Green additive lysozyme and its application

Lysozyme (lysozyme, ec 3.2.1.17) is also known as muramidase or n-acetylmuramide glycanohydralase. In 1922, British bacteriologist a. Fleming discovered that there are enzymes in the saliva and tears of humans that dissolve the bacterial cell wall. Because of its bacteriolytic activity, it was named lysozyme. Since then, the presence of lysozyme has also been found in various tissues and secretions of humans and animals, as well as certain plants and microorganisms. With the deepening of research, it has been found that not only lysozyme that dissolves the bacterial cell wall, but also species that act on the cell wall of the fungus, as well as a better understanding of its mechanism of action. In recent years, according to the bacteriolytic properties of lysozyme, people have applied it in medical treatment, food preservation, animal husbandry and biological engineering, and have certain application value.

Lysozyme is divided into two major groups according to the microorganisms it acts on, namely bacterial cell wall lysozyme and fungal cell wall lysozyme. There are two kinds of bacterial cell wall lysozyme, one is a cell wall lysing enzyme that acts on β-1,4 glycosidic bonds, and the other is a cell wall lysing enzyme that acts on the "tail" and amide part of the peptide. Fungal cell wall lysozymes include yeast cell wall lytic enzymes and fungal cell wall lytic enzymes.

Lysozyme is widely distributed in nature. It can be found in human tissues and secretions. It is also found in animal tissues. It also exists in other plant tissues and microbial cells. According to different sources, the nature and mechanism of action are slightly different.

Egg lysozyme accounts for 3.4% to 3.5% of the total egg white protein. As a typical representative of lysozyme, it is currently the focus of research and is one of the most well-known lysozymes. It consists of 18 129 amino acid residues, has 4 ss bonds, a molecular weight of 14,000, an isoelectric point of 11.1, an optimum temperature of 50 °C, and an optimal pH of 6 to 7, and its chemical properties are very stable. When the pH changes drastically within the range of 1.2 to 11.3, the structure remains stable. It is also stable under heat, and does not lose its activity when it is treated for 1 min at pH 4 to 7 and 100°C. It is a stable alkaline protein, but its thermal stability is poor under alkaline conditions.

The egg whites of other birds such as cockroach, guinea fowl, and turkey are also isolated and purified from lysozyme, which is very similar to the egg white lysozyme activity. It is also composed of 129 amino acids. Although the arrangement order is different, the amino acid of the active site is different. The arrangement is roughly the same.

Human lysozyme exists in tears, saliva, nasal mucus, milk and other secretions as well as lymph glands, white blood cells, liver, kidney, lymphoid tissue, 1ml tears contain 7mg lysozyme, 1ml milk contains 0.1 ~ 0.5mg. human lysozyme Consisting of 130 amino acid residues, it has 4 ss bonds and a molecular weight of 14600. Its bacteriolytic activity is 3 times higher than that of egg white lysozyme.

For mammalian lysozyme, lysozyme has been isolated from the milk of cattle, pigs, cats, rabbits, monkeys, horses, sheep and other animals. Its chemical properties are similar to that of human lysozyme, but its structure is not yet clear, and its bacteriolytic activity It is also about 3,000 times lower than human lysozyme. Zeng Lin (1999) used agar plates to determine the content of lysozyme in rabbit colostrum. The results showed that the content of lysozyme in colostrum was (7.96±2.01)μg/ml and the content of lysozyme in normal milk was (5.01±1.32)μg/ml. The mechanism of action of human and mammalian lysozyme was the same as that of egg white lysozyme.

At present, lysozyme has been isolated from papaya, fig, phthalocyanine, barley and other plants, and its molecular weight is relatively large, approximately 24000 to 29100. The bacteriolytic activity of plant lysozyme against E. terminalis does not exceed 1/1 of hen egg lysozyme. 3, but the decomposition activity of colloidal chitin is 10 times that of egg white lysozyme. Zhou Zewen (1994) isolated lysozyme from cabbage leaves showed that the enzyme specific activity was 3414.6u/mg and the purification factor was 197.4. Vegetable lysozyme had activity at a wide range of temperature and pH, and the optimum temperature was 60°C. The optimum pH value was 5.8. Gao Xiangyang (1997) studied radish lysozyme against Staphylococcus aureus, Staphylococcus aureus, and B. subtilis, three Gram-positive bacteria and proteobacteria, E. coli, and Salmonella typhimurium. The bacteriostatic effect of Pasteurella multocida, Salmonella pullorum, Aerogenous gram-negative gram-negative bacteria, and Saccharomyces cerevisiae, Mucliaria militaris, Rhizopus nigrum, Aspergillus niger and Penicillium sp. At the same time, the bacteriostatic effect of radish lysozyme on six phytopathogenic bacteria such as soft rot bacterium, citrus canker, tomato bacterial wilt, rice stripe, rice leaf blight and tobacco bacterial wilt was studied. Tests have shown that radish lysozyme has different degrees of bacteriostasis on the tested strains.

