LysozymeCytosolic enzyme(muramidase) orN-acetyl cytosolic glycan hydrolase（N-acetylmuramide glycanohydrlase），It is an alkaline enzyme that can hydrolyze mucopolysaccharide in bacteria.Lysozyme mainly destroyscell wallInN-acetyl muramic acidAnd N-AcetylaminoBeta-1,4 glycosidic bond between glucose, which decomposes insoluble mucopolysaccharides in cell wall into soluble mucopolysaccharidesGlycopeptide, resulting in cell wall rupture and the escape of the contents, which causes bacteria to dissolve.Lysozyme can also directly combine with negatively charged viral protein, and form a complex with DNA, RNA, and apo protein to inactivate the virus.[1]This enzyme is widely found in many human tissues, such as egg white of birds and poultry, tears of mammalssaliva, plasma, milk and other liquids, as well as microorganisms also contain this enzyme, of which egg white is the most abundant.[2]According to different sources, they can be divided into four categories, namely plant lysozyme, animal lysozyme, microbial lysozyme and egg white lysozyme.[3]
Pure lysozyme is white, yellowish or yellow crystal or amorphous powder, no odor, slightly sweet, soluble in water, insoluble in acetone and ether.Lysozyme is easy to be destroyed when encountering alkali, but in acidic environment, lysozyme is very stable to heat. When pH value is 4-7, it can still maintain its activity well after being treated at 100 ℃ for 1 min; when pH value is 3, it can withstand 100 ℃ heating treatment for 45 min.[4]
The chemical properties of lysozyme are very stable. When the pH value changes sharply within a certain range, its structure is almost unchanged.The critical point of irreversible denaturation of lysozyme is 77 ℃. With the change of solvent, the critical point of irreversible denaturation also changes. When the pH value of lysozyme solution is less than 1, the critical point of irreversible denaturation decreases to 43 ℃.[5]
The optimal pH of lysozyme is 5.3~6.4, which can be used for low acid food preservation;[7]Lysozyme aspreservativeHigh safety, can be frozen or dried, and stable vitality.[7-8]
The sensitivity of lysozyme to several denaturants is: dioxane>dimethylacetamide>dimethylformamide>acetone, and it decreases linearly with the increase of solvent dosage.[5]
Bacteriostatic mechanism
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Lysozyme can effectivelyhydrolysisBacteriacell wallOfPeptidoglycanAnd its hydrolysis site isN-acetyl muramic acid(NAM) andN-acetylglucosamineBeta-1.4 glycoside bond between the four carbon atoms of (NAG).PeptidoglycanIt's bacteriacell wallIt is composed of NAM, NAG and peptide "tail" (consisting of four amino acid residues). NAM and NAG are connected by β - 1.4 glycosidic bond. The peptide "tail" is connected to the third carbon atom of NAM by D-lactyl carboxyl group. The peptide tails are connected by peptide "bridge"（Peptide bondOr a few amino acids), NAM, NAG, peptide "tail" and peptide "bridge" together form a multi-layer network structure of peptidoglycan, which serves as the skeleton of cell wall. Any chemical bond break in the above structure can lead toBacterial cell wallDamage.aboutGram positive bacteria（G+）, such asMicrococcus luteus, Bacillus subtilis or Micrococcus muralis, andGram negative bacteria（G-）, such asEscherichia coli、Proteus、Shigella, Pneumonia, etc., in the cell wallPeptidoglycanDifferent content, G+The bacterial cell wall is almost entirely composed of peptidoglycans, while G-Only the inner wall layer of bacteria is peptidoglycan, so lysozyme can destroy G+The cell wall of bacteria is smaller than that of G-Bacteria are strong.[5]
Preparation method
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At present, lysozyme can be extracted from egg white and eggshell membrane. The common methods include affinity chromatography, ion exchange resin, direct crystallization and polyacrylic acid precipitation.[9]
Affinity chromatography
Affinity chromatography uses proteins and enzymesBiological specificityIt is a chromatographic technology designed for the specific affinity between protein or enzyme and its ligand.After the formation of enzyme substrate complex, pure enzyme can be obtained by separating the complex under certain conditions.The commonly used adsorbent is chitin and its derivatives, such as chitin powder, carboxymethyl chitin, chitin embedded cellulose, deamination chitin powder, N-acylated chitosan, deamination regenerated chitin gel.[9]
Ion exchange resin method
Ion exchange methodIs usingIon exchangerIt can be separated by the exchange reaction generated with the ions in the solution. The separation effect is good, and it is widely used for the enrichment of trace components.The general process is: fresh eggs - pretreatment - agitation adsorption - column elution - isoelectric precipitation - dialysis - spray drying - finished products.[9]
Lysozyme has positive charge in a wide range of pH values below the isoelectric point, and can be adsorbed on weak acid cation exchange resin. After elution, it can be salted out to obtain lysozyme precipitation, and then refined to obtain finished products.Egg white absorbs 724 resin, washing buffer elution, ammonium sulfate solution salting out dialysis, NaOH is used to remove alkaline protein, lyophilize lysozyme, lyophilize powder precipitation drying to obtain lysozyme.Add 540kg of fresh egg white to the treated 80kg 724 resin at 5-10 ℃, stir and absorb it for 6h, and let it stand overnight at 0-5 ℃.Pour out the upper layer of egg white, centrifuge and dry the resin, repeatedly wash the attached egg white with distilled water, then load the resin into the column, wash the resin with about 150L of 0.15mol/L, pH=6.5 phosphoric acid buffer solution, then wash it with about 600L of 10% ammonium sulfate solution, and collect the eluant.Add ammonium sulfate into the eluent, so that the final ammonium sulfate content is 40%, and white precipitate is generated, which is left in the cold place overnight.Siphon the