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Immobilised enzymes questions


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#1 electros

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Posted 07 March 2019 - 12:16 PM

Hello

 

I have some questions about enzymes and I am not sure if this is the right forum or maybe the Proteins one.

 

1) do you know any good companies that can make immobilised enzymes products to buy? preferably for point of care experiments?

 

2) if the activity of an enzyme is 200u/mg, will it convert these 200u in a minute if the reactants are in e.g. 10uM and 10nM concentrations?

 

3) can we immobilise more than one enzymes to perform coupled reactions or a cascade of reactions? will the product be stable and have good yield or will intermediate products leak and the thing won't work?

 

4) how stable are immoblised enzymes? how long can they last?

 

Thanks!



#2 bob1

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Posted 07 March 2019 - 01:20 PM

1, 3, 4) There are a number of suppliers of enzymes and the like, which ones are the best depend on the enzyme and what you are trying to do with it. Questions you need to ask yourself or your lab manager/PI: do you want the diagnostic test itself, or just the enzyme involved? Do you want the actual diagnostic enzyme used (very expensive usually) or can you get by with a product that is the same, just not certified for diagnostic tests? What form do you want the enzyme in - dried powder, liquid (i.e solubilized)?  How active do you need it? How are you going to store the enzyme (in what form and what temperature)? How long do you want to store it for? What substrate (e.g. alginate) do you want it on? A literature review for your area of research is a good idea here, it will help you know what others have done, how they have done it, and probably get you names of suppliers for some of the things you want.

 

2) 200 U/mg means that each mg of powder in a bottle/tube contains 200 U of enzyme - the powder may not be all enzyme, or some of the enzyme may not be active, so the molecular mass of the enzyme is not relevant to the calculation of concentration. I suggest that you look at the definition of the term "enzyme unit" relative to the enzymes you are investigating - it is a definition of the activity of the enzyme, nothing to do with the substrate, and often varies depending on the enzyme and the temperature of the reaction. 

 

additional 3) It depends on the enzymes, the substrates, and what you are doing with each enzyme and substrate (storage conditions etc). Some are stable, some are not.

 

additional 4) It depends on the enzyme and the storage conditions - some might work well after storing at room temp for a day in liquid, whereas others might last a year, or an hour!



#3 electros

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Posted 07 March 2019 - 01:46 PM

Thanks, activity of 200U/mg is not concentration, it means that 1mg of the enzyme can convery 200umole of substrate per minute.

 

Regarding 2) will such low concentrations of the substrates affect the substrate conversion rate?

 

Regarding 3) are there commercially available coupled enzymes or enzyme cascades and some data on them? Or is it very exotic?

 

Regarding 1) I would assume there are some big names that most people use? Someone who works with enzymes should be able to give some recommendations?



#4 mdfenko

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Posted 07 March 2019 - 02:29 PM

Regarding 2) will such low concentrations of the substrates affect the substrate conversion rate?

 

look at this wiki entry on enzyme kinetics: https://en.wikipedia...Enzyme_kinetics


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#5 bob1

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Posted 07 March 2019 - 02:47 PM

Technically correct, it's not a concentration - it is the specific activity, which is formally measured in micromol substrate min-1 mg-1, however it is equivalent to a concentration in terms of how it is handled in calculations - it allows you to simplify the mathematics so that you can convert per volume after dissolving.

 

2) It depends on the enzyme, they have a range over which they work and the minimum concentration will depend entirely on the enzyme.

3)I have no idea, I'm a virologist.

1)I rarely work with enzymes beyond that needed to do DNA/RNA manipulation and the ones expressed by my viruses. However, a list of common suppliers of reagents in the life-sciences: Sigma-Aldrich, EMDmillipore (now combined with Sigma I think), GE life sciences, Life technologies, New England Biolabs, Corning, Promega, Thermo Fisher Scientific, Novus Biologicals, R&D systems, Abcam, Biorad, Origene.  There are lots of others, if you need specific enzymes your best bet is to google and read the literature and look for the suppliers in the methodology.






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