Lysozyme, a naturally occurring enzyme with remarkable antimicrobial properties, represents a fascinating intersection of biochemistry, immunology, and modern biotechnology. First discovered in 1922 by Alexander Fleming, lysozyme has since been extensively studied for its ability to degrade the cell walls of bacteria, thereby contributing to innate immune defense mechanisms in a wide range of organisms. Found abundantly in egg whites, human tears, saliva, and various bodily secretions, lysozyme is now gaining renewed interest in the fields of medicine, agriculture, and food preservation. This comprehensive article explores lysozyme's origin, structure, biological functions, applications, and future potential as a versatile tool in human health and industry.

The Biochemical Nature of Lysozyme

Lysozyme is a glycoside hydrolase belonging to the family 22 of glycosyl hydrolases. It exhibits enzymatic activity by cleaving the β(1-4) glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of bacterial cell walls. This mechanism renders it particularly effective against Gram-positive bacteria, whose thick peptidoglycan layers are directly exposed to the extracellular environment.

Figure 1. Hydrolytic mechanism of action of lysozyme on β(1–4) linkages between NAM and NAG residues of the bacterial cell wall backbone. (Nawaz et al., 2022)

Structurally, lysozyme is a small, globular protein composed of approximately 129 amino acids, with a molecular weight of about 14.3 kDa. The enzyme's tertiary structure is stabilized by four disulfide bridges, contributing to its thermal and pH stability, characteristics that are particularly advantageous for industrial and therapeutic applications.

Figure 2. Structure of hen egg-white lysozyme (HEWL) protein built from PDB-ID 3WUN.

Natural Sources of Lysozyme

Lysozyme is widespread in nature, found in a diverse array of organisms, including mammals, birds, insects, plants, and even bacteriophages. Among its most well-known sources are:

• Egg Whites: Hen egg white lysozyme (HEWL) is one of the most studied and commercially exploited forms of the enzyme. It is highly abundant, comprising approximately 3.5% of the total protein content in egg whites.
• Human Secretions: Lysozyme is present in tears, saliva, nasal mucus, and breast milk. In these fluids, it serves as a first line of defense against microbial invasion.
• Neutrophils and Macrophages: Lysozyme is also produced by immune cells such as neutrophils and macrophages, which release it during phagocytosis to destroy engulfed pathogens.
• Marine and Insect Species: Certain fish and insect species produce lysozyme as a component of their innate immune systems, highlighting the enzyme's evolutionary conservation and importance.

Lysozyme in Innate Immunity

As a component of the innate immune system, lysozyme plays a crucial role in host defense. By hydrolyzing bacterial cell walls, it not only kills bacteria directly but also facilitates the subsequent actions of other immune molecules such as defensins and lactoferrin. Lysozyme also acts synergistically with phagocytic cells, enhancing their microbicidal activity.

In addition to its antimicrobial properties, lysozyme possesses anti-inflammatory effects. It modulates immune responses by influencing cytokine production and regulating the activity of inflammatory cells, thereby contributing to immune homeostasis.

Clinical and Therapeutic Applications

The therapeutic potential of lysozyme is being actively explored across a range of medical disciplines:

• Antibacterial Agent: Lysozyme can be used both topically and systemically to treat bacterial infections. Its mechanism of action is particularly effective against Gram-positive bacteria, due to their exposed peptidoglycan cell walls. Topically, it is incorporated into eye drops for conjunctivitis, nasal sprays for rhinosinusitis, and dermatological creams for acne and minor skin infections. Systemic applications are being investigated, particularly in synergistic formulations with antibiotics to combat resistant strains and reduce antibiotic dosage requirements.
• Antiviral and Antifungal Activity: Although primarily recognized for its antibacterial action, emerging research indicates that lysozyme also exerts antiviral and antifungal effects. It may disrupt viral capsids or interfere with viral replication by modulating host immune responses. Additionally, it can bind to fungal cell walls and affect membrane integrity. Clinical trials are underway to evaluate its efficacy in treating respiratory viral infections and fungal skin conditions, potentially broadening its therapeutic use.
• Wound Healing: Lysozyme contributes significantly to wound healing by reducing microbial load, mitigating inflammation, and supporting tissue regeneration. Its bacteriolytic activity helps prevent infection, while its immunomodulatory functions curb excessive inflammation that can delay healing. Researchers are developing advanced wound dressings and hydrogels infused with lysozyme to accelerate healing in chronic wounds, diabetic ulcers, and surgical sites.
• Oral and Gastrointestinal Health: Due to its natural occurrence in saliva and gastrointestinal secretions, lysozyme plays a vital role in maintaining mucosal integrity and preventing microbial colonization. It is being explored as a therapeutic agent for managing periodontal diseases, oral ulcers, and gingivitis. In gastrointestinal applications, lysozyme shows promise in alleviating inflammatory bowel diseases (IBD), controlling Helicobacter pylori infections, and promoting a balanced gut microbiome.
• Cancer Therapy: Preliminary investigations suggest that lysozyme may exert antitumor effects through multiple pathways. It appears to induce apoptosis in cancer cells, modulate immune surveillance mechanisms, and inhibit tumor-promoting inflammation. Some studies have reported reduced tumor growth in animal models treated with lysozyme or lysozyme-derived peptides. Although still in early stages, these findings support further exploration of lysozyme as an adjuvant in cancer therapy, particularly in immunotherapy strategies.

