Carbon Nanotubes as Activating Tyrosinase Supports for the Selective Synthesis of Catechols

Abstract

A series of redox catalysts based on the immobilization of tyrosinase on multiwalled carbon nanotubes has been prepared by applying the layer-by-layer principle. The oxidized nanotubes (ox-MWCNTs) were treated with poly(diallyl dimethylammonium chloride) (PDDA) and tyrosinase to yield ox-MWCNTs/PDDA/tyrosinase I. Catalysts II and III have been prepared by increasing the number of layers of PDDA and enzyme, while IV was obtained by co-immobilization of tyrosinase with bovine serum albumin (ox-MWCNTs/PDDA/BSA-tyrosinase). Attempts to covalently bind tyrosinase provided weakly active systems. The coating of the enzyme based on the simple layer-by-layer principle has afforded catalysts I–III, with a range of activity from 21 units/mg (multilayer, II) to 66 units/mg (monolayer, I), the best system being catalyst IV (80 units/mg). The novel catalysts were fully characterized by scanning electron microscopy and atomic force microscopy, showing increased activity with respect to that of the native enzyme. These catalysts were used in the selective synthesis of catechols by oxidation of meta- and para-substituted phenols in an organic solvent (CH2Cl2) as the reaction medium. It is worth noting that immobilized tyrosinase was able to catalyze the oxidation of very hindered phenol derivatives that are slightly reactive with the native enzyme. The increased reactivity can be ascribed to a stabilization of the immobilized tyrosinase. The novel catalysts I and IV retained their activity for five subsequent reactions, showing a higher stability in organic solvent than under traditional buffer conditions.

Carbon nanotubes supported tyrosinase in the synthesis of lipophilic hydroxytyrosol and dihydrocaffeoyl catechols with antiviral activity against DNA and RNA viruses

“…42 The activity of MWCNT/Tyr (80.5 U/mg), and the value of activity parameters, namely immobilization yield (68 %), and activity yield (48.5 %), were in accordance with our previous data (for the definition of these parameters see in the experimental part). 30 The Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) of MWCNT/Tyr were in accordance with data previously reported, and confirmed the polyelectrolyte deposition and enzyme immobilization. 30 In particular, the AFM analysis clearly showed functionalized nanotubes characterized by a smooth surface probably due to the presence of BSA in the interstitial sites between Tyr molecules.…”

“…30 The Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) of MWCNT/Tyr were in accordance with data previously reported, and confirmed the polyelectrolyte deposition and enzyme immobilization. 30 In particular, the AFM analysis clearly showed functionalized nanotubes characterized by a smooth surface probably due to the presence of BSA in the interstitial sites between Tyr molecules. The maximum height and width were higher than that observed for MWCNTs alone (experimental procedures, SEM analysis, and magnified details of the bidimensional high resolution AFM images of MWCNT/Ty and MWCNTs with the corresponding profiles, are in supplementary information SI #3, Figures 4 MWCNT/Tyr shows a performance about two times lower than the native enzyme, in agreement with the expected decrease of catalytic efficiency after immobilization.…”

“…30 Briefly, mushroom Tyr from Agaricus bisporus and BSA (BSA:Tyr ratio 3:1) were immobilized on oxidized MWCNTs, 34 by deposition of a layer of PDDA (MWCNTs-PDDA:Tyr ratio of 5:1) in sodium phosphate buffer (PBS; 0.1 M, pH 7) at room temperature ( Figure 1, step A). The excess PDDA was removed by centrifugation/re-dispersion cycles since residual PDDA in the solution can precipitate upon mixing with enzyme.…”

“…29 The efficacy of tyrosinase in the synthesis of simple catechol derivatives was successively increased by immobilization of the enzyme on multi-walled carbonanotubes (MWCNTs), using the Layer-by-Layer (LbL) procedure. 30 The LbL procedure is based on the consecutive deposition of alternatively charged polyelectrolytes onto the active species, able to protect proteins from high-molecularweight denaturing agents. 31 MWCNTs are characterized by high surface area for enzyme loading, as well as biocompatibility, and favorable electrochemical and mechanical properties.…”

“…32,33 The best catalyst (indicated in the follow as MWCNT/Tyr) was obtained by co-immobilization of Tyr and Bovine Serum Albumin (BSA) on oxidized MWCNTs, in the presence of polydiallyldimethyl ammonium chloride (PDDA). 30 Here we report the use of MWCNT/Tyr for the improved synthesis of lipophilic hydroxytyrosol and dihydrocaffeoyl catechols, and their antiviral activity against a large panel of DNA and RNA viruses, including Poliovirus type 1, Echovirus type 9, Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), Coxsackie virus type B3 (Cox B3), Adenovirus type 2 and type 5, and Cytomegalovirus (CMV). Some of these compounds were endowed with antiviral activity at sub-toxic concentrations.…”