during immobilization of enzyme on support, for example activated chitosan with glutaraldehyde, which surface residue of enzyme molecule intracte with support?
Thanks Hui, can NH2 of other residues such as arg take part in attachment?
Glutaraldehyde is a bit of a harsh homobifunctional crosslinker. Depending on your enzyme and amount of glutaraldehyde and conditions, you might lose enzyme activity, so you might try a less harsh homobifunctional crosslinker.
Thanks alot for your answers,
So we can say another NH2 of surface residues can take part in enzyme attachment, can you explain clearly what happened in immobilization reaction?!
Usually is the amino terminus of the enzyme. To interact with the NH2 of Lysine you need to work in a pH above the pKa, that is above 10. Since most of enzymes don't resist at such pH, most immobilizations are carried out at pH close to neutrality and the Lysine is little prone to interact with glutaraldehyde.
If you are able to work with a high pH you can attach the enzyme to several Lysine residues making a very stable ligation.
Chapter Improved Stabilization of Chemically Aminated Enzymes Via Mu...
Partly it will depend on the quality/purity of the glutaraldhyde: oddly when I was doing this many years ago I found the practical grade lab reagent worked far better than the purified EM grade glutathione. Depending on which enzyme you have you could try adding a competitive inhibitor (or even substrate) during the crosslinking process to help protect the active site. It is theoretically going to react with -SH, -NH or OH groups.
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