I want to perform microbial transformation of some xenobiotics with intact fungal organelles, but no cell wall or cell membrane of fungi.
So I need a way to remove the fungal cell wall and cell-membrane in a way that organelles should remain intact.
I know that detergents can work better for removing the cell membrane but there is a chance that detergents can also destroy the membrane of the other organelles.
Dear colleague!
Your question is quite general, so answering it I can just outline general direction for your search.
You can treat the young mycelium with a chitinase to get protoplasts first. And then centrifuge them to get organelle fractions. So outer membrane fraction can be removed. Exact parameters of rotation speed, enzyme treatment and other fine details hardly depend on species you study.
If you define your question concretely, I probably can give you some references for specific methods.
Dear Maria,
I am to work on species of Aspergillus, Beauveria bassiana, Cunninghamella, and Gibberella.
a couple of references:
(Isolation of cell organelles)
Common information -
http://www.ncbi.nlm.nih.gov/books/NBK21492/
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/articles/biofiles/biofiles-pdf/biofiles_v6_n5.pdf
http://www.ableweb.org/volumes/vol-13/8-padh.pdf
and a paper -
Rieder SE, Emr SD Isolation of subcellular fractions from the yeast Saccharomyces cerevisiae. Curr Protoc Cell Biol. 2001 May;Chapter 3:Unit 3.8. doi: 10.1002/0471143030.cb0308s08.
I will go on a little bit later
OK One more reference may be useful for you by the list of citations (as I'm afraid I can miss it) Vagvolgyi C., Bruckner B., Franko A. Size-dependent regeneration of Gibberella protoplasts // Acta Microbiologica Hungarica. 1990. No 37. V. 4. P. 375-378. And this: Santos T. M. C., De Melo I. S. Preparation and regeneration of protoplasts of Talaromyces flavus // Rev. Brasil Genet. 1991. N 14. V. 3. P. 835-839 Good luck!
@ Maria,
Unfortunately, I don't have access to this article:
Vagvolgyi C., Bruckner B., Franko A. Size-dependent regeneration of Gibberella protoplasts // Acta Microbiologica Hungarica. 1990. No 37. V. 4. P. 375-378.
I can search for it in my library and scan but as it takes some time I unfortunately cannot do this too soon.
I think you can try giving a high speed centrifuge (15,000 rpm+ for half hour or so to be sure) and then taking our 50% of the supernatant gently out.
It should work. I have done it long back.
Surely. But accurate microscopic control is needed to distinguish fractions and organelle conditions. There are may be some nuances in centrifuge regime and so on.
Ghazanfar,
Now I've got a photocopy of a paper: Vagvolgyi C., Bruckner B., Franko A. Size-dependent regeneration of Gibberella protoplasts // Acta Microbiologica Hungarica. 1990. No 37. V. 4. P. 375-378. I can send it (via email?) if you are interested.
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