Experiment 5

TITLE:

Isolation and partial purification of egg white lysozyme.

OBJECTIVE(s):

 To learn purification technique and to determine the lysozyme enzyme specific activity.

APPARATUS AND MATERIALS:

Egg white, vacuum pump, cheese cloth, glass wool, glass funnel, ice cold 0.01M Tris buffer in
0.02 M NaCl (pH 8.0), measuring cylinder, Amberlite, buchner funnel, three universal bottle,
filter paper, 0.5M NaCl/0.1M citrate (pH 4.5), pipette, Micrococcus lysodekticus (M.luteus)
lyophilized powder (0.3mg dry cell/mL), BSA (1 mg/ml), Distilled water:Bradford reagent
(3:2), 0.01M Tris buffer in 0.02 M NaCl.

METHODOLOGY:

Session 1:

1. The separated egg white was poured into a beaker using two layers of cheese cloth
cover in filter funnel.
2. The volume of this filtrate was recorded and cold 0.01M Tris buffer in 0.02M NaCl
(pH 8.0) was added in ratio 1:4.
3. The mixture was then swirl well for 20 minutes and then, the mixture was then pass
through the filter funnel plugged with glass wool.
4. The overall volume of the filtrate (EWF), was then measured and 5 mL of the overall
filtrate was pipette out and labeled as ‘Sample A’. This sample was kept at 4ºC for the
determination of enzyme activity and estimation of protein.
5. 0.054 g of Amberlite was mixed with 4.0 mL of EWF containing with 20 mL 0.01M
Tris buffer in 0.02M NaCl (pH 8.0).
6. The mixture is then mixed well on ice of 10 minutes.
7. After that, the mixure was filtered and applied with vacuum in buchner funnel.
8. After the first time filtration in Buchner funnel, the filter paper containing Amberlite
residue was rinsed with 5 ml cold 0.01M Tris/0.02M NaCl buffer (pH 8.0) and filtered
again in Buchner funnel. (THIS STEP WAS REPEATED TWICE)
 9. The combined volume of the filtrates was measured and 5 mL of the filtrate was
pipetted out and store in a flask at 4ºC labeled “Sample B”.
10. The filter paper from the third filtration which still contain some of the Amberlite
residue was rinse with 5 ml of 0.5M NaCl/0.1M citrate.
11. After swirling for five minutes, the mixture is again filtered in clean Buchner funnel.
12. This filtration is done twice and the combined volume of the citrate filtrate was
measured and 5 mL of the overall volume was pipetted out and label as “Sample C”
and store at 4ºC.

Session 2:

I. Assay of lysozyme activity and degree of purification

1. The stock suspension of M.lysodeikticus cells (it consists of 0.3mg/mL of 0.1M
NaCl/0.05M citrate buffer, pH 4.5) was diluted in the ratio 1: 10 with 0.1M NaCl/0.05M
citrate (pH 4.5)
2. The mixture was then mixed well and 0.1 mL of sample was added in and the
absorbance was taken at 450nm for 6 minutes at 30seconds intervals.
3. The same procedure was done for all three sample; Sample A, Sample B and Sample
C.

II. Protein Assay

TUBES BSA(1mg/ml), Distilled water, Sample, µL Bradford

µL µL reagent, mL

1 0 50 - 1.5

2 10 40 - 1.5

3 20 30 - 1.5

4 30 20 - 1.5
 5 40 10 - 1.5

6 50 0 - 1.5

Sample A - - 50 1.5

Sample B - - 50 1.5

Sample C - - 50 1.5

1. A standard curve was prepared using six samples which contain 0 to 50 µg of BSA
mixed with distilled water which the total volume was brought to 50 µL.
2. Sample A was diluted in 10x dilution, while, Sample B was diluted in 5x dilution and
sample C was not diluted.
3. Then, to all cuvettes 1.5 mL of Bradford reagent was added and mixed well and let to
stay for 10 minutes at room temperature before absorbance reading was taken at 595nm.

RESULTS AND CALCULATIONS:

Volume of egg white = 6.0 mL

Weight of egg white = 10.3 g

Volume of egg white filtrate = 17.0 mL

Weight of Amberlites = 0.05 g

Volume of sample B before 5 mL is taken = 11 mL

Volume of sample C before 5 mL is taken = 9 mL

ENZYME ASSAYS:

Table 1: The absorbance reading at 30 seconds intervals for six minutes

Time (minutes) Absorbance at 450 nm

A B C

0 0.704 0.680 0.660

0.5 0.704 0.683 0.664

1.0 0.705 0.683 0.665

1.5 0.704 0.682 0.662

2.0 0.704 0.682 0.662

2.5 0.703 0.682 0.661

3.0 0.704 0.681 0.662

3.5 0.706 0.680 0.663

4.0 0.704 0.681 0.663

4.5 0.705 0.681 0.663

5.0 0.704 0.682 0.662

5.5 0.703 0.681 0.662

6.0 0.704 0.682 0.661

Table 2: The best fit line equation, R2 value and Unit of Activity in U/mL for sample A, B and
C.

