Lysozyme, a naturally occurring antimicrobial enzyme, plays a pivotal role in innate immunity across diverse organisms, including humans, animals, and plants. This article explores the enzymatic mechanism of lysozyme, their antibacterial, antiviral, anti-inflammatory, and immunomodulatory effects. These properties have prompted its broad application in pharmaceuticals, food preservation, veterinary medicine, animal nutrition, and biotechnology.

As a specialized enzyme supplier with a commitment to biotechnology innovation and quality, Creative Enzymes provides high-purity lysozyme products tailored to the evolving needs of industries ranging from food and feed to pharmaceuticals and personal care.

Historical Perspective and Structural Overview

Lysozyme (EC 3.2.1.17) was discovered independently by Laschtschenko in 1909 and by Alexander Fleming in 1922. Fleming observed that nasal secretions and hen-egg white lysed suspensions of Micrococcus lysodeikticus, and he coined the term "lysozyme" to describe the diffusible bacteriolytic principle. In 1965, David Chilton Phillips and co-workers solved the three-dimensional structure of hen egg-white lysozyme (HEWL) to 2 Å resolution, revealing a compact 129-residue, approximately14.7 kDa α/β protein containing five α-helices and a three-stranded antiparallel β-sheet. The enzyme folds into a kidney-shaped globule with a prominent 25 Å-long active-site cleft that accommodates six consecutive N-acetyl-hexosamine residues of the bacterial peptidoglycan (PG).

Figure 1. Hen egg white lysozyme (HEWL). N terminal and C terminal are labeled. The aromatic residues that were discussed in the text are highlighted: Phe (red), Tyr (green), Trp (blue). Among Cysteine residues (yellow) the disulfide bridges take place. Tyr53, Trp62, Trp63 and Trp108 are in the active site of the enzyme. (Mangialardo et al., 2012)

Physicochemical Properties and Stability

Lysozyme is remarkably stable: it retains activity after prolonged storage in the dry state, withstands peptic digestion, and functions optimally between pH 6.0 and 7.0. Activity increases with temperature up to ~60 °C but declines rapidly above 65 °C. The hydrochloride salt form, lysozyme hydrochloride, exhibits enhanced aqueous solubility and improved shelf life in pharmaceutical and food formulations.

Substrate Specificity

The canonical substrates are the alternating N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG) residues linked via β-(1→4) glycosidic bonds in the glycan backbone of bacterial PG. Lysozyme also hydrolyses chitodextrins composed of β-(1→4)-linked NAG units.

Catalytic Mechanism: A Molecular Dissection

Substrate Binding and Distortion

Lysozyme recognizes a hexasaccharide motif within the PG strand. The D-sugar at the –1 subsite is forced into a half-chair conformation, which approximates the transition-state geometry and lowers the activation energy barrier.

Acid–Base Catalysis

Two invariant carboxylates orchestrate hydrolysis:

• Glu35 (general acid) protonates the glycosidic oxygen, facilitating departure of the NAG leaving group.
• Asp52 (electrostatic or nucleophilic catalyst) stabilizes the developing oxocarbenium ion or, in an alternative covalent route, forms a transient glycosyl–enzyme intermediate.

Kinetic isotope-effect studies and electrospray-mass spectrometry have lent support to both ionic and covalent pathways, the latter being favored in modified substrates.

Figure 2. Two possible catalytic mechanisms for HEWL. Path A; the Koshland mechanism; path B; the Phillips mechanism. R, oligosaccharide chain; R9, peptidyl side chain. (Vocadlo et al., 2001)

Product Release and Enzyme Turnover

Hydrolysis yields shorter muropeptides, weakening the PG meshwork and precipitating osmotic lysis of the bacterium.

Antimicrobial Spectrum and Limitations

Gram-Positive Bacteria

Lysozyme exhibits pronounced activity against Gram-positive bacteria, which include genera such as Staphylococcus, Streptococcus, Bacillus, and Listeria. These organisms possess a thick, multilayered peptidoglycan wall that is directly exposed to the extracellular environment, making it readily accessible to lysozyme's catalytic action.

Upon enzymatic cleavage of the β(1-4) linkages, the structural integrity of the bacterial cell wall is compromised, leading to osmotic imbalance and eventual lysis of the cell. Lysozyme's action is particularly effective during the logarithmic growth phase of bacteria when cell wall remodeling is active and the peptidoglycan matrix is more susceptible to enzymatic attack.

The potency of lysozyme against Gram-positive organisms forms the basis for its inclusion in topical antiseptics, ophthalmic solutions, and preservative formulations in the food industry.

Gram-Negative Bacteria

By contrast, Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Salmonella spp., are intrinsically more resistant to lysozyme. This resistance is primarily due to the presence of an outer membrane (OM) that overlays the thin peptidoglycan layer and acts as a formidable barrier. The OM contains lipopolysaccharide (LPS), porins, and membrane proteins that shield the underlying peptidoglycan from enzymatic degradation.

