Applications of Lysozyme in Biotechnology

Its an enzyme that helps protect the body from infection by breaking down the cell walls of certain bacteria, causing them to burst and die.

Function:

  • Bacteriolytic Activity: Lysozyme hydrolyzes the glycosidic bonds in peptidoglycan, a major component of bacterial cell walls, leading to bacterial cell lysis.
  • Immune Defense: It is found in various secretions such as tears, saliva, mucus, and human milk, where it helps protect against bacterial infections.

Sources:

  • Animal and Human Secretions: Tears, saliva, mucus, and human milk.
  • Egg White: Hen egg white contains a high concentration of lysozyme, which helps protect the developing chick from bacterial infections.

Its derived from egg white is a naturally occurring enzyme with significant antibacterial properties. Here are some key points about it:

Source and Composition:

  • Source: Egg white (albumen) of chickens.
  • Concentration: Egg white contains about 3.5% lysozyme.
  • Molecular Weight: Approximately 14.3 kDa (kiloDaltons).

Function:

  • Bacteriolytic Activity: Lysozyme breaks down the peptidoglycan layer of bacterial cell walls, leading to cell lysis. This is particularly effective against Gram-positive bacteria1.
  • Antibacterial Properties: It has both bactericidal (kills bacteria) and bacteriostatic (inhibits bacterial growth) effects.

Applications:

  • Food Preservation: Used as a natural preservative in various food products such as cheese, tofu, seafood, meats, and sausages. It helps extend shelf life by inhibiting bacterial growth3.
  • Medical Uses: Employed in wound healing creams, eye drops, and some anticancer drugs.
  • Research: Utilized in molecular biology for DNA extraction and as an external standard in mass spectrometry.

Advantages:

  • Natural and Safe: Being a natural enzyme, it is generally considered safe for consumption and use in various applications.
  • Versatility: Effective over a broad pH range (6.0 to 9.0) and stable under various conditions.

Purification:

  • Methods: Its can be purified from egg white using techniques such as dialysis, precipitation, ion-exchange chromatography, and ultrafiltration.

Additional Information:

  • Stability: Its stable at low temperatures and can be stored in a lyophilized (freeze-dried) form for extended periods.
  • Unit Definition: One unit of lysozyme will lyse 0.6 µg of Micrococcus lysodeikticus per minute under specific conditions.

Lysozyme from egg white is a versatile enzyme with a wide range of applications in food preservation, medical treatments, and scientific research

 

Mechanism:

  • Hydrolysis: cleaves the (1→4)-β-linkages between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues in peptidoglycan.
  • Induced Fit:  binds to the peptidoglycan substrate, distorting it into a half-chair conformation, making the glycosidic bond more susceptible to hydrolysis.

Applications:

  • Food Industry: Used as a preservative due to its antibacterial properties.
  • Medical Research: Studied for its potential therapeutic uses and role in innate immunity.

Lysozyme was first discovered by Alexander Fleming in 1922, and it remains a molecule of significant scientific interest due to its multifunctional properties

Lysozyme can play a supportive role in PCR, particularly in the preparation of DNA samples from certain microorganisms. Here’s how it is used:

Role of Lysozyme in PCR:

  1. Cell Wall Digestion: Lysozyme breaks down the peptidoglycan layer of bacterial cell walls, particularly in Gram-positive bacteria. This helps to release the DNA from the bacterial cells.
  2. DNA Isolation: When combined with other reagents like Proteinase K, lysozyme helps to efficiently lyse bacterial cells and isolate the DNA, which can then be used as a template in PCR.
  3. Improved Yield: By breaking down the rigid cell walls, lysozyme ensures that more DNA is released, improving the yield and quality of the DNA template for PCR.

Applications:

  • Microbial DNA Extraction: Used in protocols for extracting DNA from bacteria, especially Gram-positive bacteria, to ensure efficient lysis and high-quality DNA for downstream applications like PCR.
  • Diagnostic Assays: Lysozyme can be used in conjunction with PCR-based detection methods to identify bacterial pathogens in clinical and environmental samples.

In summary, lysozyme aids in the efficient lysis of bacterial cells, making it easier to extract high-quality DNA for PCR amplification. This is particularly useful in microbial studies and diagnostic applications.

To use lysozyme in PCR, you primarily incorporate it in the DNA extraction process from bacterial cells, particularly when dealing with Gram-positive bacteria. Here’s a step-by-step outline:

Using Lysozyme for DNA Extraction:

  1. Preparation:
    • Prepare a lysozyme solution (typically 10 mg/mL in a suitable buffer, such as 10 mM Tris-HCl, pH 8.0).
  2. Cell Lysis:
    • Take your bacterial cell pellet and resuspend it in an appropriate buffer (like TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
    • Add an appropriate volume of the lysozyme solution to the resuspended cells (e.g., 100 µL of lysozyme solution per 1 mL of cell suspension).
  3. Incubation:
    • Incubate the mixture at 37°C for 30 minutes to allow the lysozyme to digest the cell walls.
  4. Additional Lysis:
    • After lysozyme treatment, proceed with additional lysis steps such as adding Proteinase K and SDS (sodium dodecyl sulfate) to break down proteins and release the DNA.
    • Incubate the mixture at 55°C for 1-2 hours.
  5. DNA Extraction:
    • Follow standard DNA extraction protocols, such as phenol-chloroform extraction or using a commercial DNA extraction kit.
  6. Purification:
    • Purify the extracted DNA to remove any contaminants and enzymes.
  7. PCR:
    • Use the purified DNA as a template for your PCR reaction.
    • Set up the PCR reaction with the necessary components (primers, DNA polymerase, dNTPs, buffer, and magnesium ions) and run the thermal cycler.

Notes:

  • Lysozyme is particularly useful for breaking down the tough cell walls of Gram-positive bacteria, which are more resistant to lysis than Gram-negative bacteria.
  • Always ensure that lysozyme and other enzymes are of high purity to avoid introducing contaminants that could interfere with your PCR results.

By incorporating lysozyme in the DNA extraction process, you can efficiently lyse bacterial cells and obtain high-quality DNA suitable for PCR amplification.

 

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