Sample Equation R2 Abs/min = One Unit Enzyme Enzyme

value Gradient activity Activity
(min-1) (U/0.1 (U/mL)
mL)

A y= -0.000033x + 0.0064 -0.000033 -0.001 0.033 0.33

0.7043

B y= -0.000099x + 0.0396 -0.000099 -0.001 0.099 0.99

0.6818

C y= -0.00099x + 0.0214 -0.000099 -0.001 0.099 0.99

0.6626
Since one Unit (U) is equal to a decrease in turbidity of 0.001 per minutes at 450 nm, then the
Abs/min is divided by -0.001 min-1.

Enzyme activity calculation,

Sample A

Abs/min = -0.000033 min-1

Enzyme activity = -0.000033 min-1 / -0.00100 min-1

=0.033 U in 0.1 mL of Sample A

= 0.033 U × 10

= 0.33 U/mL

Sample B

Abs/min = -0.000099 min-1

Enzyme activity = -0.000099 min-1 / -0.00100 min-1

=0.099 U in 0.1 mL of Sample B

= 0.099 U × 10

= 0.99 U/mL

Sample C

Abs/min = -0.000099 min-1

Enzyme activity = -0.000099 min-1 / -0.00100 min-1

=0.099 U in 0.1 mL of Sample C

= 0.099 U × 10

= 0.99 U/mL
Protein assays

Protein concentration (mg/ml) was calculated by using this formula:

𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝐵𝑆𝐴
Protein concentration (mg/ml) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒

For example, tube 6

𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝐵𝑆𝐴
Protein concentration (mg/ml) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒

0.05 𝑚𝑔
= 0.05𝑚𝑙

= 1.0 mg/ml

Protein concentration of tube 1 to 5 were calculated by the same method and the data was
constructed as Table 1

Tube Protein concentration (mg/ml) Absorbance

1 0.0 0
2 0.2 0.401
3 0.4 0.414
4 0.6 0.511
5 0.8 0.633
6 1.0 0.713
A - 0.794
B - 0.722
C - 0.145
Table 1 shows the protein concentration (mg/ml) and the absorbance of tube 1 to C
 Graph of Absorbance at 595nm against Protein Concentration (mg/ml)
0.9

0.8
0.713
0.7 0.633 y = 0.8054x
0.6
0.511
Absorbance

0.5
0.401 0.414
0.4

0.3

0.2

0.1
0
0
0 0.2 0.4 0.6 0.8 1 1.2
Protein concentration (mg/ml)

Graph 1 shows the absorbance at 595nm against the protein concentration (mg/ml)

Based on Graph 1, the best fit equation, y=0.8054x was obtained which used to calculate the
protein concentration of Tube A, B and C

Tube A: y= 0.8054x Tube B: y= 0.8054x Tube C: y= 0.8054x

0.794 = 0.8054x 0.722= 0.8054x 0.145= 0.8054x

x= 0.986mg/ml x= 0.896mg/ml x= 0.180mg/ml

Due to the dilution factor, the final protein concentration (mg/ml) of Tube A, B and C were
calculated and the data was constructed as Table 2

Tube Protein concentration (mg/ml) Dilution factor Final protein

concentration (mg/ml)
A 0.986 10× 9.86
B 0.896 5× 4.48
C 0.180 0 0.18
Table 2 shows the dilution factor and final protein concentration (mg/ml) of Tube A, B and C.

In order to calculate the specific activity (U/mg), the value of enzyme activity (U/ml) was used
to calculate the total activity (U) and total protein (mg).