However, several strategies have been explored to overcome this barrier and sensitize Gram-negative bacteria to lysozyme:

• Permeabilization Agents: Chemical agents such as EDTA (a divalent cation chelator) and mild surfactants (e.g., Triton X-100) can disrupt the outer membrane by chelating Mg²⁺ and Ca²⁺ ions that stabilize LPS molecules. This unmasking exposes the peptidoglycan, allowing lysozyme to access and degrade it.
• Membrane-Disrupting Peptides: Cationic antimicrobial peptides (AMPs), such as polymyxins or defensins, can synergistically enhance lysozyme efficacy by compromising membrane integrity, forming transient pores or destabilizing lipid bilayers.
• Engineered Lysozymes: Recombinant or engineered variants, including goose egg-white lysozyme (GEWL) and fusion proteins with bacteriophage-derived endolysins, demonstrate improved activity against Gram-negative pathogens. These constructs often feature modular PG-binding domains, membrane-translocating peptides, or cationic extensions that facilitate traversal of the OM.
• Nanocarrier Systems: Encapsulation of lysozyme in nanoparticles or liposomes has been shown to enhance penetration into Gram-negative bacteria, offering promising avenues for targeted delivery and enhanced bactericidal effects.

Despite these advancements, the use of lysozyme against Gram-negative bacteria remains more complex and typically requires combination approaches to achieve meaningful antimicrobial outcomes.

Figure 3. SEM images of S. aureus and E. coli under the exposure of buffer (Tris-HCl, pH 7.2, control), HEWL fibril, HEWL oligomer, and HEWL after 6 h. Scale bar, 2 μm. (Nawaz et al., 2022)

Fungi and Viruses

Although not its primary targets, lysozyme exhibits limited antifungal and antiviral activity through mechanisms distinct from its peptidoglycan hydrolysis:

• Fungal Pathogens: Some studies suggest that lysozyme can bind to chitin or β-glucans in fungal cell walls, altering cell membrane permeability or interfering with nutrient uptake. While not fungicidal at physiological concentrations, lysozyme may contribute to synergistic antifungal effects when combined with other agents, such as amphotericin B or echinocandins.
• Viruses: Lysozyme lacks enzymatic activity against viruses but may inhibit viral replication indirectly by modulating host cell responses, particularly innate immunity. In the context of respiratory infections, lysozyme has been shown to reduce viral load and inflammation in animal models, possibly through immune activation, mucosal protection, or capsid destabilization.

These effects remain under investigation, and while they are less robust than lysozyme's antibacterial action, they represent a growing field of research with therapeutic implications.

Non-Enzymatic Antimicrobial Actions

In addition to its catalytic role, lysozyme exhibits non-enzymatic bactericidal properties, further expanding its antimicrobial repertoire:

• Membrane Disruption: Lysozyme is a cationic protein with positively charged residues that enable electrostatic interactions with negatively charged bacterial membranes. These interactions can lead to membrane depolarization, pore formation, and leakage of cytoplasmic contents, even in bacteria resistant to its enzymatic activity.
• Lectin-Like Properties: Lysozyme has been reported to bind carbohydrate moieties on microbial surfaces in a manner similar to lectins. This binding may facilitate opsonization or agglutination, promoting recognition and clearance by phagocytic immune cells.
• Synergy with Host Defenses: Lysozyme enhances the effectiveness of other innate immune molecules, including lactoferrin, secretory phospholipase A2, and complement proteins. These interactions contribute to the coordinated antimicrobial defense at mucosal surfaces and in phagolysosomes.

Broad-Spectrum Applications

Therapeutics and Pharmaceuticals

Lysozyme's therapeutic potential extends across numerous domains, either as a standalone agent or in synergy with other bioactives.

