Chemistry Research

Abstract:

Lyzozyme is an enzyme responsible for the hydrolysis of β-1,4-glycosidic linkage of the peptidoglycan in the cell wall of bacteria. Due to its antibacterial action, lysozyme is commonly used in food processing and also has some clinical uses. Lysozyme comes from many plant and animal sources, but is especially plentiful in hen egg whites. In this project, lysozyme was extracted from hen egg whites using ion-exchange chromatography. Buffer solutions ranging from pH 5.0 to 11.0 were used to determine what pH is optimal for the extraction of lysozyme using this method. Absorbance assays with Micrococcus lysodeikticus cell walls along with Michaelis-Menton enzyme kinetics allowed for measuring lysozyme concentrations in the filtrate.

Introduction:

Egg white consists of many proteins such as ovalbumin, conalbumin, ovomucin and lysozyme. Of these major proteins, lysozyme is the least abundant making up approximately 3.5% of egg white's protein (Gosh et al. 2000). Lysozyme is an enzyme with antibacterial properties. It has the ability to hydrolyze the β-1, 4-glycosidic linkage of the peptidoglycan in the cell wall of bacteria (Jiang et al. 2001, Li-Chan et al. 1986). The peptidoglycan layer consists of amino acids as well as sugars. Specifically, the lysozyme cleaves between the N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues. Figure 1 is a diagram of this action.

Gram-positive bacteria have a very thick peptidoglycan layer while gram-negative bacteria have a very thin peptidoglycan layer (Prescott et al. 1999). For this reason, lysozyme works mainly on gram-positive bacteria.

Being an antibacterial enzyme, lysozyme is commercially very valuable. It has many uses in food processing such as in food packaging films and in the preservation of meats such as sausages. The dairy industry relies on lysozyme as well as it is used in processing cheeses and milk. The pharmaceutical industry utilizes lysozyme as a drug to treat ulcers and infections as well as in ophthalmologic preparations (Ghosh et al. 2000, Jiang et al. 2001, Grasselli et al. 1999).

With all of these commercial uses, it is necessary to have adequate methods for the extraction of lysozyme from natural sources. The enzyme is commonly found in hen egg whites as well as in bodily secretions such as saliva, tears and urine. As stated before, lysozyme makes up only 3.5% of hen egg white's protein content. This very low content creates challenges in the purification process. It is necessary for large amounts of material to be processed to get small amounts of lysozyme (Ghosh et al. 2000). Old methods of retrieving lysozyme from hen egg whites involved direct crystallization from egg white by adding an NaCl solution at pH 9.5. One of the major problems with this procedure is that it destroys the remaining egg white making it impossible to be used for some other purpose. As an alternative to this method, scientists have moved to using other methods such as ultrafiltration, ion exchangers or affinity chromatography (Li-Chan et al.1986).

For this project, ion-exchange chromatography was used to extract lysozyme as it is a technique well adapted for a college laboratory. A method similar to that described by Li-Chan et al. (1986) was employed to determine what pH provides the optimum conditions for the extraction of lysozyme. Analysis of how much lysozyme was actually extracted was done using Michaelis-Menten enzyme kinetics and Micrococcus lysodeikticus cells. As Shugar (1952) reported, continuous monitoring of the change in absorbance in a spectrophotometer at 450nm of a solution containing Micrococcus lysodeikticus cells and lysozyme provides enough information to determine how much lysozyme is present.

Materials:

Grade A, large, white eggs, purchased at a local grocery store

freeze-dried lysozyme, purchased from Sigma Chemical Co.

freeze-dried Micrococcus lysodeikticus cells, purchased from Sigma Chemical

Amberlite IRC-50 resin beads, purchased from Sigma Chemical Co.

