Structural Features of Lysozyme and Advances in Its Applications
Structural Features of Lysozyme and Advances in Its Applications
Lysozyme (EC 3.2.1.17) is a small, basic hydrolase widely distributed in human and animal body fluids, plant tissues, and certain microorganisms. It specifically hydrolyzes the β-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in bacterial cell wall peptidoglycan, thereby disrupting the cell wall and causing osmotic lysis of susceptible bacteria. Owing to its small molecular size, structural stability, high safety, and good tolerance to pH and temperature, lysozyme is widely used in mucosal and fluid-phase innate immunity, food preservation, pharmaceutical formulations, feed additives, biomaterials, and as a lytic enzyme in laboratory applications. It has become an important technological tool for developing “natural preservatives,” antibiotic-reducing farming strategies, and functional biopreparations.
I. Overview of Lysozyme
1.1 Basic Concept and Catalytic Substrates
(1) Lysozyme is a prototypical β-1,4-glycosidase whose main catalytic substrate is bacterial cell wall peptidoglycan. Peptidoglycan consists of a polysaccharide backbone formed by alternating MurNAc and GlcNAc residues linked through β-1,4-glycosidic bonds, cross-linked by short peptide chains into a mesh-like structure that provides mechanical strength to the cell wall. Lysozyme specifically recognizes the glycosidic bond between MurNAc and GlcNAc and catalyzes its hydrolysis, thereby disrupting the continuity of the peptidoglycan backbone. As the rigidity of the cell wall is reduced, bacteria in hypotonic environments readily undergo osmotic lysis.
(2) In terms of antibacterial spectrum, lysozyme is particularly effective against typical Gram-positive bacteria (such as staphylococci, streptococci, and lactic acid bacteria). Its activity against Gram-negative bacteria with an intact outer membrane is limited due to the barrier function of the lipopolysaccharide layer. However, when combined with chelating agents, organic acids, or surfactants that increase outer membrane permeability, lysozyme can also exert notable lytic activity against certain Gram-negative bacteria.
1.2 Classification and Physiological Distribution
(1) From an evolutionary and structural family perspective, lysozymes are broadly classified into C-type (chicken-type), G-type (goose-type), and I-type (insect-type), among others. C-type lysozyme is the best-studied and the most widely used in foods, health-related products, and biochemical reagents. Although these families differ in primary sequence and certain aspects of three-dimensional structure, they share the basic ability to recognize and hydrolyze cell wall peptidoglycan.
(2) Physiologically, lysozyme is widely present in human milk, saliva, tears, nasal and respiratory tract secretions, intestinal secretions, and other mucosal fluids. It is also found in animal-derived foods such as egg white and milk, as well as in certain plant tissues and microbial secretions. Acting in concert with lactoferrin, secretory IgA, and various antimicrobial peptides, lysozyme forms an important component of the mucosal and fluid-phase antimicrobial barrier.
1.3 Biological Functions and Immunological Significance
(1) In innate immunity, lysozyme rapidly reduces local bacterial burden by directly degrading the cell walls of susceptible bacteria and is one of the key effector molecules in host defense against invading microorganisms.
(2) In mucosal environments, lysozyme reduces pathogenic bacterial adhesion and biofilm formation, thereby stabilizing epithelial barriers and indirectly attenuating local inflammatory responses. Some studies further suggest that lysozyme exerts immunomodulatory effects on macrophages and neutrophils, influencing phagocytic function and inflammatory mediator release, and may play a role in infection control and modulation of the inflammatory microenvironment.
II. Molecular Structure and Catalytic Mechanism
2.1 Structural Features
(1) Chicken egg white lysozyme is a representative member of the C-type lysozymes. It is a single-chain polypeptide of about 129 amino acid residues with a molecular mass of approximately 14.3 kDa and an isoelectric point of 10–11, thus classified as a basic protein. The molecule contains four disulfide bonds, which confer a compact globular conformation. This extensive disulfide cross-linking underlies its good thermal stability and certain tolerance to organic solvents.
(2) In three-dimensional terms, lysozyme can be roughly divided into a large domain dominated by α-helices and a smaller domain rich in β-sheets. Between these two domains lies a narrow, cleft-like groove that forms the active site for substrate binding and catalysis. This groove accommodates multiple consecutive sugar residues, allowing precise positioning of the substrate.
2.2 Catalytic Site and Substrate Recognition
(1) Conserved Glu and Asp residues in the lysozyme active center are critical catalytic groups for glycosidic bond hydrolysis. In chicken egg white lysozyme, Glu35 participates in acid–base catalysis by donating a proton to the leaving group with an appropriately tuned pKa, whereas Asp52 helps stabilize the transition state or form a short-lived covalent intermediate. Cooperation between these two residues drives hydrolysis of the MurNAc–GlcNAc glycosidic bond.
