*3.4. Oriented Immobilization of Engineered Enzymes in Functionalized Carbon Nanomaterials*

The conventional method for oriented immobilization of redox enzymes is dependent on the natural properties of redox enzymes. Engineered redox enzymes with specific residues for oriented immobilization in modified carbon nanomaterials have recently been reported. Bartlett et al. constructed a site-specific variant of *Mo*BOD S362C, in which Ser362 at the *Mo*BOD surface close to the TI Cu was replaced by a Cys residue [72]. The distance between the T1 site and Ser362 is ~1.33 nm. A thiol-maleimide click reaction between the *S362CMo*BOD variant and maleimide-functionalized MWCNT was employed to construct a stable bioelectrode. A clear DET-type bioelectrocatalytic O<sup>2</sup> reduction wave with a higher current and more sigmoidal shape was obtained in a S362C*Mo*BOD-modified electrode than that in a native*Mo*BOD-modified electrode. Such improvements in DET-type bioelectrocatalytic performance can be explained by the oriented immobilization of the enzyme with a short distance between the active site and the electrodes. A site-specific surface modification of a fungal Lac with a single covalently bound pyrene group close to the T1 site showed improved DET-type bioelectrocatalysis in the CNTs [73]. (Figure 5D) The site-specific modified pyrene was attached to the CNT through a π-π stacking interaction to control the orientation of Lac with the T1 site face to the CNT. As a result, a maximum O2-reduction current density produced by site-specific pyrene-modified Lac at a CNT electrode is 1.15 mA cm−<sup>2</sup> , which is four times higher than that

produced by native Lac at a CNT electrode (0.27 mA cm−<sup>2</sup> ). A further improved maximum O2-reduction current density of 2.75 mA cm−<sup>2</sup> was realized by immobilizing site-specific pyrene-modified Lac at β-cyclodextrin-modified gold nanoparticles(β-CD-AuNPs)-functionalized CNT [73]. The β-CD-AuNPs offer host−guest interactions between pyrene groups and β-CD moieties on the AuNPs surface to increase the number of effectively wired enzymes. A recent study on the oriented immobilization of site-specific alkylated Lac in azido-modified CNTs through copper-catalyzed click reactions was reported by the same group [74]. Interestingly, it has been found that the variant with an alkyne group in the position close to the T2/T3 site showed a higher O2-reaction current (~3 mA cm−2) than that with an alkyne group close to the T1 site (~1.8 mA cm−<sup>2</sup> ). Although several studies have reported a direct electron transfer from the electrode to the T2/T3 cluster of Lac in gold electrodes [75], more experimental evidence is needed to support this finding. − − − *β β β* ff − *β* − −

**Figure 5.** Examples of oriented immobilization of redox enzymes in modified carbon nanomaterials. (**A**) Cyclic voltammograms (CVs) of *Mv*BOD adsorbed in pristine KB (dashed line) and 4-aminobenzoic acid-functionalized KB (solid line) electrodes in an air-saturated phosphate buffer (0.1 M, pH 7.0) with a rotating rate of 2000 rpm. The dotted line shows the cyclic voltammogram of a bare KB electrode without *Mv*BOD. Reprinted from [47] Copyright (2016) with permission from Elsevier. (**B**) CVs of *Tv*Lac adsorbed in pristine CNTs (black solid line) and 1-pyrenebutyric acid adamantyl amide-functionalized CNTs (ADA-CNT, blue solid line) in a stirred oxygen-saturated McIlvaine buffer (pH 5). The dotted line shows the cyclic voltammogram of the *Tv*Lac adsorbed CNT electrode in an argon-saturated buffer. Reprinted from [62] Copyright (2016) with permission from American Chemical Society. (**C**) CVs of *Mv*BOD adsorbed in pristine KB (dashed line) and bilirubin functionalized KB (solid lines) electrodes in an O<sup>2</sup> -saturated phosphate buffer (0.1 M, pH 7) with a rotating rate of 4000 rpm. The inset shows the structure of bilirubin. Reprinted from [65] Copyright (2014) with permission from Royal Society of Chemistry. (**D**) CVs of the bare UNIK<sup>161</sup> (dotted line), UNIK161–NHS–pyrene (dashed-dotted line), and UNIK161–pyrene (solid line) adsorbed in CNT electrodes in O<sup>2</sup> -saturated 0.1 M phosphate buffer (pH 5.0). The dotted line shows bare UNIK161 adsorbed CNT electrodes in an argon-saturated buffer. UNIK<sup>161</sup> is a variant of the LAC3 enzyme (GENEBANK AAR00925.1) containing lysine, methionine, and histidine residues replacing arginine R161 (R > K161) and lysines K40 (K > M40) and K71 (K > H71) in the native sequence, respectively; in addition, UNIK161–NHS–pyrene indicates that the enzyme was modified with pyrene–NHS, which was randomly attached to UNIK<sup>161</sup> through an activated ester coupling. UNIK161–pyrene is produced through the highly selective modifications of K161 with pyrene Rreprinted from [73] Copyright (2016) with permission from American Chemical Society.
