Isolation and Purification of Lysozyme from Egg Whites: An Immune Response Ashlynn LaFlamme, Katie Carey, and Natalie Needy Department of Biology and Environmental Science, Westminster College, Fulton, MO, 65251

Abstract Reaction Mechanism of Lysozyme Results Lysozyme is a natural antimicrobial enzyme found in a wide variety of organismal • The absorbance rate for trial 2 was fastest, with a reaction velocity of immune responses. Its function is to catalyze the destruction of bacteria cell walls. This -0.0256 OD/Min. study aims to isolate and purify lysozyme from hen egg whites while maintaining the • Trial 1 had the slowest absorbance rate, with a reaction velocity of -0.0143 enzyme activity. The lysozyme was purified and isolated from egg white homogenate. Our OD/Min (Figure 2). data were analyzed by Microsoft Excel to determine how much enzyme activity was found in our isolated samples. It was concluded from our extraction and purification that we • The pure lysozyme absorbance rate was -0.0338 OD/Min (Figure 1). This is achieved isolating an extract lysozyme sample which had a 27.6% slower reaction velocity the experimental standard and represents 100% enzyme activity level. when compared to the purified lysozyme in assay. It is also noted that when the volume of • Trial 1 retained the least about of lysozyme activity, with only 42.31% of the egg extract in assay was halved, the absorbance rate decreased by 56.6%. There was no enzyme activity level (Figure 2). concluded significance in our findings, however. Further research, purification, and Figure 1. The mechanism of Micrococcus lysodeikticus cell lysing with a lysozyme catalyst. The • Trial 2 lysozyme activity was highest, with 75.74% of the total enzyme experimentation will need to be conducted on enzyme activity and the characterization of process of lysozyme degradation where hydrolyzation in the peptidoglycan layer of bacterial cell activity (Figure 2). lysozyme to achieve full isolation. membranes breaks the beta-glycosidic linkage between N-acetylmuramic acid and N-acetyl glucosamine. • The pure lysozyme assay sample was calculated to contain an concentration of 4.662x10-5 mol/L. Introduction Lysozyme plays two vital roles. The first includes protection to mammalian Experimental Enzyme Activity of Pure Lysozyme Experimental Enzyme Activity of Initial Purification and invertebrate bodies. In order to maintain proper health, lysozyme degradation of Discussion Initial Purification of Egg White Extract Assay In order to determine the activity of our experimental egg white lysozyme the cell membrane of gram-positive bacteria must occur. This process occurs due to 0.04 0.06 Pure Lysozyme Assay hydrolyzation of the beta-glycosidic linkage between N-acetylmuramic acid and N- isolation, we compared our results to the pure lysozyme absorbance velocity, 0.04 acetyl glucosamine in the peptidoglycan layer of bacterial cell membranes (Figure 1), which was our standard concentration assay (Figure 2). Only the concentration of -4 0 0.5 1 1.5 2 2.5 3 3.5 4 0.02 pure lysozyme in the assay was determined, and had a 6.993x10 M. The sample which is the natural substrate for lysozyme. (Arnheim et al. 1972). Lysozyme can be -0.01 which had the fastest absorbance rate was trial 2, which used 0.2mL of egg white found in tears, blood, mucus, human milk, and egg whites and is a common immune 0 0 1 2 3 4 5 6 7 isolated lysozyme (Figure 3). The sample in trial 1 had the slowest absorbance rate response. The second is the essential role in medical and biochemistry research. The -0.02 (Figure 3). When compared to the pure lysozyme, trial 2 was 27.6% slower and structure and characterization of lysozyme are consistent under a variety of -0.06 trial 1 was 81.0% slower (Figure 4). Trial 1 used half as much egg extract conditions (Strynadka and James 1991). Lysozyme can also be used commercially as -0.04 lysozyme as trial 2 and the reaction velocity of trial 1 was reduced by 56.6% a food preservative because it inhibits the growth of bacteria which can prolong shelf -0.06 y = -0.0143x + 0.0139 -0.11 compared to trial 2. life. It is also researched for its use in pharmaceuticals and can be used as a Absorbance at 450nm (OD) -0.08 Absorbance Absorbance at 450nm (OD) Since the substrate concentration was kept constant throughout