Peroxidase

Lactoperoxidase (EC 1.11.1.7, abbreviated as LP or LPO) is a hemoprotein and a peroxidase that is present in milk derived from animals. Lactoperoxidase, hydrogen peroxide, and thiocyanate can form a "lactoperoxidase system (LPS)." The enzyme system has antibacterial activity, can inhibit the growth of Gram-positive bacteria and negative bacteria under the condition of no refrigeration, prolong the shelf life of fresh milk, and has the effect of "cold sterilization". LPS in milk not only has an antibacterial effect, but also prevents the accumulation of peroxides, thereby avoiding cell damage caused by peroxide and protecting the breast.

Structure

LP is composed of a polypeptide chain comprising 612 amino acid residues, and is a glycoprotein with a molecular weight of approximately 78 kD with an isoelectric point of 9.6. LP contains a heme (methemoglobin) with an iron content of 0.07% and a carbohydrate content of 10%. The heme of the catalytically active center is composed of a protoporphyrin IX covalently linked to the polypeptide chain via an ester bond. Studies have found that the destruction of disulfide bonds in the LP structure and the presence or absence of heme directly affect the activity and stability of LP. LP is a common ingredient in human and bovine milk. It is one of the endogenous enzymes that can be found in the mammary gland, salivary gland, lacrimal gland and their secretions (milk, saliva and tears). The chemistry and immunological properties of LP present in the mammary gland, salivary gland, and lacrimal gland are similar.

Properties

LP participates in the natural defense system of host against invading microorganisms. LP has antibacterial activity, degrades various carcinogens and protects animal cells from peroxidation. LP is one of the thermostable enzymes in milk, and its destruction has been used as one of the indicators of pasteurization. Conventional pasteurization does not inactivate LP in milk, but the thermal stability of LP under acidic conditions (pH 5.3) may be reduced, probably due to the release of calcium from the LP which has a great impact to the thermal stability. LP is particularly sensitive to light in the presence of riboflavin.

Concentration and Activity

In bovine milk, LP is the second most abundant enzyme after xanthine oxidase, and its concentration is about 30 mg/L. It has been reported that the change in LP content is related to the strain, reproductive cycle, season, and feeding system of cattle. It is necessary to note that unlike other antibacterial proteins, the content of LP is lower in bovine colostrum, but the LP content increases rapidly to reach a maximum in 3~5 after delivery. The activity of LP in different sources of milk was different. The average activity of LP in bovine milk was 2.3 U/mL; the activity of LP in human milk was lower, 0.67-0.97 U/mL; the LP in guinea pig has the highest activity, reaching 22 U/mL. It has been reported that the enzyme activity of LP must reach 0.02 U/mL or more to have bacteriostatic activity.

Lactoperoxidase System

Lactoperoxidase system (LPS) includes three components, namely lactoperoxidase, thiocyanate (SCN¯) and hydrogen peroxide. Only when these three components work together, LPS has antibacterial activity. In practical applications, if the concentration of a certain component in the system is not enough, it needs to be added to ensure the antibacterial effect, which is called the “activation” of LPS. Among them, the concentration of lactoperoxidase should be not less than 0.02 U/mL. The concentration of natural LP in bovine milk is 1.4 U/mL, which can meet this requirement. Thiocyanate is widely present in tissues and secretions of animals. The concentration of thiocyanate in milk is only 3-5 μg/mL, which is a limiting factor for LPS activity. It has been reported that the concentration of thiocyanate required to activate LPS is about 15 μg/mL or more, so that exogenous thiocyanate needs to be added to activate LPS. In the milk that has just been extruded, the hydrogen peroxide content is only 1-2 μg/mL, and activation of LPS requires 8-10 μg/mL of hydrogen peroxide, so hydrogen peroxide must be supplied externally.

Catalytic Mechanism

Figure 1. Pathways in the lactoperoxidase-catalyzed reaction mechanism. (Seifu E. et al. 2005)

Peroxidation of H₂O₂ by LP can occur through three different cycles, resulting in divergent anti-microbial activities as follows:

A. In the presence of sufficient oxidizing halide or SCN¯ as 2-electron donor for Compound I, giving optimal activation of LP.

B. In the presence of insufficient halide or SCN¯ of appropriate redox potential, resulting in dominating 1-electron donors and accumulation of Compound II and reversible inactivation of LP.

C. In the presence of an excess of H₂O₂ resulting in the formation of Compound III, associated with irreversible inactivation of LP.

Antibacterial Function

The antibacterial properties of LPS are manifested in the bacteriostatic or bactericidal action of some microorganisms in milk. Its antibacterial mechanism is that the thiol group on the plasma membrane of the microbial cells is oxidized, resulting in the destruction of the plasma membrane structure, the leakage of potassium ions, amino acids and polypeptides. The uptake of glucose, amino acids, purines and pyrimidines by cells, and the synthesis of proteins, DNA and RNA are inhibited.

Different bacteria have different degrees of sensitivity to LPS. Gram-negative bacteria, such as Pseudomonas, Escherichia coli, and Salmonella, can not only be inhibited, but also be killed. Streptococcus and lactic acid bacteria can only be inhibited. The extent to which bacteria are affected by LPS may be related to the different cell membrane structure and characteristics of the bacteria. The cell membrane of Gram-negative bacteria is more susceptible to destruction by LPS than the cell membrane of Gram-positive bacteria. This causes some nutrients to leak, hindering the bacteria's own intake of nutrients, and ultimately leading to the death or decline of bacteria.

Application

Lactoperoxidase is an effective antimicrobial agent and is used as an antibacterial agent in reducing bacterial microflora in milk and milk products. Activation of the lactoperoxidase system by addition of hydrogen peroxide and thiocyanate extends the shelf life of refrigerated raw milk. It is fairly heat resistant and is used as an indicator of overpasteurization of milk. A lactoperoxidase system is claimed to appropriate for the treatment of gingivitis and paradentosis. Lactoperoxidase has been used in toothpaste or a mouthrinse to reduce oral bacteria and consequently the acid produced by those bacteria. A combination of lactoperoxidase, glucose, glucose oxidase (GOD), iodide and thiocyanate is claimed to be effective in the preservations of cosmetics. Antibody conjugates of glucose oxidase and to lactoperoxidase have been found to effective in killing tumor cells in vitro. In addition, macrophages exposed to lactoperoxidase are stimulated to kill cancer cells.

Reference

1. Seifu E, Buys E M, Donkin E F. Significance of the lactoperoxidase system in the dairy industry and its potential applications: a review. Trends in Food Science & Technology, 2005, 16(4):137-154.