Hi,
I am trying to lyse gram positive E. faecalis using lysozyme (final concentration: 100ug/ml). I suspend the bacterial pellet with lysozyme in buffer for 30min at 37C. Then run the supernatant on SDS-PAGE. I do see many protein bands. I am not sure if these are the proteins leaked from the cytoplasm or from the peptidoglycan layer. So my question is to know if lysozyme can be used to break open the gram positive bacteria to release cytoplasmic content?
Buffer composition: 0.2mM EDTA, 50mM Tris pH 8, 10% glycerol, 1mM PMSF, 0.1% Triton X100, 150mM NaCl
Thank you
Lysozyme is used to lyse Gram-positive bacteria, so if the treatment was sufficient, at least some of the released proteins should be cytoplasmic.
Hi Ramya
Lysozyme is used in cell disruption of both G(+) & G(-) bacteria, in our lab we use it with sonication as well (we add lysozyme then sonicate). To answer your question you need first to find out if the target protein membrane bound or cytoplasmic, although your are using detergent (Triton X-100) in the buffer that will help in cell lysis and to solubilise proteins of membrane, my advice to use lysozyme in a combination of other disruption method especiall you are using G(+) bacterium..
Good luck
Thank you for the answers. Lysozyme hydrolysis peptidoglycan residues and therefore breaks the cell wall. Adam Shapiro, do you know how the inner membrane is cleaved in gram positives by lysozyme to release cytoplasmic proteins?
Lysozyme acts on peptidoglycan only. The inner membrane ruptures once the peptidoglycan is degraded because of the internal cellular pressure, as long as the medium is not hyperosomotic.
I agree with Dr. Shapiro lysozyme only acts on peptidoglycan. Some Gram + bacteria may also need mutanolysin or lisostaphyn to disrupture brcuase the nature of peptidoglycan. There are also special techniques for the isolation of the inner membrane proteins using HEPES or other reagents and high g centrifugation
If the goal is to prevent cell lysis, you should remove Triton X-100 (solubilizes membranes) and make a buffer isotonic or weakly hypertonic (by addition of sucrose).
Is the lysozyme is stable/active during sonication process. I think it will be denature during sonication process. and also for actinomycetes cultures is this method is applicable?
If your sonication method is done properly, avoiding overheating and foaming, then lysozyme will be stable. Do not include reducing agents, since lysozyme contains disulfide bonds.
Lysozyme is thermally very stable. Its melting temperature is more than 70 degree celsius and the melting starts above 50 degree celsius
Can anyone suggest method to get the gram positive cell (streptococcus mutans) membrane intact with peptidoglycan layer.
Gram-positive bacteria synthesize several compounds that decorate their peptidoglycan exoskeleton. Based on structural differences, one can distinguish teichoic acids, teichuronic acids, lipoteichoic acids, lipoglycans, and polysaccharide modifications. For me I agree with Dr. Shapiro.
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