Egg white lysozyme — pH-adjusted dilution, CEX capture on CM-Sepharose, NaCl crystallization, and sterile filtration for food-grade and pharmaceutical applications
Lysozyme is one of the most abundant proteins in hen egg white (~3.5% of total protein) and exploits a strongly basic pI (10.7) that makes cation exchange capture highly selective over the acidic egg white proteins (ovalbumin pI 4.6, ovotransferrin pI 6.1). The classic industrial process combines a single CEX capture step with NaCl salting-out crystallization to achieve pharmaceutical-grade purity. Overall yield: 70–80% from raw egg white to crystalline lysozyme.
| Molecular Weight | 14,307 Da (129 amino acids) |
| Isoelectric Point (pI) | 10.7 (strongly basic) |
| Charge at pH 9.5 | Positive (net charge ~+4 to +6) |
| Solubility | High in low-salt buffers; crystallizes at 5% NaCl |
| Activity | Cleaves β-1,4 glycosidic bonds in peptidoglycan (bacteriolytic) |
| Source in egg white | ~3.5% of total protein (~4 g/L raw egg white) |
| Step | Key Cost Driver | Relative Cost |
|---|---|---|
| Dilution & pH Adjustment | NaCl, buffer salts, tank volume | Low |
| CEX Capture (CM-Sepharose) | Resin purchase, buffer consumption, CIP reagents | High |
| Crystallization | NaCl, chilled holding tanks, time | Low |
| Filtration & Washing | Filter membranes, wash buffer | Low |
| Sterile Filtration | 0.22 μm PES capsules, aseptic handling | Medium |
CM-Sepharose (carboxymethyl, pKa ~4) is a weak cation exchanger that retains selectivity for highly basic proteins like lysozyme (pI 10.7) at alkaline pH where strong exchangers (sulfopropyl) can over-bind. At pH 9.5, CM groups are fully ionized and lysozyme binds tightly, while all acidic egg white proteins (ovalbumin pI 4.6) are negatively charged and flow through. Elution is gentle, preserving enzymatic activity.
NaCl promotes lysozyme crystallization through a salting-out mechanism. At moderate NaCl concentrations (2–5%), electrostatic repulsion between positively charged lysozyme molecules is reduced, promoting close-packing into the tetragonal crystal lattice. Combined with cooling to 4°C (reducing solubility), the crystallization driving force is sufficient for quantitative recovery. The crystal form is highly pure because impurities are excluded from the lattice.
Food-grade lysozyme (EU additive E1105, used in cheese preservation) requires >90% purity and <10 EU/mg endotoxin. Pharmaceutical-grade (ophthalmic, dental) requires >98% purity, <0.25 EU/mg endotoxin, and sterile filtration through 0.22 μm membrane. The basic purification process is the same; pharmaceutical grade adds an additional recrystallization step and more stringent QC testing including activity assays (>20,000 U/mg).
Yes. Lysozyme is also produced recombinantly in rice (Ventria Biosciences), transgenic plants, and microbial fermentation (Aspergillus, Pichia). Recombinant sources avoid egg allergy concerns and enable consistent supply. The downstream process for recombinant lysozyme is similar (CEX capture at alkaline pH) but requires additional clarification steps for fermentation broths. Egg white remains the dominant commercial source due to very low feedstock cost (~$0.05/egg).
Drag-and-drop the CEX capture, crystallization, and filtration steps, simulate mass balance, and estimate manufacturing costs at your batch scale.
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