What is the concentration of lysozyme you are using currently? Try using proteinase K along with the lysozyme and also SDS.

What genus are you working with? In the case of S. aureus you have to use lysostaphin as lysosyme doesn't disrupt S. aureus cell wall. Heat lysis is a common method I use for plasmid isolation from S. aureus cells (80 °C / 20 min) and it works fine, I have never experienced any problems..

What of using mechanical methods like glass beads or sonication?

Gram positive bacteria naturally have large amount of peptidoglycon, lysozyme and other detergents not easily cleave, it will take additional time for breaking the cells. I advice you to carryout any one of the following protocols for Plasmid preparation.

Plasmid isolation from Gram-positive bacteria, especially lactobacilli, with mutanolysin or lysozyme (Klaenhammer, 1984) (or)

Lysis of Gram-positive bacteria with lysostaphin (Crosa et al., 1994), (or)

Isolation procedure for all plasmid sizes from all bacteria (Crosa et al.,1994)

Chech wether your lysozyme is active /inactive. Change the concentration/increasing the final concentration of lysozyme at the required. Do you chech the viability of the bacteria aftrer lysozyme treatment? Some times the bacteria remain viable means your enzyme dosent work on your bacteria. Pl. Check all these.

Plasmid isolation from Gram positive bacteria is hard job, in particular from enterococci. We were not able to purify large plasmids, while we were able to transfer them through conjugation (see Zarrilli et al, J Antimicrob Chemother 2005)

Thanks for all the kind reply .

I am using the lysosyme at the conc. Of 5mg/ml and incubated it for 1 hr heat at 100 c for 15 minute.

Pl. Try to evaluate, after the lysozyme treatment wether the bacteria has the viability. If viability is there, pl. Go with higher concentrations. Verify the bacteria/ related bacteria, having the plasmid availability.

Try with Qiaprep spin miniprep kit from Quiagen, it works really fine

What is your technique for the heating step?

I isolated a gram-positive megaplasmid with a size of approximately 30 kb.

I used the QIAGEN Plasmid Midi Prep Kit with 100 ml overnight culture. Half of the pellet was dissolved in 1 ml lysozyme solution (Tris/EDTA/Sucrose/NaCl) with 2 mg/ml lysozyme and incubated for 1 h at 37 °C with gentle shaking. After incubation, I discarded the supernatant and used the remaining pellet as starting material for the plasmid kit according manufacturer's instructions. It helped to double the volume of P1, P2 and S3 and to heat the elution buffer to 60-70 °C. I was able to see a clear and strong plasmid band on an agarose gel and purify a sufficient amount for a plasmid of this size. :) Hope it helps someone!