• Anti-Infective Therapy: Lysozyme is incorporated into oral, topical, and parenteral formulations to treat bacterial infections affecting the skin, respiratory tract, and mucosal surfaces. In nasal sprays and lozenges, lysozyme reduces bacterial colonization and supports mucosal healing in rhinosinusitis and pharyngitis. Topical creams and ointments enriched with lysozyme are used for minor burns, acne, and dermatitis, providing both antimicrobial action and inflammation control. For Gram-negative bacteria, lysozyme's effect is enhanced through combination with EDTA, which chelates divalent cations and destabilizes bacterial outer membranes, or with mild detergents like Triton X-100, facilitating entry to the peptidoglycan layer.
• Ophthalmology: Lysozyme is a key tear protein, constituting approximately 30% of total tear protein content, where it contributes to ocular surface immunity and homeostasis. Recombinant human lysozyme (rhLYZ) has shown excellent biocompatibility in artificial tear solutions, promoting corneal epithelial repair, reducing microbial colonization, and attenuating ocular inflammation. Studies indicate that lysozyme-based formulations are particularly effective for dry eye disease, post-surgical recovery, and bacterial conjunctivitis, owing to their dual lubricating and antimicrobial action.
• Wound Care: In the field of dermatology and chronic wound management, lysozyme plays a dual role as an antimicrobial and anti-inflammatory agent. Hydrogels, nanofiber mats, and biodegradable dressings infused with lysozyme are increasingly used in the management of diabetic ulcers, pressure sores, and surgical incisions. These formulations not only inhibit biofilm formation by pathogens like Staphylococcus aureus and Pseudomonas aeruginosa, but also scavenge reactive oxygen species (ROS), reducing oxidative stress and promoting tissue regeneration. Some lysozyme-based dressings are combined with silver nanoparticles or hyaluronic acid, further enhancing wound-healing dynamics.
• Oral Health: Given its natural presence in saliva, lysozyme is well-suited for dental and periodontal care. It is used in mouthwashes, toothpastes, and gels to reduce plaque biomass, gingival bleeding, and periodontal pocket depth. When paired with lactoferrin (iron-sequestering protein) and lactoperoxidase (a peroxidase enzyme), lysozyme exhibits synergistic antimicrobial effects, disrupting bacterial metabolism and improving oral microbiota balance. Lysozyme-enriched oral care products are particularly beneficial for patients with xerostomia (dry mouth) or undergoing radiation therapy, where natural salivary defense is compromised.

Food Industry

• Natural Preservative: Listed as E1105 under GRAS (Generally Recognized as Safe) status, lysozyme is used to control spoilage and pathogenic bacteria in foods such as hard cheeses, fermented sausages, wines, and seafood. It is particularly effective against Clostridium tyrobutyricum, the gas-producing bacterium responsible for "late blowing" defects in cheese. To improve thermal resistance and delivery, lysozyme is encapsulated in chitosan nanoparticles, liposomes, or protein-based matrices, enabling controlled release and extended shelf-life.
• Infant Formula: The addition of recombinant human lysozyme to cow's milk-based formulas mimics the antimicrobial profile of human breast milk. Clinical studies show reduced rates of diarrheal disease, enteric infections, and gut inflammation in infants consuming lysozyme-enriched formulas. Its inclusion also supports the establishment of a protective gut microbiome in neonates.

Biotechnology and Molecular Biology

• Cell Lysis Reagent: Lysozyme is routinely employed to digest the peptidoglycan cell wall of Gram-positive bacteria, facilitating cell disruption in plasmid isolation, protein expression studies, and metabolomic analysis. When used in combination with DNase and RNase, lysozyme prevents nucleic acid-induced viscosity during lysate preparation, ensuring higher yield and purity of biomolecules. Its use extends to automation platforms and high-throughput processing systems in clinical diagnostics and synthetic biology.
• Lysozyme-Based Endolysins: Advances in synthetic biology have led to the development of chimeric enzymes, combining lysozyme's catalytic domain with phage-derived cell wall binding domains. These engineered endolysins show remarkable bacteriolytic efficiency against multi-drug-resistant organisms such as MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin-resistant Enterococcus). They function independently of traditional membrane penetration mechanisms, offering a novel therapeutic route for combating antibiotic resistance.

Veterinary and Agricultural Use

In both aquaculture and terrestrial farming, lysozyme supports animal health and productivity by reducing microbial load without resorting to antibiotics.

• Aquaculture: Lysozyme-enriched feeds boost innate immunity in shrimp, tilapia, and carp, enhancing resistance to Vibrio spp. and other opportunistic pathogens.
• Livestock and Poultry: Supplementation in feed improves gut barrier function, reduces incidence of enteric diseases, and lowers reliance on antibiotic growth promoters.
• Transgenic Crops: Plants genetically modified to express lysozyme (typically from hen egg white or bacteriophage sources) show increased resistance to bacterial soft-rot diseases, such as those caused by Erwinia carotovora. This biotechnological strategy supports sustainable agriculture by reducing pesticide use and improving crop resilience.

Diagnostics and Biosensors

Lysozyme's specificity and catalytic action have been creatively harnessed in rapid diagnostic technologies.

• Colorimetric Biosensors: Immobilized lysozyme on gold nanoparticles (AuNPs) or graphene oxide substrates enables visual detection of bacterial contaminants. Peptidoglycan cleavage triggers aggregation or optical changes, serving as an on-site indicator.
• Electrochemical and Optical Biosensors: Lysozyme's enzymatic reaction with bacterial PG generates measurable electrochemical signals or fluorescence shifts, enabling real-time monitoring of pathogens in clinical samples, food products, and water systems.

