why EDTA is used for lysozyme action

Dear friends, I got protocol from pharmacia for Gram negative bacterial cell
lysis with lysozyme and EDTA. I have gone through several literature where
they have used EDTA with lysozyme mediated cell lysis of Gram negative
bacteria but not in case of Gram positive bacteria.
Can anybody tell me why EDTA is used specially for Gram negative bacteria,
the exact mechanism behind it
Regards.


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Chiranjit Chowdhury
Department of Biotechnology
Indian Institute of Technology, Kharagpur
Kharagpur, West Bengal, India
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Am 30.10.2007, 14:11 Uhr, schrieb chiranjit chowdhury <[Only registered users see links. ]>:


The function of EDTA is to complex metal ions, which are required as
cofactors for some proteases and other hydrolases. Heavy metals may also
inactivate enzymes e.g. by reacting with sulfhydryl groups.

chiranjit chowdhury wrote:

EDTA chelates divalent cations like calcium and magnesium. IIRC, these
divalent cations are important for maintaining the structures on the
cell surface. Removing them destablises these cell surface structures
and makes it easier to lyse the cells.

As far as I can remember, having too much as well as too little calcium
and magnesium can change the cell surface structures which, if I am
remember correctly, is I think one reason why you use calcium when you
prepare competent cells to make the cells more permeable to DNA (calcium
also help screen out the charges allowing the DNA to stick to the cell
surface).

I doubt whether it has to do with the cell wall..probably
lipoppolysaccharide and protein outermembrane is succeptible to EDTA
disruption, than the peptidoglycan layer of gram positive bacteria.

Correct me.

Sudheendra
NBRC


On 11/1/07, ChenHA <[Only registered users see links. ]> wrote:



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Sudheendra Rao N R wrote:

Who said anything about cell wall?

On Nov 1, 8:42 am, "Sudheendra Rao N R" <[Only registered users see links. ]> wrote:

That's pretty close. The substrate for lysozyme is peptidoglycan,
which is the major component of the cell wall in gram-positive
bacteria. Gram negs also have a (thin) peptidoglycan layer, but it is
surrounded by an outer membrane that has LPS in it. Without additional
treatment, this outer membrane protects gram neg bacteria from
lysozyme. EDTA can disrupt this layer and allow lysozyme to have
access to the peptidoglycan.

Nick


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