i have to calculate activity of immobilized (using sodium alginate) lipase using pNPP assay
Prepare a series of beads with different amount of the immobilized enzyme. Run timed reactions with high excess of substrate. As the enzyme amount per surface are increases the product vs. enzyme amount curve will have negative curvature and reaches maximum at a certain enzyme amount. The maximum of the product formation is limited by the amount of enzyme immobilized and the activity of the enzyme. If you know the amount of of enzyme immobilized, and the reaction time you can estimate activity. The maximum also depends on the total surface area of the solid support. Make sure you use the same type and amount of bead for each of the reactions.
For more information try this web site:
http://www1.lsbu.ac.uk/water/enztech/
or this book:
'Enzyme Technology' by Martin Chaplin and Christopher Bucke (Cambridge University Press, 1990)
Thank for your reply. but i need more specific answer with equations. Because 1000u lipase (pnpp assay) when immobilized into sodium alginate bead the activity reduced into 25u.
Agitation is also important, as film diffusion is another limiting factor. The bead suspension needs to be placed in a shaker for your assay. You will need try various shaking speeds and choose one where the activity with excess substrate seems to level off, that is, where film diffusion limitations are minimized.
If you immobilize too much enzyme on the surface, the rate limiting step becomes the diffusion of the substrate across the stagnant solvent layer near the surface, not the immobilized enzyme amount. Do not expect the same enzyme activity per amount of enzyme as with homogeneous enzyme reactions. If you want to make the enzyme amount the rate limiting step, you have to spread out the enzyme on a larger surface area by using more beads or use beads with smaller particle diameter.
can i compare the activity of crude and immobilized enzyme based on the volume (of lipase) taken for pNPP assay?
We developed a method for anthrax (attached pdf). You can also find some theoretical background in it as well.
If you have enzyme solution with known concentration, then make a dilution series of it. Use equal volume of the dilution series and bind each to the same amount of beads. Bind you unknown solution using the same volume of sample and amount of beads. Add the same concentration and volume of the substrate to all. Incubate them together for the same time. quench the reaction and measure the products.
Based on the dilution series you can construct a calibration curve. Use the curve to determine the concentration in the unknown.
Hi Activity of immobilized enzyme would always be less than native free enzyme.Generally the expressed activity in immobilization is 30-40% of the enzyme loaded.
For immobilized enzyme assay, you may take the enzyme on weight basis for assay and carry out the reaction and calculation as for native enzyme wherein you substitute volume by weight. The activity obtained should be determined on dry basis determining the dry weight of enzyme seperately.
All the best
THANK YOU FOR THE REPLY. THEN WHY PEOPLE GIVING PRIORITY TO THE IMMOBILIZED ENZYMES IN INDUSTRIES?
Advantage? Immobilized enzymes are reusable in continuous processes yielding superior space-time production costs. This does not matter very much when one is doing research with mg amounts but it can significantly reduce costs in production-scale situations.
It can also make the quantitative analysis large number of samples more economical. If you are interested check out our just accepted manuscript.
On-column trypsin digestion coupled with LC-MS/MS for quantification of apolipoproteins
http://www.sciencedirect.com/science/article/pii/S1874391916304225
please suggest me a method to degrade mycolic acid of bacterial cell wall with out degrading DNA?
Hi. For immobilized lipase, use weighed qty of sample and use the weight in calculation as you use volume for liquid enzyme.
Paralley determine dry weight of immobilized beads and calculate the dry equivalent of enzyme in your sample to calculate activity in units on dry basis
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