I am looking to differentiate isoenzymes on lysing gram-negative bacteria.
Hen white-egg lysozyme is used to lyse gram-negative E. coli intact cells the highest lysis rate occurred at pH 8.9 in the system containing 0.03 M NaCl. Digestion of cell wall is achieved by the addition of lytic enzymes to a cell suspension. Enzymes are highly selective, gentle and most effective. Lysozyme is widely used to lyse bacterial cells. The enzyme hydrolyses α-1,4 glycosidic bond in the mucopeptide moiety of bacterial cell wall of gram positive bacteria. The final rupture of the cell often depends on the osmotic pressure of the suspending medium. In case of gram negative bacteria like Escherichia coli pretreatment with detergent such as Triton X-100 or addition
of EDTA is necessary. EDTA is used to destabilize the outer membrane
thereby making the peptidoglycan layer accessible to lysozyme.Yeast cell
lysis requires a mixture of different enzymes such as glucanase, protease,
mannanase or chitinase. Sequential disruption of microbial cells for selective
product release involves the use of lytic enzymes based on the type of
microbial cells and process conditions. Importnat materials required are
Overnight grown bacterial culture, Alkaline CuSO4 solution, Folin’s reagent,
Lysis buffer containing Lysozyme.
I routinely lyse E. coli cell pellets for 2-ml samples of cultures at OD600 = 1-3 by resuspending in 200 µl 50 mM phosphate buffer + 0,4 M NaCl (EDTA is optional) and adding 10 µl of a 20 mg/ml solution of lysozyme in the same buffer. Make sure that the pellet is well dispersed, otherwise DNA released from incipient cell lysis will turn clumps into a goo that is hard to disperse. Incubate at 37 °C for 10 min, then add 10 µl of a 20% (v/v) solution of Triton X-100 and incubate for a further 5 min at 37 °C.
I am using 3 isozymes which I already know are serine-proteases to lyse bacterial cells. I need a quantitative method for stating differences between the 3 isozymes on lysing bacterial cells. Somebody suggested treating a bacterial cell culture with the isozymes and counting the lysed cells on an electron microscope field, is this the best way?
Patrick, I am solely considering that the proteases will hydrolyse the membrane proteins of the gram-negative bacterial cells and thus lyse them. I am still working on the methodology I will use, I still need suggestions, thanks!
We used to use release of alkaline phosphatase activity as an indicator of cell lysis for Legionella bacteria. A similar enzyme activity could be tested for as released only by lysis of the cells? could you just test for loss of viability in the cultures?
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