Lysozyme | ChemTalk

Core Concepts

In this article, you will explore the protein lysozyme through its structure, classification and antibacterial function.

What is a Lysozyme?

Lysozyme is an antibacterial enzyme naturally present in animals and humans as a component of the innate immune system to fight against microorganisms. It is a glycoside hydrolase also known as muramidase or N-acetylmuramic acid hydrolase. As the latter name indicates, it functions by hydrolyzing/breaking the glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in the peptidoglycan of bacterial cell walls. This hydrolysis affects the integrity of the protective cell wall of bacteria, thereby causing their lysis and death. 

Lysozyme was discovered by Alexander Fleming in 1922, the identifier of penicillin, when he accidentally recognized that a drop of his nasal mucosa caused the lytic death of bacteria. He named this antibacterial element “lysozyme”. Interestingly, lysozyme was the first enzyme and the second protein to have its structure elucidated through X-ray diffraction techniques. 

Structure and Function

Lysozyme is a small monomeric protein with a molecular weight of 11-22 kDa. The wide range in its mass is attributed to the different types of lysozymes found in various species. Its structure was first characterized in the 1960s by X-ray crystallography. The particular structure of lysozyme imparts it the antibacterial function. A type of lysozyme, chicken lysozyme, is made of a single polypeptide chain of 129 amino acids stabilized by disulphide bonds among its eight cysteine residues. The enzyme’s N- and C-terminal residues constitute lysine and leucine, respectively. The six helix regions along with disulphide linkages confer this protein a high thermal stability. The binding site exists between the α and β domains in a large cleft, where the amino acids glutamate (Glu35) and aspartate (Asp52) constitute the active site. These amino acids are crucial for lysozyme’s catalytic activity against the substrate peptidoglycan. 

Primary structure of lysozyme showing amino acids in blue and disulphide linkages in red (Left). 3D model of chicken lysozyme with Glu35 and Asp52 in its active site (Right).

As mentioned earlier, lysozyme is an antibacterial agent. For this, it targets the main constituent of bacterial cell walls i.e. peptidoglycan. Peptidoglycan is a polymeric layer of sugars and amino acids in the cell wall surrounding the plasma membrane. It comprises polysaccharide chains made of alternating N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) sugar residues attached by β-1,4 glycosidic linkages. With each NAM, a pentapeptide (chain of five amino acids) is attached to allow cross linking between the polysaccharide chains. This way, peptidoglycan forms a protective meshwork, which contributes to the bacterial cell shape and integrity. 

Structural organization of peptidoglycan in a bacterial cell wall

Upon exposure to a bacterium, lysozyme as a part of the immune response, comes into action. The active site of lysozyme binds the substrate peptidoglycan (its polysaccharide component) in its cleft. There, the amino acids Glu35 and Asp52, the crucial elements of the active site structure and function, initiate the hydrolytic antibacterial activity of lysozyme. The reaction mechanism involves the transfer of a proton from Glu35 to the oxygen of β-1,4 glycosidic bond between NAM and NAG. This cleaves the C-O glycosidic bond, hence releasing NAG side of the polysaccharide chain, and forming oxocarbenium ion of NAM. The latter interacts with Asp52 producing a glycosyl-enzyme intermediate. A water molecule gives its hydroxyl ion to the oxocarbenium ion to release the NAM side of the polysaccharide chain from the enzyme. This yields a hydrolyzed/cleaved peptidoglycan chain and a freely available lysozyme ready to attack another glycosidic bond. 

Hydrolytic activity of lysozyme’s active site residues on the β-1,4 glycosidic bonds in polysaccharide chain of peptidoglycan (substrate)

Mechanism of Antibacterial Action

Bacteria mostly grow in hypotonic environments (less solute concentration outside than inside the cell). Under such conditions, water moves inside the bacterial cell by a process termed as osmosis. The inward flow of water generates a high internal osmotic pressure, which could burst the cell. However, the crosslinked peptidoglycan mesh in the cell wall of bacteria protects them from bursting/lysis of the cell. This allows bacteria to survive and reproduce in such environments.

Upon lysozyme action, the degradation of peptidoglycan weakens the bacterial cell wall. In case of gram positive bacteria (a type of bacteria having a thick but single layer of peptidoglycan as their cell wall), hydrolysis by lysozyme completely removes its cell wall resulting in a protoplast. A protoplast is a cell without a protective cell wall around the plasma membrane. Such a cell is osmotically sensitive, which when placed in a hypotonic solution, experiences an uncontrolled influx of water. Increased osmotic pressure inside the cell causes its lysis and ultimately the death of bacterium. 

The antibacterial activity of lysozyme is most effective against the gram positive bacterial cell walls. On the contrary, gram negative bacteria (another bacterial type having a sandwiched peptidoglycan between two cell membranes) are less affected by lysozyme action. This is due to the presence of lipopolysaccharides on their cell wall, which to some extent inhibit the enzyme from attacking their peptidoglycan.

Mechanism of antibacterial activity of lysozyme by hydrolysis (against gram positive cell wall)

Sources and Types

Lysozyme is found in tears, saliva, mucus, blood serum, human and cow milk, egg-white of birds as well as in invertebrates and plants. It is also present in the cytoplasmic granules of macrophages and polymorphonuclear neutrophils (types of white blood cells). Among all these sources, lysozyme is most abundant in chicken egg-white constituting 3.5% of the total egg-white proteins. Despite its hydrolytic nature, this enzyme is non-toxic to humans and animals as it is only specific to peptidoglycan, a characteristic of bacterial cell walls. 

On the basis of origin, structural features and function, lysozymes have been classified into three main families. These include:

• C-type: This family refers to the chicken or conventional type, including the human and chicken egg-white lysozymes. Among the two, human lysozyme has a three-fold greater antibacterial activity than the chicken lysozyme. C-type lysozymes constitute a single ~14.3 kDa polypeptide chain containing alpha and beta strands as well as disulphide linkages. Their active site residues involved in the catalysis of β-1,4 glycosidic bond include Glu35 and Asp52.
• G-type: Lysozymes in the goose egg-whites as well as those found in ostrich, black swan and some fish species, belong to this type. These have a greater molecular weight of ~21 kDa with a different folding of the polypeptide than the c-type. G-type lysozymes also cleave the same β-1,4 glycosidic linkage but use Glu73 as the catalytic residue.
• I-type: This family includes the lysozymes from invertebrates, plants and bacteriophages. These range in a mass of ~11-15 kDa due to variations in their amino acid sequence in different invertebrates as well as in the same species (isoforms). Unlike the c-type lysozymes, their structure does not contain disulphide linkages. Their catalytic amino acids vary e.g. Glu and Asp residues. Interestingly, i-type lysozymes also possess chitinase activity by digesting chitin.

What are its Applications?

The antibacterial role of lysozyme and the large number of its available natural sources have made this enzyme a centre of interest in different applications. Chicken egg-white being its richest and easily accessible source, is widely exploited for practical uses. Lysozyme is employed in medicine as an alternative antibiotic to treat and prevent traditional antibiotic-resistant bacterial infections. During wound healing, dressings coated with lysozymes help prevent infection to the wound. It also serves biomedical applications as in the removal of biofilms accumulated on the body implants. In lab settings, lysozyme is useful for collecting the periplasmic proteins of bacteria e.g. E. coli, by lysis. Moreover, many food and beverage industries utilize lysozymes as a food preservative to increase the shelf-life of their products. In addition, lysozyme also finds its applications in the agriculture sector for crop protection.