(2) When a peptidoglycan fragment enters the active groove, it is precisely positioned by multiple hydrogen bonds and hydrophobic interactions, with successive sugar residues occupying distinct subsites within the binding cleft. This spatial matching, combined with finely tuned amino acid side-chain interactions, allows lysozyme to cleave specific β-1,4-glycosidic bonds with high efficiency and specificity.
2.3 Catalytic Mechanism and Physicochemical Properties
(1) The catalytic process of lysozyme can be summarized as substrate binding, protonation and cleavage of the glycosidic bond, formation of an intermediate, entry and activation of a water molecule, hydrolysis of the covalent or high-energy intermediate, and product release. Overall, lysozyme operates via a typical glycosidase mechanism involving cooperative general acid–base catalysis and nucleophilic catalysis. After turnover, the enzyme returns to its original conformation and is ready for the next catalytic cycle.
(2) Lysozyme generally exhibits maximal activity in the pH range of 5.0–7.0, where the charge states of the active-site residues favor catalysis. Its optimal reaction temperature is typically 30–40 °C. Short-term exposure to 60–70 °C may still retain partial activity, but prolonged high temperature or high humidity can irreversibly disrupt disulfide bonds and overall conformation, leading to inactivation.
(3) Under appropriate ionic strength, lysozyme shows good solubility and stability. However, high concentrations of heavy metal ions and strong oxidants can damage disulfide bonds and key side chains. In organic solvent systems, enzyme activity is strongly influenced by solvent polarity, water content, and temperature; high-water-content systems are generally more favorable for maintaining activity.
III. Sources and Production Technologies of Lysozyme
3.1 Conventional Extraction Processes
(1) Industrially, chicken egg white is the most common raw material for lysozyme production; lysozyme can also be extracted from animal body fluids or certain plant tissues. Typically, raw materials are first pretreated and centrifuged to remove lipids and coarse impurities. Ammonium sulfate precipitation is then used for initial enrichment, followed by ion-exchange chromatography to separate lysozyme based on charge differences. Final polishing is often carried out using gel filtration chromatography to remove low-molecular-weight impurities and aggregates. The purified enzyme solution is then converted into lyophilized powder or dry powder through freeze-drying or spray-drying.
(2) This extraction-based method is technically mature and reliable, and the resulting product has a favorable safety profile that meets requirements for food applications and general pharmaceutical excipients. However, overall yield is limited by raw material availability, extraction efficiency, and purification cost, making this route more suitable for medium- to high-value products or applications with specific requirements for source.
3.2 Genetic Engineering and Recombinant Expression
(1) Genetic engineering approaches clone lysozyme-encoding genes into suitable expression vectors and introduce them into engineered strains such as Escherichia coli, Pichia pastoris, or Bacillus subtilis for heterologous expression. Through codon optimization, signal peptide design, promoter strength tuning, and optimized fermentation conditions, high-yield strains can be developed that facilitate downstream purification.
(2) Recombinant production offers clear advantages of high output and easier cost control. It also enables rational modification of the enzyme through site-directed mutagenesis, domain fusion, and other protein-engineering strategies to adjust antibacterial spectrum, enhance stability, or reduce potential allergenicity. Moreover, recombinant human lysozyme or human-like variants can be generated for applications requiring higher safety margins.
(3) Compared with extraction-based methods, recombinant production imposes higher demands on fermentation scale-up, correct folding, disulfide bond formation, and stringent control of impurities such as endotoxins and host-cell proteins. These challenges must be addressed through process optimization and robust quality systems.
3.3 Comparison of Production Methods
Production Method | Raw Material/Source | Key Process Steps | Advantages and Disadvantages |
Extraction | Egg white, animal tissues (e.g., tears, saliva) | Centrifugation, ammonium sulfate precipitation, ion-exchange chromatography, gel filtration | Advantages: mature processes, high product safety; Disadvantages: limited raw materials, low yield, relatively high cost |
Genetic engineering | Engineered strains such as E. coli, Pichia pastoris, B. subtilis | Gene cloning, vector construction, fermentation of engineered strains, purification | Advantages: high yield, scalable production, tunable enzyme properties; Disadvantages: requires optimization of fermentation and more complex purification |
3.4 Quality Control and Activity Assessment
(1) Quality evaluation of lysozyme preparations typically includes assessment of protein purity and molecular weight (e.g., by SDS-PAGE or HPLC), enzyme activity, microbial limits, and basic physicochemical parameters such as moisture and ash content. These indicators together determine whether the product meets the requirements for food, dietary supplements, or pharmaceutical excipients.
