I am carrying out research on enzyme immobilization on Fe3O4 by covalent bonding method.Firstly, Fe3O4 is functionalized with 3-Mercaptopropyltriethoxysilane and then it will be activated by glutaraldehyde cross linking reagent for enzyme immobilization. I want to know about the reaction mechanism between 3-Mercaptopropyltriethoxysilane, glutaraldehyde and enzyme molecuels. How to make covalent bonding between enzyme and 3-Mercaptopropyltriethoxysilane. so please describe well.
Hi Baskar,
commercial glutaraldehyde is usually sold as aqueous concentrates (25 and 50% are the commonest). In fact, these solutions contain large amounts of oligomers - arising from aldolic/crotonic condensation reactions - leading to new polyaldehydes, containing a number of -CH=C-CHO groups. The double bonds of the alpha-beta insaturated aldehydes easily undergo Michael-type nucleophilic additions by -SH and -NH2 functions, leading to rather stable covalent bond. The old hypothesis of reactions based on Schiff chemistry should be ruled out, as other dialdehydes incapable of forming alpha-beta-insaturated aldehydes are unable to immobilize anything. Glutaraldehyde coupling is best performed at slightly basic or neutral conditions. In the case of -SH groups, when working at acidic pH values the reaction could go through a different mechanism - common to all other aldehydes - and stable thioacetals are formed. This is not noxious to the immobilization, as thioacetals are rather stable and inert towards hydrolysis. In the case of your second step (enzyme immobilization) on the contrary acidic pH values must be avoided: in such a case, protonation of the epsilon -NH2 of the lysines (involved in the covalent bond formation) will prevent the nucleophilic addition to the activated double bonds.
Best regards
Enrico
Dear Enrico
Thank you so much for your valuable answer.
I can easily understand the reaction mechanism between NH2 group and glutaraldehyde. I am unable to get the reaction mechanism between SH and glutaraldehyde. I want to know clearly about bond formation between these functional groups. so could you please describe the reaction mechanism between SH and Glutaraldehyde in equation form?
Thanking You
Dear Baskar, happy to have been of any help...
The reaction between aldehydes (and ketones also) and thiol groups is well known in organic chemistry and strictly parallels that corresponding of aldehydes (and ketones) and alcohols. Under acidic catalysis, hemiacetals and acetals are formed. Depending on the particular structures and properties, formation of both hemiacetals and acetals could become particularly easy (see for example the Haworth structures of sugars, which are spontaneously formed). In the case of thio-hemiacetals and thio-acetals, the formation reaction is even more favoured, and the formation of thioacetals is practically irreversible. In the case of your interest, two thiol groups could react with one aldehyde (–CHO) group coming from glutaradehyde, leading to water elimination and thioacetal formation:
2 R–SH + R'-CHO –> (R–S)2CH–R' + H2O
Once formed, the obtained thioacetal is quite inert and stable against hydrolysis. Obviously, the remaining unreacted aldehyde groups on the glutaraldehyde oligomers could well react with the aminogroups of your enzyme, thus affording the desired immobilized enzyme preparation.
Hoping to have answered your question.
Best regards
Enrico
Dear Enrico
Thank you so much for your valuable help.
I got clearly understand well and form the reaction mechanism.
Just a further point...
Enz–NH2 + –CH=CR'-CHO –> Enz–NH–CH-CHR'–CHO
I apologize for the incompleteness of the structure (this box is not suitable for chemical structure drawing). Good luck!
Enrico
Dear Enrico
I had drawn clearly the reaction mechanism as per as your valuable guidance.
Thank you so much.
I am working on enzyme immobilization on functionalized magnetic nano particle as a biocatalyst for biodiesel production.
If I have any doubt arises related to my research, I would like to share with you.
I would like keep in touch with you..
Sincerely Yours
Baskar Thangaraj
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