At present, lysozyme produced by microorganisms is divided into 7 categories: 1 within the n-acetyl ketamine enzyme, this enzyme is the same as the egg lysozyme, destroy the β-1,4 glycosidic bond in the bacterial cell wall peptidoglycan; 2 amidase, Cut off the n-acetyl muramic acid-l-alanine bond between the nam of the bacterial cell wall peptidoglycan and the peptide "tail"; 3 Endopeptidases break the peptide bond in the peptide "tail" and the peptide "bridge" ;4β-1,3,β-1,6 glucanase and mannanase, this enzyme breaks down the cell wall of yeast cells; 5 chitinase, together with glucanase, can decompose mold and yeast; 6 Phosphomannosidase, in conjunction with glucomannanase, can decompose protoplasts; 7 chitosanase, mainly decompose mucor and rhizopus.

The enzyme is a specific enzyme that is infected and induced by phage, but the enzyme is not present on uninfected host cells.

Lysozyme is an alkaline hydrolase that hydrolyzes mucopolysaccharides. This type of mucopolysaccharide is one of the main components of the bacterial cell wall. The enzyme catalyzes the hydrolysis of n-acetylmuramic acid and n-acetylglucosamine in the cell wall. The β-1,4 glycosidic bond breaks down insoluble polysaccharides of the cell wall into soluble glycopeptides, and the bacterial contents escape to dissolve the cell walls. Lysozyme can directly hydrolyze gram-positive bacteria. In the presence of secretory immunoglobulin a and complement, it can also hydrolyze gram-negative bacteria such as E. coli. In addition, it can also be combined with a variety of acidic substances that induce inflammation, inactivated, and can enhance the efficacy of antibiotics and other drugs, improve the mucopolysaccharide metabolism of the tissue matrix, so as to achieve the purpose of anti-inflammatory and tissue repair.

Liang Aihua (2001) investigated the effects of egg lysozyme and β-lactam antibiotics on the growth and endotoxin release of E. coli at different temperatures. RESULTS: At different culture temperatures, ampicillin or cefotaxime 50 μg/ml could all lead to bacterial lysis and induce escherichiacoli to release large amounts of endotoxin into the culture supernatant. These culture supernatants were cultured in macrophages in vitro. A large number of tumor necrosis factor (tnfα) and interleukin 6 (il-6) production can be induced on the cells. The combination of hen egg white lysozyme and β-lactam antibiotics can prevent bacteria from lysing, reduce the release of bacterial endotoxin, and reduce the production of macrophages tnf- and il-6.

Lysozyme can interact directly with negatively charged viral proteins and form double salts with DNA, RNA, and apoproteins, inactivating viruses. The enzyme can also prevent and treat viral hepatitis, especially the effect on post-transfusion hepatitis and acute hepatitis. It also has anti-influenza virus activity in the body, and its complex with cholate can strongly inhibit the growth of influenza virus and adenovirus, and can prevent herpes virus infection.

As one of the body's non-specific immune factors, lysozyme participates in various immune responses of the body, and plays an important role in maintaining the physiological balance of the body in the normal defense function and non-specific immunity of the body. It can improve and enhance macrophage phagocytosis and digestive function, activate leukocyte phagocytosis, and can improve leukocyte reduction caused by cytostatic agents, thereby enhancing the body's resistance.

Lysozyme also has the function of activating platelets, which can improve the local blood circulation disorders, secrete pus, and enhance local defense functions, thereby reflecting its role in stopping bleeding and swelling. It can also act as a host resistance factor that protects tissues locally.

Lysozyme in infants can directly or indirectly promote the proliferation of infant intestinal bacteria Lactobacillus bifidus, promote infant digestion and absorption, can promote the normalization of artificial feeding infants intestinal bacteria; can enhance the serum sterilized protein (properdin), γ- The ability of in vivo defense factors such as γ-globulin to resist infection, especially for preterm infants, has the effect of preventing weight loss, preventing digestive diseases, and increasing body weight.

The bacterial cell wall is composed of murein. The murein is a polymer composed of n-acetylglucosamine and n-acetylmuramic acid alternately. The acid residues on the wall can be The linked polypeptide is called peptidoglycan. Peptidoglycan is the main component of bacterial cell wall. It is composed of nam, nag and peptide "tail" (usually 4 amino acids). nam and nag are linked by β-1,4 glycosidic bond, peptide "tail" is passed The d-lactoyl carboxyl group is attached to the 3rd carbon atom of nam and the peptide tails are connected by a peptide "bridge" (peptide bond or a few amino acids), and nam, nag, peptide "tail" and peptide "bridge" The multi-layer network structure that constitutes peptidoglycan, as the skeleton of the cell wall, can cause damage to the bacterial cell wall by breaking any chemical bond in the above structure. Lysozyme efficiently hydrolyzes peptidoglycans of the bacterial cell wall whose hydrolysis site is between the 1 carbon atom of n-acetyl muramic acid (nam) and the 4 carbon atom of n-acetylglucosamine (nag). 1,4 glycosidic bond, as a result, the bacterial cell wall becomes loose, loses its protective effect on the cell, and finally the cell lyes and dies.

For g+ bacteria and g-bacteria, the content of peptidoglycan in the cell wall is different. The g+ bacterial cell wall is almost entirely composed of peptidoglycan, whereas the g-bacteria has only peptidoglycan in the inner wall layer. Therefore, lysozyme can only destroy the g+ bacteria. The cell wall, but little effect on g-bacteria.