I have immobilized pectinase on magnetic nanoparticles and want to measure the kinetics in a stirred tank reactor (batch). But my worry is:
1. How is it possible to get a vigorous mixing of the nanoparticles with the substrate, given the impossibility of using magnetic stirrers and my doubts that a shaker might not give vigorous mixing?
2. Every time I take a sample, I end up with a reduced amount of nanoparticles in the reaction mixture. Is there an easy way of avoiding particle loss, given that we need to take samples every 15 or 30 seconds?
We have used magnetic beads in enzymatic reactions and have used rotators that turn the tube head over head. This seems to work fine. We also did no shaking at all and got the same results. For magnetic particles, if you use a magnet to pellet the beads and leave the tube next to the magnet during withdrawal this should minimize bead loss and I would use a large enough volume so as not to perturb the beads when pipetting.
We have used magnetic beads in enzymatic reactions and have used rotators that turn the tube head over head. This seems to work fine. We also did no shaking at all and got the same results. For magnetic particles, if you use a magnet to pellet the beads and leave the tube next to the magnet during withdrawal this should minimize bead loss and I would use a large enough volume so as not to perturb the beads when pipetting.
In my opinion if it’s possible you should not use one medium reaction but many small medium reactions. Each one could be stopped at the needed time of reaction. By this way you have the same enzyme concentration in all your medium reactions. Good Luck
As you mentioned "stirred tank reactor", I suppose you will work with a big volume of substrate. You coul use a stirred tank as described in:
http://www.essentialchemicalindustry.org/processes/chemical-reactors.html
Scrol down the page for more information.
One of the "reactant inlets" could be used for taking samples. Use a very thin tube with a small filter at one end. The porosity of the filter has to be such as to retain the nanoparticles. This end will be introduced into the reaction vessel. The other end will be adapted to a seringe. At sampling times, with the seringe create the vacum necessary for sampling, collect the sample and uncouple the seringe. Close the tube with a clip or, after dispensing the sample into a container, put the seringe right back in place for next sampling.
Good luck!
You can dilute your reaction substrate concentration to take enough volume from the medium for enzyme detection to void the huge error in small samples of broth medium.
In this case and since the particles density is relatively high, use a mixer rather than a magnetic mixer. Use a syringe to take small samples. To measure the kinetic parameters and assuming good mixing and no external and internal mass transfer resistances, use relatively low substrate concentration. follow the substrate concentration with time, and construct a curve between the substrate concentration with time. evaluate the rate at time equal zero which is the slope of the curve at time equal zero. repeat the experiment several times with other initial substrate concentrations and in each time evaluate the initial rate data. construct the double reciprocal plot (lineweaver-Burk plot to obtain a straight line between 1/rate and 1/s
for michaelis Menten equation. You will then obtain Ks and Mmax for the enzyme. assuming no inhibition.
There have been quite a lot of vauable comments. For mme, there is one decicive question. There might be to extreme situations:
(1) Assumed-mechanisms-base kinetics; in this way you might be able to distinguish between several mechanisms. All mass transfer limitations should be aoided..
(2) Kinetics for reactor design including transport limitations.
M. Barns
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