Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation

Figures & data

Table 1.  Different molar ratio mPEG-SS 5000/NH₂.

Lysozyme (mg)	10	10	10	10	10
mPEG-SS 5000/LZ (m/m)^a	2	1	0.5	0.25	-
mPEG-SS 5000/NH2^b	0.86	0.43	0.21	0.11	-

^aWeight ratio mg/mg.

^bMolar ratio mPEG-SS 5000/NH₂. Each molecule of lysozyme own 7 anime group (6 ϵ-lisinas and 1 N-terminal).

Table 2.  Purification of hen egg white lysozyme.

Sample	Volume (mL)	Total unit (U)	Total protein (mg)	Specific activity (U/mg)	Yield (%)	Fold purification
Egg white	90	882000	7605	116	100	1
Eluate (IRC-50)	100	680000	460	1478	77	12
Permeate	100	540000	80	6750	61	58
Egg white	80	1088000	6997	155	100	1
Eluate (IRP-88)	100	900000	527	1706	83	11
Permeate	100	680000	122	5574	62	36

Figure 1.  Polyacrylamide 12% SDS-PAGE electrophoresis of the products of the purification steps of LZ purification. P, molecular mass standard; 1, egg white (20 µg); 2, eluate of IRC-50 (20 µg); 3, permeate IRC-50 (20 µg); 4, eluate of IRP-88 (20 µg); 5, permeate IRR-88 (20 µg). The arrow indicates the LZ band around 14.4 kDa.

Figure 2.  Electrophoresis in polyacrylamide SDS-PAGE 10%. Kinetics of the PEGylation reaction of LZ with mPEG-SS 5000 for 2 h at 30°C. P, standard of molecular mass; 1, 3, 5, 7, 10, 20, 30, 60 and 120 minutes, indicate the time interval in minutes of aliquots collected and submitted to electrophoresis.

Figure 3.  Residual enzyme activity (%) of LZ-mPEG-SS 5000 in different molar ratio (mPEG-SS 5000/NH₂) of 0.11, 0.21, 0.43, 0.86 toward substrate. (-•-) M. lysodeikticus; (-▪-) Glycol chitosan. Results are means ± SD, n = 3.

Figure 4.  Evaluation of the enzymatic activity (A) and stability (B) of native LZ (-○-) and LZ-mPEG-SS 5000 (-▪-). The activity was determined in different buffers varying the pH from 4–12 (4–5, 50 mM acetate; 6–8, Hepes 50 mM and 9–12 Tris-HCl 50 mM) at 25°C. The stability was determined after incubation by 1 h in the same buffers at 50°C. At the end of this time the enzymatic activity was determined against substrate M. lysodeikticus as described in methods. Results are means ± SD, n = 3.

Figure 5.  Resistance to proteolysis of native lysozyme (-○-) and LZ-mPEG-SS 5000 (-▪-), digestion as assessed by enzyme activity upon incubation with Proteomix^® (A), Protex 6L^® (B) and fungal protease (C). Results are means ± SD, n = 3.

Figure 6.  Antimicrobial activity of lysozyme, lysozyme-mPEG-SS 5000 and free mPEG-SS 5000 against E. coli. The survial of E. Coli was expressed as CFU/mL. Values are the means from three independent experiments.