Dear everyone,
Recently, the immobilization of β-D-Glucosidase on Fe3O4@SiO2 magnetic nanoparticle has been carried out in our lab. The Fe3O4@SiO2 was modified with 3-aminopropyl triethoxysilane to obtain amino functionalized nanoparticle,then activated with 2 % (v/v) glutaraldehyde according literature. However, the immobilization efficiency of β-D-Glucosidase protein is relatively low(~10%),leading to very low enzyme activity. Can someone show me some advice to improve the immobilization efficiency?
It depends on the cleavage of protein. Even though every protein has two sites (amine and carboxylic) but you could imagine if one site is in the inside of protein. The reaction may not happen properly. Actually you should observe this. At the moment, you try to anchor protein via aldehyde-amine interaction, probably you could try another site, which is carboxylic group. You could use EDC/NHS. Or you would probably like to try p-aminobenzamidine coupling. This is the article how to anchor trypsin via PAB coupling.
https://pubs.rsc.org/en/content/articlelanding/2013/cc/c3cc41701h/unauth
EDC/NHS will provide worst results, steric effects always matter. 10% immobilization with respect to what? The surface?
The immobilization efficiency should be calculated with respect to the surface of the particles. The same is true for the concentration of glutaraldehyde. Are you quantifying the concentration of amine groups on the surface? You could estimate your efficiency using this concentration.
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