These sensors are increasingly used in resource-limited settings due to their low cost, portability, and rapid turnaround time, playing a role in point-of-care diagnostics, outbreak surveillance, and industrial quality assurance.

Immune-Modulatory and Anti-Inflammatory Roles

Lysozyme's biological functions extend far beyond its well-known bacteriolytic activity. Increasing evidence supports its role as a multifaceted immunomodulator, capable of shaping both innate and adaptive immune responses while offering protection against inflammation-induced tissue damage.

• Macrophage Activation and Chemotaxis: Lysozyme enhances the recruitment and activation of immune cells, particularly macrophages. It promotes chemotaxis, guiding these cells toward infection sites, and stimulates phagocytic activity, thereby accelerating the clearance of pathogens and cellular debris. This makes lysozyme a vital component in the early-phase inflammatory response.
• Peptidoglycan-Derived Immune Signaling: Upon hydrolyzing bacterial cell walls, lysozyme releases muropeptides, the soluble fragments of peptidoglycan. These fragments serve as ligands for NOD-like receptors (NOD1 and NOD2)—intracellular pattern recognition receptors that activate NF-κB and MAPK pathways, priming immune cells for a heightened inflammatory response and improving microbial recognition. This pathway is particularly relevant in gut immunity, where bacterial sensing must be balanced with tolerance to the microbiota.
• Antioxidant Activity: Lysozyme exhibits significant ROS-scavenging capabilities, particularly against superoxide anions (O₂^-) and hydroxyl radicals (•OH)—two of the most damaging reactive oxygen species generated during inflammation. By mitigating oxidative stress, lysozyme helps preserve tissue integrity, especially in inflammatory conditions like colitis, sepsis, and chronic wounds. Its antioxidant action also synergizes with other protective proteins such as glutathione and catalase in maintaining redox balance.

Collectively, these immunological functions position lysozyme not merely as an antimicrobial effector but as a bridge between microbial sensing and immune homeostasis, with promising implications for treating autoimmune disorders, chronic inflammation, and mucosal immune dysfunction.

Protein Engineering and Next-Generation Lysozymes

With advances in protein engineering, lysozyme has been reimagined into a new generation of designer enzymes tailored for enhanced functionality, expanded target range, and improved pharmacokinetics. Two primary strategies—directed evolution and rational design—have yielded a growing library of lysozyme variants optimized for diverse applications.

• Thermostable Variants: Through site-directed mutagenesis and high-throughput screening, thermostable lysozyme mutants with melting temperatures (T₅₀) exceeding 80 °C have been developed. These robust variants retain enzymatic activity under extreme heat, making them suitable for pasteurization processes in the food and beverage industry, where typical lysozymes would denature.
• Acid-Stable Mutants: Rational engineering has produced lysozymes that maintain activity at low pH levels, allowing for oral or gastric delivery in therapeutic settings. These acid-tolerant variants are particularly promising for targeting gastrointestinal pathogens, enabling lysozyme to survive the stomach's harsh conditions and act directly in the gut lumen.
• Chimeric Lysins: By fusing lysozyme's catalytic domain with cell wall-binding domains (CBDs) from bacteriophages, researchers have created modular chimeric lysins that selectively bind and lyse target bacteria. These engineered proteins offer pathogen specificity and are being investigated as precision antimicrobials for drug-resistant bacteria, such as Streptococcus pneumoniae and Clostridioides difficile.
• PEGylated Lysozyme: Covalent attachment of polyethylene glycol (PEG) chains to lysozyme enhances its circulatory half-life, reduces immunogenicity, and improves tissue penetration. PEGylated lysozyme formulations are being evaluated for systemic use, particularly in sepsis, bacterial pneumonia, and inflammatory disorders, where prolonged therapeutic presence is desirable without triggering immune rejection.

These bioengineered variants exemplify the versatility of lysozyme as a customizable therapeutic scaffold, opening avenues for its use in precision medicine, nanomedicine, and next-generation antimicrobial therapies. As research continues, lysozyme may evolve from a naturally occurring enzyme into a tailor-made molecular weapon against an increasingly resistant microbial world.

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In summary, lysozyme exemplifies nature's ingenuity in host defense: a single, small enzyme that cleaves a single glycosidic bond yet exerts profound and multifaceted effects. From its historical discovery in Fleming's laboratory to its present role as a cornerstone of antimicrobial therapeutics, food preservation, and biotechnology, lysozyme continues to inspire innovation. Ongoing advances in protein engineering, nanotechnology, and systems biology promise to unlock even broader applications, reinforcing lysozyme's status as a quintessential broad-spectrum bioprotective agent.

At Creative Enzymes, we supply high-quality lysozyme products for research, therapeutic, and industrial use. Trusted for their purity and performance, our lysozymes support innovation across diverse applications. Explore our lysozyme portfolio today to accelerate your next breakthrough. Contact our team today—we're here to help.