Glucose oxidase reconstitution

I. Willner, V. Heleg-Shabtai, R. Blonder, E. Katz, and G. Tao, Electrical wiring of glucose oxidase by reconstitution of FAD-modified monolayers assembled onto Au-electrodes. J. Am. Chem. Soc. 118, 10321-10322 (1996). 

Au NPs (1.2 nm) that include a single /V-hydroxysuccinimide-active ester functionality were modified with 2-amino-ethyl-flavin adenine dinucleotide, (5), and apo-glucose oxidase was reconstituted on the FAD cofactor units to yield the Au NP-GOx hybrid (Fig. 12.6a). The resulting hybrids were linked to the Au surface by different dithiol bridging units (8), (9), and (10). The resulting NP-functionalized glucose oxidase, GOx, exhibited electrical contact with the electrode surface, and the Au NPs... 

Zayats M, Katz E, Willner I. Electrical contacting of glucose oxidase by surface-reconstitution of the apo-protein on a relay-boronic acid-FAD cofactor. Journal of the American Chemical Society 2002, 124, 2120-2121. 

To optimize the photoswitchable bioelectrocatalytic features of the protein, site-specific functionalization or mutation of the active site microenvironment is essential. This was accomplished by a semisynthetic approach involving the reconstitution of the flavoenzyme-glucose oxidase with a semisynthetic photoisomerizable FAD cofactor (Scheme 9).1511 The photoisomerizable nitrospiropyran carboxylic acid (24) was covalently coupled to N6-(2-aminoethyl)-FAD (25), to yield the synthetic photoisomerizable nitrospiropyran-FAD cofactor 26a (Scheme 9(A)). The native FAD cofactor was removed from glucose oxidase, and the synthetic photoisomeriz-able-FAD cofactor 26a was reconstituted into the apo-glucose oxidase (apo-GOx), to yield the photoisomerizable enzyme 27a (Scheme 9(B)). This reconstituted protein... 

A further approach to controlling electrical communication between redox proteins and their electrode support through a photo-command interface includes photo stimulated electrostatic control over the electrical contact between the redox enzyme and the electrode in the presence of a diffusional electron mediator (Scheme 12).[58] A mixed monolayer, consisting of the photoisomerizable thiolated nitrospiropyran units 30 and the semi-synthetic FAD cofactor 25, was assembled on an Au electrode. Apo-glucose oxidase was reconstituted onto the surface FAD sites to yield an aligned enzyme-layered electrode. The surface-reconstituted enzyme (2 x 10-12 mole cm-2) by itself lacked electrical communication with the electrode. In the presence of the positively charged, protonated diffusional electron mediator l-[l-(dimethylamino)ethyl]ferrocene 29, however, the bioelectrocatalytic functions of the enzyme-layered electrode could be activated and controlled by the photoisomerizable component co-immobilized in the monolayer assembly (Figure 12). In the... 

Scheme 12 Surface reconstitution of apo-glucose oxidase on a mixed monolayer associated with an electrode consisting of an FAD cofactor and photoisomerizable nitrospiropyran units, and reversible photoswitching of the bioelectrocatalytic functions of the enzyme electrode.

Figure 8.11 Electrical coupling of AuNP-reconstituted glucose oxidase to a Au electrode by ...

Figure 12.4 Photoinduced bioelectrocatalyzed oxidation of glucose to gluconic acid by glucose oxidase (COD) reconstituted with a nitrospiropyran-modified FAD cofactor (Sp-FAD) assembled as a monolayer on the Au electrode. Fhe Sp-FAD reveals reversible photoisomeriz-able properties yielding nitromerocyanine-FAD isomer (MRH+-FAD) [34]...

Willner I, Blonder R, Katz E. Reconstitution of apo-glucose oxidase with a nitrospiro-pyran-modified FAD cofactor yields a photoswitchable biocatalysts for amperometric transduction of recorded optical signals. J Am Chem Soc 1996 118 5310-11. 

Figure 19. (A) The reconstitution of apo-glucose oxidase on a PQQ-FAD monolayer assembled on an Au electrode. (B) Cyclic voltammograms of the PQQ-FAD-reconstituted glucose oxidase on an Au electrode (a) in the absence of glucose (b) with glucose, 80 mM. Recorded in 0.1 M phosphate buffer, pH 7.0, under Ar, at 35°C potential scan rate, 5 mV s. Inset calibration curve corresponding to the amperometric responses (measured by chronoamperometry, = 0.2 V vs. SCE) of the PQQ-FAD-reconstituted glucose oxidase enzyme electrode at different concentrations of glucose.

Willner, I., Katz, E., Willneg B., Blondei R., Heleg-Shabtai, V., and Buckmann, A. F. Assembly of functionalized monolayers of redox proteins on electrode surfaces novel bioelectronic and optobioelectronic systems. Biosens. Bioelectron. 1997,12, 337-356. Blonder R., Katz, E., Willneg I., Wray, V., and Buckmann A. F. A lication of a nitro-spiropyran-FAD reconstituted glucose oxidase and cfuurged electron inediators as optobioelectronic assemblies for the amperometric transduction of recorded cortical signals control of the ON - OFF direction of the photoswitch. J. Am. Chem. Soc. 1997, 119, 11747-11757. 

Blonder, R., Willner, I., and Buckmann, A. F. Reconstitution of apo-glucose oxidase on nitrospiropyran and FAD mixed monolayers on gold electrodes Photostimulation of bioelectrocatalytic features of the biocatalyst. /. Am. Chem. Soc. 1998, 120, 9335-9341. 

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