Lysozyme in the Food and Agriculture Industries

Lysozyme's antimicrobial properties make it a valuable additive in food preservation and agricultural practices:

• Food Preservation: HEWL is approved as a food additive in many countries, used to prevent spoilage and extend the shelf life of cheese, meat, and beverages. It inhibits spoilage-causing bacteria such as Clostridium tyrobutyricum in cheese production.
• Animal Feed Additive: Lysozyme supplements in animal feed can reduce reliance on antibiotics by promoting gut health and resistance to infections in livestock and poultry.
• Crop Protection: Genetically modified plants expressing lysozyme genes have shown increased resistance to bacterial pathogens, representing a potential strategy for sustainable agriculture.

Figure 3. Use of lysozyme as a food preservative. Lysozyme inhibits the growth of deleterious microorganisms, thus prolonging food shelf life. Lysozyme has been used to preserve fresh fruits and vegetables, seafoods, meats and sausages, and varieties of semihard cheeses such as Edam, Gouda, and some Italian cheeses. (Bergamo and Sava, 2024)

Pharmaceutical Formulations and Delivery Systems

The development of novel drug delivery systems has enhanced lysozyme's therapeutic efficacy. Nanoparticles, liposomes, and hydrogels loaded with lysozyme are being explored for controlled release and targeted delivery. These formulations improve bioavailability, stability, and tissue-specific action, broadening the scope of lysozyme-based therapies.

Figure 4. Four kinds of delivery systems for lysozyme in wound healing. (Zhao et al., 2023)

Biotechnological and Diagnostic Applications

Lysozyme's enzymatic properties lend themselves to various biotechnological applications:

• Cell Lysis in Molecular Biology: Lysozyme is a staple in molecular biology protocols for breaking down bacterial cell walls to release intracellular contents. This step is essential for the extraction of high-quality DNA, RNA, and proteins in laboratory research. Its specificity for peptidoglycan and ability to lyse Gram-positive bacteria make it a preferred choice in many genomic and proteomic workflows.
• Biosensors: Innovative biosensor platforms have been developed by immobilizing lysozyme onto various substrates to detect bacterial contamination in clinical samples, food products, and environmental water sources. The presence of bacterial peptidoglycan can trigger enzymatic activity, which is then translated into measurable electrical or optical signals. These biosensors offer rapid, sensitive, and cost-effective solutions for public health monitoring and industrial quality assurance.
• Pharmaceutical Quality Control: In pharmaceutical manufacturing, lysozyme assays are utilized to monitor microbial contamination and to evaluate the effectiveness of antimicrobial agents. Quantifying lysozyme activity can serve as a proxy for bacteriolytic potency in drug formulations, ensuring product safety and consistency. Additionally, lysozyme is used as a reference enzyme in the standardization of antimicrobial testing methods.

Safety, Allergenicity, and Regulatory Considerations

While lysozyme is generally recognized as safe (GRAS) for use in foods and pharmaceuticals, certain concerns persist:

• Allergenicity: As a protein derived from egg whites, lysozyme may elicit allergic reactions in susceptible individuals, particularly those with egg allergies.
• Immunogenicity: Repeated systemic administration of non-human lysozyme may provoke immune responses, necessitating careful formulation and patient monitoring.

Regulatory agencies such as the FDA and EFSA have established guidelines for lysozyme usage, including permissible concentrations and labeling requirements to ensure consumer safety.

Future Directions and Research Frontiers

Ongoing research is expanding the horizon of lysozyme applications:

• Engineered Lysozymes: Protein engineering techniques are being used to enhance lysozyme's stability, spectrum of activity, and resistance to inhibitors.
• Combination Therapies: Lysozyme is being tested in combination with antibiotics, antimicrobial peptides, and immunotherapies to enhance efficacy and reduce drug resistance.
• Role in Microbiome Modulation: Investigations are underway to elucidate lysozyme's impact on the human microbiome and its potential in treating dysbiosis-related diseases.
• Theranostics: Integration of lysozyme with diagnostic and therapeutic platforms (theranostics) could allow for real-time monitoring and targeted treatment of infections and inflammatory diseases.

From its humble beginnings in egg whites to its current role as a promising therapeutic and biotechnological agent, lysozyme exemplifies the power of nature-inspired molecules in modern science. Its broad-spectrum antimicrobial activity, immunomodulatory functions, and versatility across diverse applications underscore its potential as a medical marvel. Continued research and innovation will undoubtedly unlock new dimensions of lysozyme's utility in addressing global challenges in health, agriculture, and biotechnology.

At Creative Enzymes, we are proud to offer high-quality lysozyme products that meet the rigorous demands of both research and industrial applications. Explore our lysozyme offerings today or contact us for more information!