4.     A DNA extraction is performed using a kit the epicenter QuickExtract Bacterial DNA Extraction Kit (QEB0905T) and E.Z.N.A. Bacterial DNA Kit from Omega bio-tek (D3350-00) as controls

5.      A DNA extraction is performed using both kits but replacing the kit provided lysozyme with the student prepared egg white (EW) lysozyme.

Name	I/D/C	Units	Comment
Eggs	C	-	-
Lysozyme	D	-	-
Partial Purification of Lysozyme Protocol	I		Refer to Protocol

Is it possible to extract lysozyme from egg whites? My hypothesis is that the extraction of lysozymes from egg whites will be possible. The results I may discover are that the purity of the lysozyme from egg white may not be as pure as we need for a DNA extraction.

References

·        Alderton, G., Ward, W.H., & Fevold, H.L. (1945). Isolation of Lysozyme From Egg White [PDF]. Journal of Biological Chemistry, (157), 43-58. Retrieved from http://www.jbc.org/content/157/1/43.full.pdf

·        Fakharany, E. E., & Hassan, M. (2016). A universal method for extraction of genomic DNA from various microorganisms using lysozyme. New Biotechnology, 33(1), S210. http://dx.doi.org/10.1016/j.nbt.2016.06.1445

·        Ganong, B. (2007, June 11). Isolation of Lysozyme from Egg White. Retrieved October 20, 2016, from Barry Ganong's Homepage website: http://faculty.mansfield.edu/bganong/biochemistry/lysozyme.htm

·        QuickExtract™ bacterial DNA extraction kit [pdf]. (2012, December). Retrieved from http://www.epibio.com/docs/default-source/protocols/quickextract-bacterial-dna-extraction-kit.pdf?sfvrsn=8

·        Xue-li, C., Ting, L., & Ito, Y. (2007). Separation of Chicken Egg-White Lysozyme by High-Speed Countercurrent Chromatography using a Reverse Micellar System. Journal of Liquid Chromatography & Related Technologies, 30(17), 2593-2603. http://dx.doi.org/10.1080/10826070701540555