Lysozyme
Lysozyme, also known as muramidase or N-acetylmuramide glycanohydrolase, is an alkaline enzyme that can hydrolyze mucopolysaccharides in bacteria.
Mechanism of Lysozyme
Lysozyme mainly destroys the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decomposing the insoluble mucopolysaccharides of the cell wall into soluble glycopeptides, leading to the rupture of the cell wall and the escape of contents, thus dissolving the bacteria.
Sources of Lysozyme
This enzyme widely exists in various tissues of the human body, the egg white of birds and poultry, tears, saliva, plasma, milk and other fluids of mammals, as well as microorganisms, among which egg white has the most abundant content. According to different sources, it can be divided into four categories: plant lysozyme, animal lysozyme, microbial lysozyme and egg white lysozyme.
Figure 1: Protein Structure of Lysozyme
Discovery Process of Lysozyme
|
Person |
Time |
Contribution |
|
Charles Nicolle |
1907 |
First reported bacterial lysis phenomenon and put forward the hypothesis of cell wall degradation |
|
Lashchenko |
1909 |
Discovered that the antibacterial effect of egg white comes from enzymes |
|
Alexander Fleming |
1922 |
Discovered and named lysozyme, and confirmed its wide existence and antibacterial mechanism |
|
Robert Robinson |
1937 |
Achieved the initial purification of lysozyme |
|
David Phillips |
1965 |
Analyzed the structure of lysozyme, revealed the catalytic mechanism,and promoted the development of enzymology and protein chemistry |
Production Process of Lysozyme
1. Classic Process Flow for Extracting Lysozyme from Egg White
(Taking 1 kg of fresh egg white as raw material, which can be scaled up to ton level)
1）Pretreatment
- Breaking eggs → filtering → diluting with 1–2 times deionized water to reduce viscosity.
- Adjusting pH to 9.5 (with 1 mol/L NaOH) to fully swell egg white protein.
2）Heat-salt precipitate
-
Adding 5% NaCl (w/v) while stirring, then continuing to heat to 70–75 °C and maintaining for 10 min.
(Lysozyme is heat-resistant and does not denature under this condition)
(Impurities such as ovalbumin flocculate and precipitate due to thermal denaturation) - Immediately cooling to 25 °C in an ice bath, standing at 4 °C for 2 h, centrifuging the precipitated precipitate at 8000 rpm for 15 min to obtain the supernatant.
3) Isoelectric Point Crystallization
- Adjusting the supernatant to pH 10.5 with 1 mol/L HCl → saturating with 10% NaCl → standing at 4 °C for 48–72 h.
- Lysozyme crystals precipitate, collect the crystals by centrifugation, and then dissolve them in 0.1 mol/L phosphate buffer (pH 6.5).
4）Recrystallization and Decolorization
- Adjusting the pH of the solution to 9.5 again, repeating steps 2-3 to remove pigments and miscellaneousproteins.
- After secondary crystallization, the purity can reach 95% (SDS-PAGE), with a yield of 0.35–0.45 g/kg egg white.
5) Ultrafiltration/Dialysis for Desalination
Desalting with a 10 kDa ultrafiltration membrane or dialysis bag for 24 h, and freeze-drying to obtain white powder.
6）QC
- Specific activity: ≥9000 U/mg (Micrococcus lysodeikticus method)
- Chloride: <3.5%
Application of Lysozyme
1. Bacterial DNA Extraction
1）Take 0.5-2 mL of bacterial culture solution (no more than 2×109 cells at most), centrifuge at 10,000 rpm for 2 min, and discard the supernatant.
[Note]: The amount of bacterial cells should not be excessive, as excessive amount will seriously reduce the yield. The initial processing amount is related to bacterial density, species, etc.
2）First prepare lysozyme buffer: 20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton X-100, then add a certain amount of lysozyme dry powder to the lysozyme buffer to a final concentration of 20 mg/mL.
3）For bacteria, especially Gram-positive bacteria, add 180 μL of 20 mg/mL Lysozyme, oscillate to resuspend, and place in a 37°C water bath or metal bath for more than 30 min.
4）Add 20 μL of Proteinase K (20 mg/mL) and oscillate to mix well.
5）If residual RNA affects subsequent experiments, add 5 μL of RNase A (100 mg/mL) solution, oscillate to mix well, and place at room temperature for 5-10 min.
6）Add 200 μL of binding solution BD, immediately oscillate to mix well, and place in a 70°C water bath or metal bath for 10 min.
7）Perform column equilibration on the DNA adsorption column.
8）Add 100 μL of isopropanol to the above cooled sample pretreatment mixture, immediately oscillate to mix well, and briefly centrifuge to collect the liquid in the tube cap.
9）Add the above mixture to the DNA adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.
10）Add 500 μL of deproteinizing solution, centrifuge at 12,000 rpm for 30 sec, and discard the waste liquid.
11）Rinse twice.
12）Add 30-100 µL of eluent to the center of the DNA adsorption column, centrifuge, and recover the DNA solution.
2. Bacterial RNA Extraction
1）First prepare lysozyme buffer: 20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton X-100, then add a certain amount of lysozyme dry powder to the lysozyme buffer to a final concentration of 1 mg/mL.
2）For Gram-negative bacteria: add 100 μL of 1 mg/mL lysozyme working solution. Vortex for 10 sec, incubate at room temperature for 2 min. Repeat the oscillation and incubation 3 times.
3）For Gram-positive bacteria: add 100 μL of 1 mg/mL lysozyme working solution. Vortex for 10 sec, incubate at room temperature for 2 min. Repeat the oscillation and incubation 6 times.
[Note]: When the number of bacteria is more than 5×108 cells, the amount of lysozyme working solution added is 200 μL.
4）Briefly centrifuge to collect the cells, and discard the supernatant.
5） Vortex to resuspend and disperse the cells. Add 500 μL of lysis buffer, repeatedly pipette to mix well, and vigorously vortex until there are no obvious insoluble substances.
6）Add the above lysate to the DNA removal column, centrifuge at 12,000 rpm for 1 min, and retain the filtrate.
7）Add 0.5 times the volume of absolute ethanol, and immediately pipette to mix well.
8）Add the above mixture to the RNA adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.
9）Add 500 μL of deproteinizing solution, centrifuge at 12,000 rpm for 30 sec, and discard the waste liquid.
10）Rinse twice.
11）Add 30-50 µL of enzyme-free water to the center of the RNA adsorption column, centrifuge, and recover the RNA solution.
Ordering Information
|
Product Name |
Product Number |
|
Lysozyme |
10402ES |
|
10412ES |
|
|
10406ES |
|
|
MolPure™ Bacterial DNA Kit |
18806ES |
|
19301ES |