Written and maintained by CASRAI Editorial Board
Last updated
Short answer
Lysozyme is an antimicrobial glycoside hydrolase (EC 3.2.1.17) that cleaves β-(1,4)-glycosidic bonds in bacterial peptidoglycan cell walls. Predominantly sourced from chicken egg white, it possesses a molecular weight of 14.3 kDa and an isoelectric point of 11.35. Bench researchers utilize lysozyme primarily for bacterial cell lysis, recombinant protein extraction, and protoplast formation.
Definition and scope
Lysozyme, systematically designated as mucopeptide N-acetylmuramoylhydrolase or muramidase (EC 3.2.1.17), is a monomeric globular enzyme. In biochemical research, the standard commercial preparation is hen egg-white lysozyme (HEWL, Lysozyme C, UniProt P00698), encoded by the LYZ gene in Gallus gallus.
The mature polypeptide comprises 129 amino acids, yielding an exact sequence-calculated molecular weight of 14,307 Da (standardly referenced as 14.3 kDa; sedimentation equilibrium values indicate ~14.4 kDa). Four intramolecular disulfide bridges (Cys6–Cys127, Cys30–Cys115, Cys64–Cys80, Cys76–Cys94) provide high conformational stability.
Lysozyme exhibits a strongly basic isoelectric point (pI) of 11.35, imparting a net positive charge at physiological pH. Its optical extinction coefficient is E1% (281.5 nm) = 26.4 (absorbance of 2.64 for 1 mg/mL in a 1 cm cuvette). Researchers can reference the protein molarity calculator to convert mass concentrations into precise micromolar values.
How it works or how it is used in practice
Lysozyme hydrolyzes the β-(1,4)-glycosidic bonds between alternating N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG) residues in peptidoglycan. The active-site cleft accommodates a hexasaccharide segment across subsites A through F, with cleavage occurring between subsites D and E.
Catalysis is mediated by two conserved residues: Glutamic acid 35 (Glu35) and Aspartic acid 52 (Asp52). Glu35 sits in a hydrophobic pocket (pKa ~6.2), acting as a general acid proton donor. Asp52 resides in a polar environment (pKa ~3.7), remaining ionized to electrostatically stabilize the oxocarbenium ion transition state before water regenerates the enzyme.
In hypotonic buffers, enzymatic digestion weakens the cell wall, causing rapid osmotic lysis. In isotonic or hypertonic solutions (e.g., 0.5 M sucrose or sorbitol), the enzyme digests the peptidoglycan wall while leaving the cytoplasmic membrane intact, generating osmotically fragile bodies known as protoplasts or spheroplasts. Bench procedures for wall-deficient cells are covered in the guide on protoplast formation.
Turbidimetric activity assay and unit definitions
Enzymatic activity is standardly measured using the turbidimetric clearance assay from Shugar (1952), documented in the Worthington Enzyme Manual, using dried Micrococcus lysodeikticus (ATCC 4698):
- Substrate suspension: 9 mg of dried M. lysodeikticus suspended in 0.1 M potassium phosphate buffer (pH 7.0) to 30 mL (0.03% w/v), yielding an initial A450 of 0.60 to 0.70.
- Assay execution: 2.9 mL of substrate suspension is equilibrated at 25°C in a 1 cm cuvette for 4 to 5 minutes. A 0.1 mL aliquot of diluted enzyme is added and mixed.
- Shugar unit definition: One unit produces a decrease in absorbance at 450 nm (ΔA450) of 0.001 per minute at 25°C and pH 7.0.
- Linear range: Accurate measurements require dilutions yielding 150 to 500 units/mL (a linear rate of 0.015 to 0.040 ΔA450/min).
Types or related concepts
Commercial lysozyme preparations differ by purity, formulation, and intended application. The table below summarizes standard laboratory grades verified from manufacturer specifications:
| Formulation Type | Physical Format | Typical Specific Activity | Reconstitution Medium | Storage Conditions | Primary Bench Application |
|---|---|---|---|---|---|
| Salt-Free 2x Crystallized (Worthington LYSF) | Lyophilized dialyzed powder | ≥ 8,000 Shugar U/mg dry weight | Cold reagent-grade water or 10 mM Tris-HCl | 2–8°C (desiccated) | Enzyme kinetics, structural crystallization, sensitive lysis |
| Standard Lyophilized Powder (Sigma L6876 / L7651) | Lyophilized powder with trace NaCl | ≥ 40,000 units/mg solid | 10 mM Tris-HCl (pH 8.0) or water at 10 mg/mL | –20°C (desiccated) | Routine bacterial lysis, plasmid purification, DNA extraction |
| Ready-to-Use Liquid Solution (Thermo Cat. 90082) | 50 mg/mL aqueous liquid | Functional lysis grade | Pre-dissolved (direct 1:100–1:200 addition) | –20°C (stable liquid) | High-throughput screening, mass spectrometry sample prep |
| Recombinant / Animal-Origin-Free | Microbially expressed powder | Equivalent to native HEWL | 0.1 M potassium phosphate or 10 mM Tris-HCl | –20°C | Biomanufacturing, cGMP processes, animal-free workflows |
Where it matters for labs, clinics or supply decisions
Lysozyme is widely used during cell disruption for recombinant protein extraction and downstream protein purification. Because lysozyme targets peptidoglycan bonds without harsh denaturants or prolonged sonication, it preserves the native tertiary structure of sensitive target proteins.