(2) Enzyme activity is commonly measured using the rate of turbidity decrease in a suspension of Micrococcus lysodeikticus, with the change in absorbance over a defined time converted into U/mg or U/mL. Alternatively, colorimetric or fluorometric assays with synthetic substrates can be used for high-throughput measurement. Continuous monitoring of enzymatic activity during formulation development and stability studies helps evaluate how well the formulation and storage conditions preserve lysozyme structure and function.
IV. Major Application Fields of Lysozyme
4.1 Preservation and Shelf-Life Extension in the Food Industry
(1) In food applications, lysozyme serves as a natural preservative that effectively suppresses growth of susceptible bacteria in various products. For example, in yogurt and cheese, lysozyme can reduce contamination by pathogens such as Listeria monocytogenes and Staphylococcus aureus, thereby extending shelf life while exerting relatively little impact on the starter lactic acid bacteria (which nonetheless should be evaluated case-by-case depending on strain and dosage).
(2) In meat and aquatic products, lysozyme can be combined with refrigeration, salting, and smoking to inhibit spoilage bacteria, slow deterioration of color and flavor, and thus reduce the use of conventional chemical preservatives while enhancing product safety perception. In beverages and condiments, moderate addition of lysozyme can lower the risk of microbial overgrowth and turbidity-related spoilage, improving overall product stability.
4.2 Pharmaceutical Uses and Mucosal Protection
(1) In the pharmaceutical field, lysozyme is frequently incorporated into local formulations for the eye, oral cavity, nasal cavity, and respiratory tract, such as eye drops, lozenges, sprays, and topical ointments. By specifically hydrolyzing cell wall peptidoglycan, lysozyme exerts an auxiliary antibacterial effect and helps clear bacterial debris, thereby reducing local inflammatory load. For mild infections and inflammatory discomfort, lysozyme-containing products can serve as adjuvant interventions.
(2) As a mucosal protective factor, exogenous lysozyme supplementation may strengthen the intrinsic defenses of the oral, pharyngeal, and upper respiratory tract mucosa by reducing pathogenic bacterial adhesion and biofilm formation, and by acting synergistically with secretory IgA and lactoferrin to maintain local microbial balance. In certain wound dressings and gels, lysozyme is added to suppress wound infection and promote granulation tissue formation and wound healing.
4.3 Applications in Feed and Animal Production
(1) In feed and animal production, lysozyme is viewed as a “green” feed additive that can partially substitute antibiotic growth promoters. By modulating intestinal microbiota composition, inhibiting overgrowth of harmful bacteria, and supporting colonization of beneficial microbes, lysozyme helps maintain gut health and enhances nutrient absorption efficiency.
(2) In young or highly stressed animals such as piglets, chicks, and fish or shrimp, inclusion of lysozyme in feed or drinking water may help reduce the incidence of diarrhea and enteritis, improve survival rates, and enhance performance. This provides a biotechnological option for reduced-antibiotic or antibiotic-free production systems.
4.4 Experimental and Diagnostic Uses
(1) In molecular biology and microbiology, lysozyme is a standard tool enzyme for lysis of Gram-positive bacterial cell walls and is widely used for plasmid preparation, genomic DNA extraction, RNA isolation, and recombinant protein production. In certain Gram-negative bacteria, combining lysozyme with EDTA to increase outer membrane permeability can also enhance lysis efficiency and facilitate preparation of protoplasts or analysis of cell wall components.
(2) In diagnostics and formulation quality control, lysozyme activity assays can serve as indicators of how well a given formulation preserves protein structure. Some novel biosensors also exploit the specificity of lysozyme for bacteria or peptidoglycan to develop platforms for detecting viable bacterial loads or assessing antimicrobial efficacy.
4.5 Biomaterials and Tissue Engineering
(1) In biomaterials and tissue engineering, lysozyme can be immobilized or encapsulated in polymer matrices, surface coatings of metallic implants, or hydrogels to confer antimicrobial properties and inhibit biofilm formation. Surface modification of implants with lysozyme may reduce postoperative infection risk and extend device lifespan.
(2) By combining lysozyme with chitosan, gelatin, or biodegradable polymers, wound dressings and tissue-engineering scaffolds with integrated antimicrobial, moisturizing, and pro-healing functions can be developed, offering potential benefits for chronic ulcers, burn wounds, and other hard-to-heal lesions.
