I am trying to isolate laccase enzyme from Cryptococcus neoformans which is a cell wall associated enzyme. Is there any method which I can use for efficient cell lysis and obtain the enzyme?
I have use French Press in my enzyme assays using E.coli. It basically turns the cell membrane up-side-down to expose the intracellular enzyme (Complex I). Works fine. Example https://homogenizers.net/products/french-press-g-m
Best Pilvi
ISOLATION, PURIFICATION AND CHARACTERISATION OF ENZYMES Steps involved in isolation of a protein or enzyme: 1) Cell disruption (which can done via a number of number of different processes of choice e.g Detergents lysis, Osmolysis, freezethaw cycles, enzymatic lysis, ultrasonication, Homogenisation) 2) Centrifugation (at a specific speed depending on the organ, tissue, organelle or fluid). 3) Removal of supernatant (Decantation to obtain supernatant) You can see this article
Two General Methods for the Isolation of Enzyme Activities by Colony Filter Screening
The issue is that Cryptococcus neoformans possesses a capsule which is making it hard to break down the cells mechanically. One study had been conducted which suggested that incubation in B Mercaptoethanol can release the proteins in the cell wall by breaking S-S bonds. Will the presence of the capsule affect the activity of B mercaptoethanol?
Hi Pal
Freeze the biomass of yeast and crush it with ceramic mortar with glass balls, then added a few amount of buffer, after that use centrifugal.
good luck
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