Bench stock solution preparation and handling
To ensure enzymatic potency, follow standard preparation guidelines from manufacturer datasheets:
- Dissolution: Dissolve lyophilized powder at 10 mg/mL in sterile 10 mM Tris-HCl (pH 8.0) or molecular-biology-grade water, yielding a clear to faintly hazy, colorless solution.
- Refrigerated shelf life: Neutral aqueous solutions remain stable at 2–8°C for at least one month. Mildly acidic solutions (pH 4.0–5.0) retain activity for several weeks refrigerated.
- Long-term storage: Aliquot the 10 mg/mL stock into single-use tubes (50–100 µL) and store frozen at –20°C. Avoid repeated freeze-thaw cycles, which promote protein aggregation and activity loss.
Differential lysis: Gram-positive versus Gram-negative bacteria
Lysis requirements depend on bacterial cell wall structure. Gram-positive organisms (such as Micrococcus and Bacillus) expose peptidoglycan directly, lysing efficiently with 1 to 2 mg/mL lysozyme at 37°C in 30 to 60 minutes. Dormant spores, however, possess specialized proteinaceous coats that resist enzymatic lysis, as discussed in the guide on formation of spores in bacteria.
Gram-negative bacteria (such as Escherichia coli) possess an outer membrane with lipopolysaccharide cross-linked by divalent cations that prevents lysozyme from reaching peptidoglycan. Effective lysis requires supplementing lysozyme (0.2–0.5 mg/mL) with 1 to 2 mM EDTA to chelate stabilizing cations, often alongside non-ionic detergents like Triton X-100 or Tween-20.
Common misunderstandings
- Expecting Gram-negative lysis without permeabilizers: Lysozyme cannot penetrate an intact outer membrane alone; chelating agents like EDTA are mandatory for E. coli.
- Treating commercial unit definitions as identical: Shugar turbidimetric units (0.001 ΔA450/min) do not equal FIP or international micromolar turnover units. Always check the supplier’s assay method.
- Subjecting stock solutions to freeze-thaw cycles: Repeated freeze-thaw cycles denature lysozyme and cause precipitation. Always freeze in single-use aliquots.
- Overlooking lysate viscosity: Extensive bacterial lysis liberates high-molecular-weight genomic DNA, creating high viscosity. Treat lysates with a universal nuclease or gentle shearing.
Frequently asked questions
What is lysozyme?
Lysozyme is an antimicrobial enzyme (EC 3.2.1.17) that hydrolyzes β-(1,4)-glycosidic linkages in bacterial peptidoglycan. Sourced primarily from chicken egg white, it is widely used in laboratories for gentle enzymatic bacterial cell lysis.
What is the molecular weight of lysozyme?
Chicken egg-white lysozyme has a sequence-calculated molecular weight of 14,307 Da (standardly referenced as 14.3 kDa), with sedimentation equilibrium measurements indicating an apparent mass of ~14.4 kDa.
What is the isoelectric point of lysozyme?
The isoelectric point (pI) of hen egg-white lysozyme is 11.35. Because of its basic amino acid composition, it carries a strong net positive charge in neutral and physiological buffers.
How do you prepare and store a lysozyme stock solution?
Dissolve lyophilized lysozyme powder at 10 mg/mL in 10 mM Tris-HCl (pH 8.0) or sterile water. Store refrigerated at 2–8°C for up to one month, or dispense into single-use aliquots at –20°C to prevent freeze-thaw degradation.
Sources
- Worthington Biochemical Corporation. “Lysozyme.” Worthington Enzyme Manual. Accessed 2026-11-07. https://www.worthington-biochem.com/products/lysozyme
- Sigma-Aldrich / Merck. “Lysozyme from chicken egg white.” Product Information Sheet (Cat. L6876 / L7651 / SAE0152). Accessed 2026-11-07. https://www.sigmaaldrich.com/
- Thermo Fisher Scientific. “Lysozyme Solution (50 mg/mL).” Instructions for Use (Cat. 90082 / 89833). Accessed 2026-11-07. https://www.thermofisher.com/
- UniProt Consortium. “Lysozyme C – Gallus gallus (Chicken).” UniProtKB Entry P00698. Accessed 2026-11-07. https://www.uniprot.org/uniprotkb/P00698/entry
