General Protocols on enzyme assays and purification

[R Annie O'NieL]

hello everybody! was just wondering if there are any good general protocols or books for general enzyme assay, purification and extraction,from bacteria and fungi? I may be starting a new project for extraction of enzymes from bacteria to produce a new easily absorbable nutritional feed (just from the top of my head). Ideas and/or feedback will be much appreciated. 

[Mega [Andreas Stuermer]]

Would be interesting what exactly you are looking for? Signal peptides? or his tags? maltose binding tag?

[R Annie O'NieL]

I'm  interested in isolating out various kind of hydrolytic enzymes from bacterial strains to use it in the production of weaning food. I am not entirely sure of how to get about the project, as its still just a rough concept. It should be a biochemistry project according to my mentor so i thought of putting in an enzyme assay as well. I'm not certain of its feasibility and I also have a time constraint of about 3 months. Its either that or... i thought of some kind of fermented weaning food (only problem is that i am not sure it will be a biochemistry project). So i am a bit discombobulated.

[Josiah Zayner]

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

[R Annie O'NieL]

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.

[poli]

It'll be tough to do this in 3 months without a bio background and lab but at least isolation may be doable. My suggestion would be to make solid agar media that contains the macromolecule you wish to break down and spread the fermented food on it, see what grows and how robustly. Assays for each type of macromolecular would consist of exposing the macromolecule to the cells for a set length of time and separating and quantifying the digested fraction, don't forget to include a negative control without the active ingredient.