supernatant, filter and drain the sediment, then dissolve the sediment in a paste form with twice as much distilled water, put it into a dialysis bag, dialysis the distilled water for about 24 hours at about 5 ℃, and change the water for 2-3 times in the middle.Centrifuge to remove the sediment, wash the sediment with a small amount of water once, and combine the washing solution with the centrifugal solution.Then slowly add 1mol/L into the dialysatesodium hydroxideAt the same time, continuously stir the solution to make the pH value rise to 8.0-9.0. If there is white precipitate, remove it by centrifugation.Then adjust pH=5.0 with 3mol/L hydrochloric acid and freeze dry to obtain white flake lysozyme.Alternatively, adjust the pH of the centrifugal solution to 3.5 with 3mol/L hydrochloric acid, slowly add 5% solid sodium chloride under stirring, and leave it at about 5 ℃ for 48h. Centrifuge, wash the precipitation with 0 ℃ acetone, and dry it to obtain lysozyme.[9]
Direct crystallization of salt
Add a certain amount of iodide or carbonate and other salts into the egg white, and adjust the pH value to 9.5-10.0. The lysozyme will slowly precipitate in the form of crystal, while most of the protein is still in the solution.After ultrafiltration concentration and desalting, in order to obtain a more pure product, crystal purified enzyme solution is used for ultrafiltration treatment, pH 9.5 is adjusted with NaOH, and centrifugation is used for removal.The supernatant was stirred slowly, NaCl was added to 5%, and left for a week to obtain coarse crystals.The crystal is dissolved in pH 4.6 acetic acid water, and the insoluble matter is separated and recrystallized. The final yield of crystallization is about 60%.That is, nearly 1.3 g of recrystallized product can be obtained per kilogram of egg white, and the activity is determined to be 8000U/mg.[9]
Polyacrylic acid precipitation method
The polyacrylic acid precipitation method is to extract and filter the enzyme containing eluate, first go through the adsorption step, that is, adjust the pH of the filtered clear solution to 6.0 with 20% NaOH solution, and keep stirring in the process of adjusting the pH value.A small amount of milky white precipitate is precipitated, and then 15% polyacrylic acid is added, and white precipitate is precipitated immediately. Add it until the pH value is 3.0, and let it stand for 17 hours for static precipitation to obtain milky white colloidal substance adhered to the bottom.The second step is to dissociate the lysozyme polyacrylic acid condensate obtained in the previous step.Add a small amount of distilled water and use 0.5 mol/L sodium carbonate to dissolve it. After transfer, adjust the pH value to 9.5.Add 5% CaCltwoThe solution dissociates lysozyme polyacrylic acid until there is no precipitation, and then filters to obtain a clear solution.The third step is salting out. Add 5% NaCI solution to the obtained clarified solution and stir it evenly.Put it in the refrigerator and adjust the temperature to 0 ℃.After crystal precipitation, useAnhydrous ethanolWash several times, setConstant temperature incubatorDry and weigh at 40 ℃.[9]
Storage method
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It is recommended to store in a cool and dry environment away from light. Storage temperature: below zero.[3]
Over storage or unfavorable storage conditions will reduce the enzyme activity to varying degrees;If the temperature and humidity are too high, it is necessary to properly increase the usage.[3]
application area
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Medical field
It can be used as a natural anti infective substance with bactericidal effect.It has antibacterial, antiviral, hemostatic, detumescent and analgesic effects, and can accelerate tissue recovery.Lysozyme Buccal Tablets For acute and chronic pharyngitismouth ulcerEtc.[10]
side effect
Occasionally, there are mild allergic reactions, rashes, etc.[10]
Food field
It is effective against gram-positive bacteria, aerobic spore forming bacteriaBacillus subtilisLichen typeBacillusAll of them have antibacterial effect, but they are not effective for those without cell wallHuman cellsThere will be no adverse effects.Therefore, it is suitable for anticorrosion of various foods.[11]In addition, the enzyme can also kill intestinal putrefactive cocci, increase intestinal anti infectivity, and promote infant intestinal bifidosisLactobacillusProliferate and promote the coagulation of casein, which is beneficial to digestion, so it is also a good additive for baby food and drinks.[12]
Bioengineering
The characteristics of lysozyme specific hydrolysis of bacterial cell wall are helpful to understand the structure of bacterial cell wall:Protoplast, available forMicrobial classificationas well asMicrobial breedingAnd other academic research.In recent years, lysozyme has become an indispensable tool enzyme for cell engineering and genetic engineering, which is used to extract and manufacture enzymes, nucleic acids and active peptides.[4]
urine Lysozyme
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principle
Lysozyme frommonocyte、NeutrophilsIt is an enzyme that can dissolve certain bacteria.FermentableGram-positive cocci MuralAcetylaminoPolysaccharide component, which can break the cell wall.Its molecular weight is 14000-15000 Da, which can be obtained fromGlomerular basement membraneAfter filtration, more than 90% can be reabsorbed by renal tubules, so there is little or no lysozyme in urine.Use a bacterial suspension as the matrix, add the sample to be tested and keep it warm for a certain time. If the sample contains lysozyme, the bacteria will be dissolved, and the turbidity of the bacterial suspension will decrease or become clear. The photoelectric turbidimetry can be used to measure its turbidity change, or the plate method can be used to measure the size of its lysosphere.[13]
Reference value of urinary lysozyme: the concentration in urine is 0-2mg/L.[13]