Tube A

Total volume = 17 ml, Enzyme Activity = 0.33 U/ml

𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒(𝑚𝑙)
Total Activity (U) =
𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝑈 /𝑚𝑙)

17
=
0.33

= 51.52 U

Total protein (mg) = Total Volume (ml) × Protein Concentration (mg/ml)

= 17 mL × 9.86 mg/ml

= 167.62 mg

𝑇𝑜𝑡𝑎𝑙 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑈)

Specific Activity (U/mg) =
𝑇𝑜𝑡𝑎𝑙 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 (𝑚𝑔)

51.52
=
167.62

= 0.307 U/mg

Tube B

Total volume = 11 ml, Enzyme Activity = 0.99 U/ml

𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒(𝑚𝑙)
Total Activity (U) =
𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝑈 /𝑚𝑙)

11
=
0.99

= 11.11 U
Total protein (mg) = Total Volume (ml) × Protein Concentration (mg/ml)

= 6 mL × 4.48 mg/ml

= 26.88 mg

𝑇𝑜𝑡𝑎𝑙 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑈)

Specific Activity (U/mg) =
𝑇𝑜𝑡𝑎𝑙 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 (𝑚𝑔)

11.11
=
26.88

= 0.413 U/mg

Tube C

Total volume = 9 ml, Enzyme Activity = 0.99 U/ml

𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒(𝑚𝑙)
Total Activity (U) =
𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝑈 /𝑚𝑙)

9
=
0.99

= 9.09 U

Total protein (mg) = Total Volume (ml) × Protein Concentration (mg/ml)

= 9 mL × 0.18 mg/ml

= 1.62 mg

𝑇𝑜𝑡𝑎𝑙 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑈)

Specific Activity (U/mg) =
𝑇𝑜𝑡𝑎𝑙 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 (𝑚𝑔)

9.09
=
1.62

= 5.61 U/mg

To calculate the fold purification and overall yield of Tube A, B and C, let sample A as
crude extract,

Tube A Fold Purification

 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑇𝑢𝑏𝑒𝐴
= 0.307𝑈/𝑚𝑔

0.307 𝑈/𝑚𝑔
= 0.307 𝑈/𝑚𝑔

=1

Overall Yield

𝑇𝑜𝑡𝑎𝑙 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑇𝑢𝑏𝑒 𝐴

= × 100 %
51.52 𝑈

51.52 𝑈
= 51.52 𝑈 × 100 %

= 100
Tube B

Fold Purification Overall Yield

𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑇𝑢𝑏𝑒 𝐵 𝑇𝑜𝑡𝑎𝑙 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑇𝑢𝑏𝑒 𝐵

= = × 100 %
0.307 𝑈/𝑚𝑔 51.52 𝑈

0.413 𝑈/𝑚𝑔 11.11 𝑈

= = 51.52 𝑈 × 100 %
0.307 𝑈/𝑚𝑔

= 1.35 = 21.56%

Tube C

Fold Purification Overall Yield

𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑇𝑢𝑏𝑒 𝐶 𝑇𝑜𝑡𝑎𝑙 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑇𝑢𝑏𝑒 𝐶

= = × 100 %
0.307 𝑈/𝑚𝑔 51.52 𝑈

5.61 𝑈/𝑚𝑔 9.09 𝑈

= 0.307 𝑈/𝑚𝑔 = × 100 %
51.52 𝑈

= 18.27 = 17.64%
Overall data of partial purification of egg white lysozymes was constructed as Table 3.

Tube Total Enzyme Total Total Specific Fold Overall

Volume Activity Activity Protein Activity Purification Yield
(ml) (U/ml) (U) (mg) (U/mg) (%)
A 17 0.33 51.52 167.62 0.307 1 100

B 11 0.99 11.11 26.88 0.413 1.35 21.56

C 9 0.99 9.09 1.62 5.61 18.27 17.64

Table 3 shows the partial purification of egg white lysozymes.

DISCUSSIONS:

