I'm working with a very poorly studied fungi that I suspect releases extracellular enzymes, since I don't know the nature of these enzymes how do I solate them?
To start with, if these enzymes you are targeting are extracellular as you say..it means that they will be present in the growth medium. You will have to grow the fungus in a broth/liquid medium and thereafter separate the fungal thallus from the growth medium which you can do by filtration..or by centrifugation. If you centrifuge...the pellets will be fungal cell components because they are heavier and will sediment....so the supernatant will contain the enzymes and other soluble cell components in suspension. discard the pellets and use the supernatant for purification of the crude enzyme extracts
Fungi produce a different kind of enzyme,at first, u know which enzyme u need? what is the benefit? then chek the activity assay,,,,,,,,,,after that if u want to isolate that enzyme use sodium sulphate precipitation to precipitation proteins with their corresponding solubility,,,,,,,,,,,,,,then from that fracture try to isolate pure enzyme,,,,,,,and it goes on
After following the steps outlined by Becky Aloo, what do you do next? It depends on what you are trying to accomplish. Do you want to purify the enzymes? Do you just want to know which enzymes are there?
If you want to know which enzymes are in the culture supernatant, a modern way would be to use proteomics. You would digest the proteins from the culture supernatant with trypsin and subject the resulting mixture of peptides to mass spectrometry-based proteomics methods to obtain amino acid sequences that could be compared with a database of sequences derived from the genomic sequence of the organism.
If you want to purify the enzymes, you need to start with an idea of what catalytic activity they possess. Then you can subject the culture supernatant to a variety of purification procedures (ammonium sulfate precipitation, various types of chromatography) and use an assay for a specific enzyme activity to test fractions for the presence of the enzyme in order to follow each enzyme through the purification steps.
If your enzyme is present in the broth filtrate you check for its activity and total protein content. You subject your sample to several purification steps (precipitation, dialysis, chromatography etc. checking after each step activity and protein concentration to estimate purity. In ideal situation you could find some affinity chromatography purification procedure to isolate pure sample of your enzyme. I hope this helps as I can not do more since you provide no data which protein you are attempting to isolate.
I think you have got many ideas how to proceed. You may check common hydyrolase activities by mixing their substrates in agar and pour your extract in a well made in centre of plate after solidification.
Rather I wish to knew how you suspect that it release the enzymes ?.........that may b clue for pure answer to your query
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