[DIYbio] Re: General Protocols on enzyme assays and purification

yes exactly ! It would be super if i could isolate out a good bacterial/fungal strain form the environment, perhaps lactobacilli or aspergillus,which produce a rich source of extracellular enzymes. With regards to a general assay for enzymes that sounds brilliant but i how would i get about that? The hydrolases i want to isolate are your normal  general amylase, protease, lipase, phytase, etc.

On Sunday, 20 April 2014 23:59:58 UTC+5:30, Josiah Zayner wrote:

Trying to do this you will run into two major problems:
1. Acquiring enough of any given protein
2. Isolating the protein

Your best bet is probably to find a hydrolytic protein(http://en.wikipedia.org/wiki/Hydrolase) and express it recombinantly and purify it.
You can find sequences at NCBI (http://www.ncbi.nlm.nih.gov/)
And order them from IDT (http://www.idtdna.com/site)
Clone it into an expression vector and purify using affinity chromatography. Then test it's activity using a colorometric or spectroscopic assay. Assay for different types of hydrolases will be different as they each have different chemical activity. I suggest searching http://scholar.google.com for what assay would be good for whatever enzyme you choose or want to look for.

That said you can always isolate colonies of bacteria and grow up 5mL cultures or so and use Ammonium sulfate precipitation (http://en.wikipedia.org/wiki/Ammonium_sulfate_precipitation) to isolate the proteins but that is probably very drastic. Even if you isolate the protein fraction identification with proteolytic LC MS will probably be outside the scope of your work and length of time you are there.

What you probably want to do is develop some sort of growth or visual assay and first attempt to just isolate bacteria from the environment and then grow up the winners and attempt to isolate the protein if you opt against the recombinant protein approach. Again, this will probably depend on which hydrolases you plan on looking for but being creative you might be able to come up with a more general assay.

Josiah