In this experiment, we used the separation of the enzyme lysozyme from egg white to demonstrate
the process of purification of proteins. The purity of the lysozyme at each stage (sample A,B and
C) were determined using enzyme assays and spectrophotometry. Based on this experiment, two
sessions have been carried out to partially purify the lysozyme and quantify the degree of
purification. First session was isolation and partial purification of egg white lysozyme. Meanwhile,
second session was assay of lysozyme activity and degree of purification.
The systematic name for lysozyme is "mucopeptide N-acetylmuramylhydrolyase." Lysozyme of
egg white was chosen to hydrolyze bacterial cell walls of Micrococcus lysodeikticus. Lysozyme
catalyzes the hydrolysis of β-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”). Lysozyme surface was dominated by its basic side chain therefore it bears a net
positive charge at neutral pH.
Lysozyme has a lower molecular mass about 14.6 kDa therefore considerable purification can be
achieved by simple gel- filtration, which separates proteins on the basis of size. This filtrate was
known as “EWF” (egg white filtrate) and kept in low temperature (4℃) to maintain active enzyme.
It was then labelled as “Sample A” to determine its enzyme activity and estimate the protein.
Sample A is knows as crude extract because it consisted 167.62 mg. For sample B, 4.0mL of EWF,
buffer solution and Amberlite were mixed together and swirled gently on ice to allow complete
binding and to avoid the changes in enzyme activity. Amberlite Resin contains a stoichiometric
equivalent mixture of uniform particle size gel polystyrene cation exchange resins (Praha, 2017).
Thus, amberlite is a cation exchanger. It was then homogenate and filtrated out the sample and
labelled as “Sample B”. The filtration was done by rinsing the amberlite with NaCl of pH 8.0 to
remove proteins of high pI (pI>8.0). Based on the calculation, we calculated the total protein in
sample B was 26.88 mg which is relatively very low compared with the crude extract. Sample C
was prepared by filtration with Amberlite(Ion-exchange chromatography) from previous sample
and rinsed with citrate buffer at pH 4.5 to remove proteins with lower pI (pI>4.5). The total protein
of sample C is 1.62 mg stored in 4℃ to be used in second session.
Second session was carried out to figure out the assay of lysozyme activity and degree of
purification. The assay method is based on observing changes in turbidity of a suspension of dried
cell walls of Micrococcus lysodeikticus. This is a spectrophotometric assay in which the digestion
of the cell wall suspension can be measured at 450 nm. As digestion takes place, the turbidity of
the cell wall suspension decreases. This decrease is used to measure enzyme activity
(Acad.carleton.edu, 2017). When measuring enzyme activity in, continuous assay is carried out.
The absorbance of the cell wall suspension was measured every 0.5 minutes up to 6.0 minutes.
From the results obtained, it was observed that absorbance decreases per time. This shows that
enzyme activity has hydrolysed the cell walls of Micrococcus lysodeikticus as time increases. The
total enzyme activity of proteins after each purification steps were calculated. The total activity of
crude egg white lysosyme is 51.52 U and after first and second purification steps, the total enzyme
activity reduces to 11.11 U and 9.09 U respectively.
Besides enzyme activity assay, protein assay was also carried out to determine the total protein,
specific enzyme activity, fold purification and overall yield of desired protein from enzyme
concentration. In this assay, a protein standard curve is plotted to determine the concentration of
enzyme in sample A, B and C. The final protein concentrations are 9.86 mg/ml, 0.48 mg/ml and
0.18 mg/ml respectively for samples A, B and C. The total protein decreases after each steps of
purification. Furthermore, the specific activity and fold purification of enzyme increases.
However, the overall yield of protein is 17.64% which is quite little. Therefore, to further improve
the experiment, proteins should be pretreated with dialysis before being purified since lysozyme
is a large macromolecules. Another approach of purification can be used such as affinity
chromatography using polypropylene column with IV-acetyl-fl-D-glucosaminide-Sepharose-4B
being modified and used as its matrix which will bind to lysozyme (JUNOWICZ & CHARM ,
1975). Purification of lysozymes is widely used in the food industries. One of the application is to
control lactic acid bacteria in different foods. In addition, lysozyme is also used to control
malolactic fermentation (MLF) during winemaking. (K.Liburdi, 2014)

QUESTIONS:

1. Can you suggest another technique (recall lecture material) that you could try to
further purify the lysozyme found in your most active fraction.

- Another way to further purify the protein is by using the Affinity chromatography. Affinity
chromatography is a method that used to separate the protein based on its highly specific
interaction. This method is quick and specific but then the resin and ligands can be expensive. So,
the glycosidic linkages of chitin between the N-acetyl-D-glucosamine residues are able to be
hydrolysed by lysozyme thus selective adsorption and purifying is possible. An affinity ligand
such as pH-responsive polymer PMMDN with l-thyroxin and buffers with pH 5.5 such as
Phosphate is used. Furthermore, the buffer is used to allow the optimal adsorption. Then, we could
purify the protein by first washing out the unadsorbed protein with 0.05m Tris-HCl(pH8.2) and
0.1M NaCl, then further purify it with 0.05M Na-carbonate-bicarbonate. Since we already purified
the impurities, now we are allowed to elute out the bound protein by using 0.1M Na-carbonate
bicarbonate (pH 9.6) and 0.3M NaCl.

2. On the basic of the evidence you now have, can you compute the % purity of your
most active fraction? (100% means no other protein present). Explain.