V. Safety, Limitations, and Application Strategies
5.1 Safety and Potential Allergenicity
(1) Overall, lysozyme is of natural origin and exhibits a favorable safety profile in toxicological evaluations, with a long history of use in food and topical formulations. However, egg-white–derived lysozyme is inherently an egg protein and may carry a risk of hypersensitivity in individuals allergic to egg proteins. Therefore, when used in foods or oral formulations, ingredient labeling must comply with relevant regulations, and product design should take the characteristics of the target population into account.
(2) For highly sensitive populations and scenarios requiring high doses or long-term administration, recombinant human lysozyme or human-like variants produced via genetic engineering may reduce immunogenicity associated with heterologous proteins. Nonetheless, their safety and tolerability must still be systematically verified in preclinical and clinical studies.
5.2 Spectrum Limitations and Synergistic Strategies
(1) Because the primary target of lysozyme is cell wall peptidoglycan, it is highly effective against Gram-positive bacteria, whereas its direct bactericidal activity against Gram-negative bacteria with intact outer membranes, mycoplasmas, and intracellular pathogens is limited. In complex microbial environments, lysozyme alone is often insufficient to cover the full spectrum of target organisms.
(2) In practice, synergistic strategies are commonly employed, combining lysozyme with chelating agents, organic acids, nisin, antimicrobial peptides, or plant-derived bioactives. By disrupting outer membranes, lowering pH, interfering with metabolism, and disturbing biofilms through multiple mechanisms, these combinations can enhance overall antimicrobial and preservative efficacy while reducing individual component dosage and resistance risk.
5.3 Formulation Considerations and Practical Use
(1) In formulation development, the sensitivity of lysozyme to pH, temperature, and ionic strength must be carefully considered. For foods or products that undergo high-temperature sterilization, lysozyme is preferably added after cooling, or introduced as a sterile-filtered post-addition solution to avoid significant activity loss during heat processing. Neutral or mildly acidic buffers are typically chosen for dissolving lysozyme, and long-term storage is recommended at low temperature, protected from light, and with minimal freeze–thaw cycles.
(2) When used in complex matrices, attention must be paid to the influence of excipients and matrix components on both activity measurements and practical efficacy. High salt, high sugar, or high protein content may interfere with lysozyme interaction with bacterial cell walls and reduce lytic efficiency. Therefore, in activity assessment and formulation optimization, suitable assay methods and evaluation criteria should be established that reflect the actual application system.
VI. Related Aladdin Products
Catalog No. | Product Name | Category | Source/Application | Remarks |
Egg White Lysozyme, Ultra Pure Grade | Native enzyme/protein | Derived from chicken egg white; preparation of high-purity lysozyme | Ultra-pure enzyme preparation suitable as a reference standard and for experiments requiring high purity | |
Lysozyme from Egg White | Native enzyme/protein | Derived from chicken egg white; bacterial cell wall lysis and lysozyme assays | High activity; commonly used for preparation of protoplasts, cell lysis, and antimicrobial studies | |
Lysozyme from Egg White | Native enzyme/protein | Derived from chicken egg white; general lysozyme and cell wall degradation | Suitable as a routine lytic tool enzyme in microbiology and biochemical experiments | |
Lysozyme from Egg White (Purified, Salt-Free) | Native enzyme/protein | Derived from chicken egg white; purified and desalted | Suitable for systems sensitive to ionic background, such as structural, biophysical, or kinetic studies | |
Recombinant Human Lysozyme Protein | Recombinant protein | Recombinant human lysozyme for functional and mechanistic studies | Applicable to receptor-binding assays, antimicrobial activity evaluation, and in vitro models as a substitute for native human lysozyme | |
Lysozyme from Human Neutrophils | Native protein | Human neutrophil-derived lysozyme close to physiological state | Suitable for studies of innate immunity, inflammation, and neutrophil granule proteins |
Overall, lysozyme is a classical natural antibacterial enzyme and a model protein for structure–function studies, with a solid foundation of research in molecular structure, catalytic mechanism, safety, and application patterns. Thanks to its well-defined target, favorable physicochemical stability, and high safety, lysozyme has established mature application systems in food preservation, pharmaceutical formulations, feed additives, biomaterials, and laboratory tool enzymes, and holds irreplaceable advantages in the development of natural preservatives, antibiotic-reducing farming strategies, and functional biopreparations. Looking ahead, advances in protein engineering, materials science, and delivery technologies will continue to drive engineering optimization of lysozyme, the design of combination formulations, and the construction of smart functional materials. These developments will further unlock the potential of this traditional natural antibacterial enzyme in high value-added scenarios and provide important technological support for building safer, greener, and more sustainable biological control systems.
Aladdin: https://www.aladdinsci.com/