Lysozyme Solution

Product No. L3790

From chicken egg white
Storage Temperature –20 °C

EC Number: 3.2.1.17
CAS #: 12650-88-3
Synonyms: Muramidase, Peptidoglycan N-acetylmuramoylhydrolase
E1%281.5 nm: 26.4 in 0.1 M KCl1
EmM (280 nm): 362
Isoelectric Point (pI): 11.353
Optimum pH: 9.22

Product Description

This product is a ready-to-use solution of lysozyme. The lysozyme has been purified from chicken egg white. This lysozyme solution can be used for the purification of both DNA and protein from bacteria.

Lysozyme is widely used in the enzymatic lysis of microbial cells.4,5 It hydrolyzes the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in the polysaccharide backbone of peptidoglycan present in bacterial cell walls. Gram-positive bacterial cell walls contain a high proportion of peptidoglycan and are quite susceptible to hydrolysis by lysozyme. Gram-negative bacteria are less susceptible to hydrolysis since they have a lower proportion of peptidoglycan and an outer membrane. They may be made more susceptible to lysis by the addition of EDTA, which chelates metal ions in the outer bacterial membrane. This optimizes the lysis of the bacterial cell wall with lysozyme.6 Lysozyme will also hydrolyze chitin oligosaccharides.7,8

Lysozyme consists of a single chain polypeptide containing 129 amino acid residues which is cross-linked with 4 disulfide bridges.9 Its molecular mass is 14,307 based upon amino acid sequence. Lysozyme is inhibited by indole derivatives, which bind to and distort the active site, and imidazole, by formation of a charge-transfer complex.10 It is also inhibited by
surface-active agents such as sodium dodecyl sulfate, sodium dodecanate, and dodecyl alcohol. Other compounds of these types will inhibit lysozyme provided that the carbon chain present is at least 12 or more carbons in length.11 Lysozyme is also inhibited by N-acetylglucosamine (NAG) and lactone analogs of peptidoglycan.12

Reagent

This solution is supplied as 10 mg/ml lysozyme in 25 mM sodium acetate pH 4.5 with 50% glycerol.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Storage/Stability

This product is stable for at least 2 years when stored at –20 °C in the unopened container.

Procedure

Below is a protocol for the extraction of proteins from E. coli using this lysozyme solution. Addition of nucleases, such as benzonase (Product No. E1014), may help reduce the viscosity of the released chromosomal DNA. Protease inhibitors may also be added to prevent breakdown of proteins during cell lysis (Protease Inhibitor Cocktail for Poly-His proteins, 5 ml, Product No. P8849).

  1. Collect the cells that express the protein of interest by centrifuging at 5,000 x g for 10 minutes.
  2. Carefully remove the media from the cell pellet. The cell pellet may be frozen or used fresh.
  3. Use 10 ml of CelLytic B (B3553) plus 0.2 ml of lysozyme solution (final concentration of 0.2 mg lysozyme/ml) per gram of cell paste. Mix the sample well to completely resuspend the cells.
  4. Incubate the extraction suspension with shaking at room temperature for 10-15 minutes to fully extract the protein from the cells.
  5. Centrifuge the extract at 1,900 x g for 15 minutes to pellet the insoluble material. For very viscous extracts, centrifuge at 25,000 x g for 15 minutes.
  6. Carefully remove the supernatant containing the soluble proteins. Approximately 90 to 95% of the soluble proteins will be found in this fraction.

Materials

     

 References

  1. Aune, K.C., et al., Biochemistry, 8, 4579-4585 (1965).
  2. Davies, R.C., et al., Biochim. Biophys. Acta, 178, 294-305 (1969).
  3. Wetter, L.R., et al., J. Biol. Chem., 192, 237-242 (1951).
  4. Maniatis, T., et al., Molecular Cloning, A Laboratory Manual, 2nd ed., 1.29 (1989).
  5. Haas, M.J., et al., Methods Enzymol., 43, 620-628 (1975).
  6. Schutte, H. et al., Biotechnology and Applied Biochemistry, 12, 599-620 (1990).
  7. Rupley, J.A., Biochim. Biophys. Acta, 83, 245-255 (1964).
  8. Holler, H., et al., Biochemistry, 14, 2377-2385 (1975).
  9. Jolles, P., Angewandte Chemie, International Edition, 8, 227- 239 (1969).
  10. Swan, I.D.A., J. Mol. Biol., 65, 59-62 (1972).
  11. Smith, G.N., et al., Archives of Biochemistry and Biophysics, 21, 383-394 (1949).
  12. Horton, H.R., et al, Principles of Biochemistry, Third Edition, Chapter 6, pp. 9.9 (Prentice Hall, Inc., NJ, 2002).

 

RM,AC,TMS 07/16-1

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