How much enzyme should I use to...

2 posts / 0 new
Last post
scocioba
scocioba's picture
How much enzyme should I use to...

How can I figure out how much enzyme (concentration) I need for a given experiment. For example, I want to find out how much enzyme I need to degrade cell walls of a piece of plant tissue. I want to use the optimal amount of reagent and complete the process in a reasonable amount of time. How can I find out how much enzyme I require without running controlled experiments to test for best concentration and wasting reagents, which defeats the purpose. A general equation for this process? Anything??  Thanks! 

Sami Tuomivaara
Sami Tuomivaara's picture
scocioba,

scocioba,

It is not clear from your post what is your ultimate goal of the degradation, to prepare spheroplasts, or something else. Anyway, if you want total degradation of some type of polysaccharide in the cell wall, here's an train of thought that you can use to estimate the amount of enzyme.

First of all, estimate the percentage of your target molecule in the tissue. All the numbers I give are my estimates and approximations, you need to plug in the values you think are good estimates for your tissue and experiment.

For argument's sake, let's say you want to degrade xylan. It's percentage in the cell wall is 20% by weight. If you have 1 g of fresh plant tissue, 10% might be cell wall. Hence, you have 20 mg of xylan in your sample. In terms of xylose, there are

n(moles) = m(g) / M(g/mole) = 20 mg / 150 g/mol = 0.133 mmoles = 133 micromoles

of xylose and same number of glycosidic bonds between the xylose units.
Complete degradation of xylan doesn't require the hydrolysis of every bond in the wall, let's say that 10% degradation is enough and you can get rid of the xylan (average length of xylan oligosaccharide is then 10 which can be for example dialyzed out). That means 13 micromoles of bonds needs to be hydrolyzed.

Definition: 1 Unit of enzyme activity degrades 1 micromole of glycosidic bonds per minute.

If you add 0.1 Unit of enzyme, it takes 133 minutes for the degradation to complete, assuming all the xylan is freely available for the enzyme (and that the enzyme is saturated all the time). But in practice I'd leave the reaction proceed longer, maybe overnight with 0.1 Unit of enzyme. If you have lot of enzyme available (and it is cheap), go ahead and use more enzyme.

I work with plant cell walls as well! As you know, plant cell wall (or its fragments) don't form homogenous solution but rather a suspension, so there are issues such as enzyme penetration and substrate presentation so it is impossible to give final answer to your question, so you have to, and you can estimate. Note that you should somehow pre-process the tissue for effective enzymatic degradation, unless you are concerned about the viability of the cells. If you ball-mill or otherwise grind the tissue, the substrate availability is of course better, and the degradation is over faster with less amount of enzyme.

Cheers