KH2PO4 and K2HPO4, purchased from Fisher Scientific

pH meter

sonicator

stirring plates

vacuum filtration supplies

micropipette

disposable pipettes

balance

Varian DMS 100S UV/Visible Spectrophotometer

spectrometric tubes

glass pipette and pipette bulb

Methods:

Before the extraction procedure can begin, several buffer solutions must be prepared. 0.5M solutions of K₂HPO₄ and KH₂PO₄ were made and mixed together to reach the desired pH. A stock solution of 0.5M K₂HPO₄ was made by adding 114.115g to a 1000mL volumetric flask and diluting up to the mark with distilled water. A stock solution of 0.5M KH₂PO₄ was prepared by adding 68.04g to a 1000 mL volumetric flask and once again diluting up to the mark with distilled water. The pH6.5 buffer used for rinsing off the excess egg white was prepared by adding approximately 100mL of KH₂PO₄ to 650mL of K₂HPO₄ and adjusting the pH while monitoring with a digital pH meter. The other solutions with differing pH used in the extraction were prepared by mixing the two stock solutions and adjusting the pH while monitoring the pH with a digital pH meter.

0.1M solutions of K₂HPO₄ and KH₂PO₄ were also needed to prepare a pH 7.0 buffer for use in the standard curve assay. 1.36g of KH₂PO₄ and 1.75g of K₂HPO₄ were added to two separate 100mL flasks and dissolved in distilled water. The 7.0 buffer was made by mixing approximately equal parts of the two stock solutions together and monitoring with a pH meter.

After the buffer solutions were prepared, the standard curve assay was completed. A stock lysozyme solution was made by adding 100mg of freeze dried lysozyme to a 100mL volumetric flask and dissolving in distilled water. A stock solution of Micrococcus lysodeikticus cells was prepared by combining 9 mg of cells with 30mL of the 0.1M pH7.0 phosphate buffer. Various amounts of M. lysodeikticus cells and pH7.0 buffer were added to the spectrometric tubes. The tube was placed in the spectrometer set at 450 nm and the absorbance was set at 1.000. At time 0, the desired amount of lysozyme was added to the tube and the tube was shaken for a few seconds. The tube was then immediately place back in the spectrophotometer and the absorbance was read every 15 seconds for a period of two minutes. The amounts of each solution added to each tube are shown in Table 1.

The initial rate, V_o, could have been determined by plotting the absorbances versus time and determining the slope of the line. Since only the initial rate is of importance, the graphical method was not used to determine V_o, rather the change in absorbance between 0 and 15 seconds was divided by the change in time, 15 seconds. The result of this mathematical equation is V_o. The V_o values for each spectrometric tube were then used in a plot of 1/V_o versus 1/concentration of lysozyme. The slope of this standard curve was used to determine the amount of lysozyme extracted from the egg whites.

The first step in the extraction procedure was to separate the egg white from the yolk and the shell, place it in a 250mL beaker and record the mass of the egg yolk. The beaker was then placed in a sonicator for 30 minutes. 7.5g of Amberlite resin beads were then added to the beaker and the egg white/resin mixture was placed on a magnetic stirring plate for one hour. The excess egg white was then rinsed from the resin beads three times with approximately 50mL portions of 0.5M pH 6.5 phosphate buffer solution. A disposable pipette was used to remove any remaining egg white and buffer from the resin. 10mL of buffer solution at the experimental pH was then added to the resin and allowed to set for 15 minutes. After this time period, the buffer/lysozyme solution was removed from the resin using vacuum filtration. Analysis of the amount of lysozyme was conducted as within minutes after the extraction procedure.

To determine the amount of lysozyme, 9mg of M. lysodeikticus were added to 30mL of 0.1M pH7.0 phosphate buffer solution and 2.8mL of this solution was placed in the spectrometric tube. The tube was then placed in the spectrophotometer set at 450nm and the absorbance was set at 1.000. 200mL of lysozyme solution was then added to the tube at time 0, shaken and placed back in the spectrophotometer. Absorbance readings were taken every 15 seconds for two minutes. The procedure was repeated three times for each extraction. V_o was calculated as described above and the amount of lysozyme was determined using the standard curve. This amount of lysozyme is for 200mL. To find the total amount extracted, it is necessary to use a ratio to determine the amount of lysozyme in 10mL (the amount of buffer added).

Results:

The V_o values for the known concentrations of lysozyme were calculated as stated in the methods section and the results are shown in Table 2.

The actual standard curve is shown in Figure 2.

The equation of the line is y = 7334.6x - 6.7986. Rearranging this equation and substituting 1/V_o for Y and 1/mg for X, results in equation 1.

