Photoswitchable enzyme

Photonic activation of redox enzymes results in the light-induced bioelectrocataly-tic activation of an enzyme cascade. This permits application of photoswitchable enzymes as amplifiers of weak photonic signals. Alternatively, sensitive actino-meters, measuring low irradiation doses, might be envisaged. 

Redox enzymes are the active component in many electrochemical enzyme electrode biosensor devices.1821 The integration of two different redox enzymes with an electrode support, in which one of the biocatalysts is photoswitchable between ON and OFF states, can establish a composite multisensor array. The biomaterial interface that includes the photoswitchable enzyme in the OFF state electrochemi-cally transduces the sensing event of the substrate corresponding to the nonphoto-switchable enzyme. Photochemical activation of the light-active enzyme leads to the full electrochemical response, corresponding to the analysis of the substrates of the two enzymes. As a result, the processing of the signals transduced by the composite biomaterial interface in the presence of the two substrates permits the assay of the... 

Photoswitchable enzymes could have an important role in controlling biochemical transformations in bioreactors. Various biotechnological processes generate an inhibitor, or alter the environmental conditions (pH, for example) of the reaction medium. Photochemical activation of enzymes that adjust environmental conditions or deplete the inhibitor to a low concentration may maintain the bioreactor at optimal performance. More specifically, integration of the photoswitchable biocataly-tic matrix with a sensory electrode might yield a feedback mechanism in which the sensor element triggers the light-induced activation/deactivation of the photosensitive biocatalyst. 

The enzyme GOx was transformed into a photoswitchable enzyme by its chemical modification with nitrospiropy-ran photoisomerizable units [382]. The... 

Another application for photoswitchable enzymes attached to gold surfaces is the cytochrome c-mediated biocatalysed reduction of O2 by cytochrome oxidase, using a functional pyridine-nitrospiropyran photoiso-merisable mixed monolayer electrode (see Sect. 6.1, Fig. 39) [8,16,17]. 

Scheme 8 Assembly of a photoisomerizable glucose oxidase monolayer electrode and the reversible photoswitchable activa-tion/deactivation of the bioelectrocatalytic functions of the enzyme electrode.

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... 

Photoswitchable activation of enzymes provides a means to trigger a biocata-lytic transformation ON and OFF . This feature shows two important functions ... 

The concept of photoswitchable biomaterial can be extended to different biological functions, such as cofactors, inhibitors, enzymes, receptors, hormones, antigens/antibodies, DNA, etc. This opens a broad spectrum of applications in different biomaterial science disciplines. 

Table 2 summarizes different possible applications of photoswitchable biomaterials, while detailing the nature of the biomaterial, the area of application, and, when possible, specific examples. Reversible light-induced activation and deactivation of redox proteins (enzymes) corresponds to write - read - erase functions. The photonic activation of the biomaterial corresponds to the write function, whereas the amperometric transduction of the recorded optical information represents the read function of the systems. Switching off of the redox functions of the proteins erases the stored photonic information and regenerates the photosensory biomaterial. These integrated, photoswitchable redox enzyme electrode assemblies mimic logic functions of computers, and may be considered as first step into the era of biocomputers. 

Photoswitchable redox-enzymes 1. Amperometric transduction of optical information - biocomputers 2. Amplification of weak optical signals -photonic amplifiers 3. Multisensor arrays — biosensor and bioelectronics 4. Photoelectrochemical systems Enzyme immobilized on electronic transducer... 

Photoswitchable sub strate-protein / cofactor- enzyme/antigen-antibody interactions Light-stimulated affinity chromatography Immobilization of photoactive separation component on solid matrices... 

Photoswitchable electrical communication between enzymes and electrodes has also been achieved by the application of photoisomerizable electron-transfer mediators [195, 199]. DilTusional electron mediators (viologen or ferrocene derivatives) were functionalized with photoisomerizable spiropyran/merocyanine units. These mediators can be reversibly photoisomerized from the spiropyran state to the merocyanine state (360 < A < 380 nm) and back (A > 475 nm). An enzyme multilayer array composed of glutathione reductase or glucose oxidase was electrically contacted only when the photoactive group linked to the redox relay (viologen or ferrocene derivative, respectively) was in the spiropyran state. 

FIG. 7.8 Electronk transduction of photoswitchable bioeiectrocatal)rtic functions of redax enzymes by the tethering of photoisomerizable units to the protein. (R is a dtffusional electron mediator that electrically contacts the redox site of the protein with the electrode support.)... 

The photoisomerizable enzyme monolayer electrode also revealed photoswitchable bioelectrocatalytic activity (Figure 7.10). In the presence of ferrocene carboxylic acid (5) as a diffusional electron transfer mediator, the nitrospiropyran-tethered GOx (4a) revealed a high bioelectrocatalytic activity, reflected by a high electrocatalytic anodic current. The protonated nitromerocyanine-GOx (4b) exhibited a two-fold lower activity, as reflected by the decreased bioelectrocatalytic current. By the reversible photoisomerization of the enzyme electrode between the 4a- and 4b-states, the current responses are cycled between high and low values (Figure 7.10, inset). 

FIG. 7.9 Ass mbix of a photoisomerizabie glucose ackfase monofayer elecirophotoswitchable activation/deactivation of the Uoelectrocatalyttc functions of the enzyme electrode. 

It also was found that the direction of the photobiocatalytic switch of the nitrospiropyran-FAD-reconstituted enzyme is controlled by the electrical properties of the electron transfer mediator. With ferrocene dicarboxylic acid as a diffusional electron transfer mediator, the enzyme in the nitrospiropyran-FAD state (10a) was found to correspond to the OFF state bio-catalyst, while the protonated nitromerocyanine state of the enzyme (10b) exhibits ON behavior. In the presence of the protonated 1-[1-(dimethyl-amino )ethyl]ferrocene, the direction of the photobioelectrocatalytic switch is reversed. The nitrospiropyran-enzyme state (10a) is activated toward the electrocatalyzed ox idation of glucose, while the protonated nitromerocyanine enzyme state (10b) is switched OFF, and is inactive for the electrochemical oxidation of glucose. This control of the photoswitch direction of the photoisomerizable reconstituted enzyme was attributed to electrostatic interactions between the diffusional electron mediator and the photoisomefizable unit... 

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