at 0.30g/L of potentiating agent for antibiotics (Proctor and Cunningham, 1988). Due to the suspended Micrococcus lysodeikticus in buffer and water, we can assume that the stability of lysozyme, it is one of the most researched enzymes. -0.1 -0.16 y = -0.0256x + 0.0497 quantity of enzyme is directly proportional to the velocity of the reaction. We can The characterization of lysozyme is well known. The average molecular -0.12 also assume that the percent activity is directly related to the amount of lysozyme weight is 14,300 Daltons and it is located in the cell membrane (Alderton, 1944). The y = -0.0338x - 0.056 -0.14 in the samples since this was the only variable altered. This allows us to correlate structure is a singular polypeptide chain consisting of 129 amino acids with Trial 1 Trial 2 Time (minute) -0.21 the decrease in enzyme activity with the decrease of lysozyme concentration additional no subunits (Berg, 2019). The thermal stability occurs at a pH of 5.0 and Time (minute) added to the assay. This explains why trial 1 was much slower and expressed less the isoelectric pH range is between 10.5 pH and 11.0 pH (Alderton, 1944). The Figure 2. The rate of absorbance (optical density at 450nm) per minute for Figure 3. The rates of absorbance (optical density at 450nm) per minute for enzyme activity than trial 2. Lysozyme only has one subunit, therefore it cannot active site has the architecture of a deep crevice with two domains, one for beta-sheet the reaction of 100% pure lysozyme. This curve is the standard for our the egg white isolated lysozyme catalyzed assay reactions . Trial 1 assayed bind to more than one substrate at a time. The increase of egg white extract structures and one for helical structures, linked by an alpha helix (Strynadka and experiment. It represents the rate at which pure lysozyme facilitates the substrate 0.1mL of isolated lysozyme, and trial 2 assayed 0.2mL of isolated lysozyme. The lysozyme was the only variable altered, indicating that a higher concentration of James, 1991). Micrococcus lysodeikticus in lysing the cell walls of the bacterium in a buffer two slopes represent the reaction rate of lysing Micrococcus lysodeikticus cells for lysozyme in trial 2, under the conditions of our assay, was more effective at This characterization information allows for a beginning knowledge of the solution. each reaction. catalyzing the reaction than trial 1. The increase of enzyme activity compared to correct buffer pH, dialysis bag, and various equipment and methods needed to begin the increase of lysozyme concentration can be seen in figure 3 and appears to be isolating and purifying lysozyme from hen egg whites to achieve the maximum Enzyme Activity Results directly related. I would suggest further testing of various isolated enzyme activity of lysozyme. concentrations in order to determine which yields the greatest amount of activity Lysozyme Activity vs. Lysozyme Concentration Pure Lysozyme and calculating the significance of these findings. Purification Method 1 In order to determine if there is a significant comparison between Trial 2 our isolated lysozyme and the purified lysozyme, further purification methods Prepare 1.5mL of 2% bentonite 0.8 are needed. These methods would include dialysis, gel chromatography, suspension in 1% KCl pH 4.0 0.6 and lyophilization. Further purification would promote higher activity levels of Trial 1 0.4 the enzyme. In addition to purification, a Bradford test is needed to determine the exact protein concentration of lysozyme. This concentration of lysozyme Add 100mL of egg white and 0.2 stir by hand for 5 minutes Activity (OD/Minute) % can be compared to the purified lysozyme. Throughout the isolation 0 0.00E+00 5.00E-06 1.00E-05 1.50E-05 2.00E-05 2.50E-05 3.00E-05 3.50E-05 4.00E-05 4.50E-05 5.00E-05 and purification process, it is important to note that lysozyme can be inhibited

Lysozyme Concentration (M) by surface-active reagents, such as dodecyl sulfate, alcohols and fatty acids. It is also essential to store the enzyme in its optimal conditions, such as Centrifuge with 4,000g Figure 4. The percent of lysozyme activity graphed over the concentration of for 20 minutes as 4ºC maintaining the pH and temperature. Keeping the enzyme in its optimal conditions lysozyme using reaction velocity for all three assay samples. Each point represents will prevent the enzyme from degradation, thus inhibiting activity. the activity and concentration of lysozyme in the reaction, representing pure lysozyme, Pour off excess liquid trial 2, and trial 1 values. The amount of enzyme present positively correlates directly References with the percent activity of the reaction. 1. Alderton G, Ward WH, Fevold HL. 1944 Oct 22. Isolation of lysozyme from egg white. The Western Regional Research Laboratory:44–58. [cited 2020 Feb Wash clay with 0.5M Assay Assay Procedure 2]. https://www.jbc.org/content/157/1/43.full.pdf?sid=3a75259b-7e00-41f3-bf4b- K3(PO4) pH 7.0 5ddbcbd4f402 Lysozyme 2. Berg JM. Biochemistry. 8th ed. New York: Macmillan International Higher Education; 2019. Micrococcus lysodeikticus Cells (Intact) → Micrococcus lysodeikticus Cells (Lysed) Preparation for procedure: Dekina SS, Romanovska II, Ovsepyan AM, Bodyul MG, Toptikov VA,. 2015 Dec 1. Wash clay with 30mL of 5% Directory of Open Access Journals. Biotechnologia Acta. [cited 2020 Feb Active Enzyme Suspend Micrococcus 11]. https://doaj.org/article/d0ed5907a3ad4a3c80d3ae6842de43fc pyridine pH 5. Repeat 3x. Stock Solutions Blank Stir solution and keep Test Sample lysodeikticus Dilute to a final 3. Shugar D. 1952. The measurement of lysozyme activity and the ultra-violet inactivation in an ice bucket and of lysozyme. Biochimica et Biophysica Acta 8:302–309. lyophilized cell in volume of 30mL keep covered until 4. Worthington V. 1993. Worthington enzyme manual: enzymes and related biochemicals. Centrifuge with 4,000g Buffer 25mL of K (PO ) pH with K (PO ) pH 7.0 3 4 3 4 added to the assay New Jersey: Worthington Biochemical Corporation. p250-253. for 20 minutes as 4ºC 0.1M Potassium Phosphate buffer at pH 0.5mL 0.3mL 7.0 5. Strynadka, N., and James, M.: Lysozyme Revisited - Crystallographic Evidence for 6.94 Distortion of an N-Acetylmuramic Acid Residue Bound in Site-D, J Mol Biol 220, 401, 1991 Assay Procedure: 6. Arnheim N. et al., Chemical studies on the Enzymatic Specificity of Goose Egg White Add (NH₄)₂SO₄ to obtain a Substrate Suspension Lysozyme, JBC Vol. 248, No 1 pp. 233-236 (1973) 1. Set spectrophotometer-20 to 450 nm and 25ºC. concentration of 2.6M 9.0mg of Micrococcus lysodeikticus cells, 2.5mL 2.5mL 7. Oliver WT, Wells JE. 2015 Aug 13. Lysozyme as an alternative to growth promoting antibiotics in swine production. Journal of animal science and biotechnology. [cited 2020 Mar 25mL buffer, and 5mL H O 2. Pipette 2.5 ml of Micrococcus lysodeikticus lyophilized cell suspension into 2 a cuvette and incubate for 4-5 minutes to achieve temperature equilibrium. 10]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535397/ Centrifuge with 4,000g 8. Proctor VA, Cunningham FE. 1988. The chemistry of lysozyme and its use as a food Lysozyme 3. Prepare the blank sample by pipetting 0.5 ml of buffer solution to the cuvette for 20 minutes as 4ºC preservative and a pharmaceutical. Critical reviews in food science and nutrition. [accessed 0.002g of pure lysozyme per 200mL H2O or 0.0mL 0.2mL to give a total volume of 3.0 ml. 2020 Mar 8]. https://www.ncbi.nlm.nih.gov/pubmed/3280250 0.1mL of egg extract lysozyme 4. Zero spectrophotmoeter-20 using the prepared buffer-substrate blank. Acknowledgements Collect supernatant 5. Once ready to record enzyme activity, add 0.1 mL of diluted enzyme to the Table 1. The contents of the table organize and describe what materials the assay We would like to thank the Westminster College Biology and cuvette and immediately begin recording the absorbance. consists of. It lists the stock solutions, concentrations, and volumes used in each Chemistry departments, and more specifically, Dr. Johanna Morrow for her Follow assay procedure to solution and test sample. The reaction which lysozyme facilitates during the assay 6. Record the absorbance value in 15 second intervals for the first 120 seconds, invaluable help and knowledge during this arduous process. We would also like assess lysozyme activity is also included, which lowers the activation energy to lyse Micrococcus then in 30 second intervals until the reaction has reached 5 minutes. to thank Alana Funk for managing lab supplies and materials, as well as assisting lysodeikticus cells. us in our waste processing and disposal.