Tube Total Enzyme Total Total Specific Fold Overall

Volume Activity Activity Protein Activity Purification Yield
(ml) (U/ml) (U) (mg) (U/mg) (%)

A 17 0.33 51.52 167.62 0.307 1 100

 B 11 0.99 11.11 26.88 0.413 1.35 21.56

C 9 0.99 9.09 1.62 5.61 18.27 17.64

- Based on the Fold Purification. We could conclude that the method that used in this experiment
is a suitable method to purify a protein because as the table above shown, the fold purification is
increased from 1 to 1.35 then finally to 18.27, it is getting more purer after each steps but the purify
(fold purification) does not increase a lot. Moreover, the recovery yield (17.64%) is too low.

3. What technique would offer a sensitive way to detect minor contamination by other
proteins (with physical properties difference from those of lysozyme) that might be
present in your most active fraction?

- A sensitive tool such as Electrospray ionization(ESI-MS) allow us to detect minor contamination

by other protein. Another method which can be used is affinity chromatography. Affinity
chromatography offer a very specific purification because only the desired molecules will be abled
to interact at the stationary phase and became trapped. Other minor contamination will elute out
while the one stayed inside the column will continue bind to the resin through interaction such as
hydrogen bonding, ionic bonding, disulphide bonding and the hydrophobic bonding. In order to
elute other bonded the protein, buffer with difference pH or concentration can be used to elute out
the bonded protein.

CONCLUSION:

Based on the lysozyme physical properties, it provided the information for us to know which
purification method is suitable to use to purify a contaminated protein. In this experiment, ion
exchange chromatography is used to purify the contaminated protein. Ion exchange
chromatography could separate the impurities by the charge it carried. For an example, if the
protein is negative charge, a positive charge such as CM-cellulose is used as a resin. When the
contaminated protein solution is poured into the column, the negative charge protein will bind to
the resin while other impurities will elute out (STRANG, 2017).

Based on the result, Ionic exchange chromatography is not a suitable method for this experiment
because the fold purification and recovery yield is too low. From tube A, the fold purification is 1,
then the protein is further purified and second sample is obtained and labelled as tube B, the fold
purification is 1.35. The protein then purify again and sample C is obtained and the fold
purification is 18.27. From the fold purification result, we could observe that the protein is getting
more purer but then the purification does not provide tremendous purity result and in order to get
more purer result, more reagent and sample is required thus ionic exchange chromatography is not
a suitable method for this experiment.

In order to get the best result, another technique such as Affinity Purification, Centrifugation, SDS-
page and so on can be used but in this experiment, affinity purification can be used to further purify
the protein. After eluting out the solution from the ionic chromatography, the solution can be
further purify by pouring the solution into the column filled with pH-responsive polymer PMMDN
with l-thyroxin. Thus, the contaminated protein is allowed to be further purify based on its specific
interaction with the resin (Enrique Junowicz, Stanly E. Charm, 2017).

REFERENCES:

1. Acad.carleton.edu. (2017). Lysozyme Assay, Antibacterial Response, Research Link 2000.

[online] Available at:
http://www.acad.carleton.edu/curricular/Biol/resources/rlink/lab1p5.html [Accessed 16
Apr. 2017].

2. Colby.edu. (2017). BC 367 Experiment 3 Purification and Characterization of the Enzyme

Lysozyme. [online] Available at:
http://www.colby.edu/chemistry/CH367/laboratory/expt3.pdf [Accessed 16 Apr. 2017].

3. JUNOWICZ , E. & CHARM , S. E., 1975. PURIFICATION OF LYSOZYME BY AFFINITY

CHROMATOGRAPHY. FEBS LETTERS, 7 July, 57(2), pp. 219-221.

4. K.Liburdi, I. &. M., 2014. Lysozyme in Wine: An Overview of Current and. Comprehensive
reviews in Food Science and safety, Volume 13, pp. 1062-1073.
5. Praha, Z. (2017). AMBERLITE IRN217 - Ionex : ZSE Praha s.r.o.. [online] Zsepraha.cz.
Available at: http://www.zsepraha.cz/ionex/amberlite-irn217.html [Accessed 16 Apr.
2017].

6. Enrique Junowicz, Stanly E. Charm. (2017, April 10). Purification of Lysozyme by affinity
chromatography. Retrieved from
http://www.sciencedirect.com/science/article/pii/0014579375807204

7. STRANG, R. H. (2017, April 10). Purification of Egg-White Lysozyme By Ion-exchange

Chromatography. Retrieved from http://onlinelibrary.wiley.com/doi/10.1016/0307-
4412(84)90003-7/pdf
PRELAB: