Immobilization of Cysteine-Tagged Proteins on Electrode Surfaces by Thiol-Ene Click Chemistry.
Thiol-ene click chemistry can be exploited for the immobilization of cysteine-tagged dehydrogenases in an active form onto carbon electrodes (glassy carbon and carbon felt). The electrode surfaces have been first modified with vinylphenyl groups by electrochemical reduction of the corresponding diazonium salts generated in situ from 4-vinylaniline. The grafting process has been optimized in order to not hinder the electrochemical regeneration of NAD(+)/NADH cofactor and soluble mediators such as ferrocenedimethanol and [Cp*Rh(bpy)Cl](+). Having demonstrated the feasibility of thiol-ene click chemistry for attaching ferrocene moieties onto those carbon surfaces, the same approach was then applied to the immobilization of d-sorbitol dehydrogenases with cysteine tag. These proteins can be effectively immobilized (as pointed out by XPS), and the cysteine tag (either 1 or 2 cysteine moieties at the N terminus of the polypeptide chain) was proven to maintain the enzymatic activity of the dehydrogenase upon grafting. The bioelectrode was applied to electroenzymatic enantioselective reduction of d-fructose to d-sorbitol, as a case study.
Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), UMR7564 CNRS - Université de Lorraine , 405, rue de Vandoeuvre, F-54600 Villers-lès-Nancy, France.
Lin
L
Zhang
Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), UMR7564 CNRS - Université de Lorraine , 405, rue de Vandoeuvre, F-54600 Villers-lès-Nancy, France.
Neus
N
Vilà
Microbiology, Saarland University , Campus, Geb. A1.5, D-66123 Saarbruecken, Germany.
Tobias
T
Klein
Microbiology, Saarland University , Campus, Geb. A1.5, D-66123 Saarbruecken, Germany.
Gert-Wieland
GW
Kohring
Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), UMR7564 CNRS - Université de Lorraine , 405, rue de Vandoeuvre, F-54600 Villers-lès-Nancy, France.
Ievgen
I
Mazurenko
Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), UMR7564 CNRS - Université de Lorraine , 405, rue de Vandoeuvre, F-54600 Villers-lès-Nancy, France.
Alain
A
Walcarius
Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), UMR7564 CNRS - Université de Lorraine , 405, rue de Vandoeuvre, F-54600 Villers-lès-Nancy, France.
Mathieu
M
Etienne
D057930:Click Chemistry; D003545:Cysteine; D004566:Electrodes; D010088:Oxidoreductases; D013438:Sulfhydryl Compounds
D013438:Sulfhydryl Compounds; D010088:Oxidoreductases; D003545:Cysteine
NAD; cysteine-tag; dehydrogenase; diazonium electrografting; thiol−ene click-chemistry