The average V_o value for each extraction was used in equation 1 to obtain the amount of lysozyme extracted. Table 3 shows the results of using this equation as well as the amount of lysozyme in the entire egg white.

Figure 3 gives a graphical representation of the average amounts of lysozyme extracted.

Each egg white had a different mass, and since the amount of lysozyme in each egg white is proportional to it's mass, it is necessary to compare the amounts of lysozyme extracted in terms of percentages. The average percentage of lysozyme extracted is shown in Figure 4.

From the above results, one can conclude that the optimum pH for the extraction of lysozyme from egg whites using ion-exchange chromatography is between pH 7.0 to pH 10.0, the median being approximately 8.5. Between pH 5.0 and pH 11.0, a definite parabola is seen in the graph. The optimum conditions are in the middle and the pH values on either side show that much less functional lysozyme was extracted at these extreme pH values. It is important to note, however, that none of the percentages, even under optimum conditions, are very high.

Discussion:

The isoelectric point, pI, of a molecule is the pH at which that molecule is neutral. At a pH higher than the pI, the molecule has an overall negative charge while at a pH lower than the pI, the molecule is positively charged (Boyer 2000). The isoelectric point of lysozyme is about 11.0. Many other proteins make up egg whites as well and most of their isoelectric points are less than 6.0 (Jiang et al 2001, Lui et al 2000, Grasselli et al 1999, Ghosh et al 2000, Ghosh and Cui 2000). This being the case, the results obtained in this study are more understandable. At any pH less than 11.0, lysozyme carries an overall positive charge, thus binds to the negatively charged resin. At a pH less than 6.0, the other proteins in the egg white become positively charged as well. This becomes a problem in the extraction of lysozyme as now the other proteins are "sticking" to the resin as well as the lysozyme. As a result, the lysozyme yields decrease considerably. Another possible explanation for the decrease in lysozyme amount at low pH is that under acidic conditions proteins are not very stable and tend to denature.

There are many other aspects, other than pH, that can be altered in this procedure. At the beginning of this project there were hopes of being able to experiment with different resins to see if something worked better than the Amberlite IRC-50 resin. Unfortunately, due to time constraints this was not possible. Another idea for future work would be to follow the same procedure using the entire pH range. One would expect that virtually no lysozyme would be obtained under extremely basic or extremely acidic conditions due to reasons stated above.

The procedure for the extraction of lysozyme using ion-exchange chromatography is one that could very easily be incorporated into the biochemistry course taught at Heidelberg College. There are several basic techniques used such as Michaelis-Menten kinetics, and ion-exchange chromatography that are very important for biochemistry students to know and understand. During one lab period students could create the standard curve using known concentrations of lysozyme. A second lab period could be used for extraction and analysis.

Work with lysozyme is constantly being completed because of how commercially useful it is. One current study is interested in enabling lysozyme to work on gram-negative bacteria as well as those that are gram-positive. Lipophilization of the enzyme, making the protein hydrophobic and liposoluble, enables it to penetrate through the outer membrane of the gram-negative bacteria and reach its peptidoglycan layer (Liu et al. 2000). Experimentation with this technique as well as many others continue and will do so until the many questions about the production and applications of lysozyme are answered.

Boyer, Rodney. Modern Experimental Biochemistry, 3rd ed. Benjamin Cummings: New York 2000.

Ghosh, Raja; Silva, Saliya Sudarshana; Cui,Zhanfeng. Biochemical Engineering Journal 2000, 6, 19-24.

Ghosh, R.; Cui, Z.F. Biotechnology and Bioengineering 2000, 68, 191-203.

Grasselli, Mariano; Camperi, Silvia A.; Navarro del Canizo, Agustin A.; Cascone, Oscaldo. Journal of the Science of Food and Agriculture 1999, 79, 333-339.

Jiang; Wang; Chang; Chang. Journal of Food Science 2001, 66, 1089-1092.

Li-Chan; Nakai; Bragg; Lo. Journal of Food Science 1986, 51, 1032-1036.

Lui; Azakami; Kato. Nahrung 2000, 44, 407-410.

Prescott, Lansing M., John P. Harley, and Donald A. Klein. Microbiology, 4th ed. McGraw-Hill: Boston, MA 1999.

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Last modified: 9 January 2003. SEK.