Immobilization of laccase over carbon nanotubes for biocatalysis applications

The use of oxidative enzymes has received great attention due to their efficiency and selectivity. Laccase is an oxidative enzyme that is used to catalyze a wide range of phenolic compounds. Laccase is increasingly used in oxidative processes, such as: oxygen delignification, color removal, bioremediation, degradation of polycyclic aromatic hydrocarbons (PAH), biosensors, biofuel cells, among others. However, the low stability in the free form and the impossibility of reuse has limited its application at industrial level. Thus, the immobilization of laccase is an effective method of continuously applying oxidation reactions. In this work, laccase was immobilized on multi-walled carbon nanotubes (MWCNTs) modified using different functionalization approaches: with i) carboxylic acid; ii) 3- aminopropyl)triethoxysilane (APTS); iii) glutaraldehyde; iv) both APTS and glutaraldehyde; and v) N-ethyl-N-(3-(dimethylamino)propyl) carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS). The main objective was to understand the behavior of the enzyme when immobilized on functionalized MWCNTs and find out the more efficient functionalization method. The enzyme-MWCNT conjugate with enhanced immobilization efficiency, catalytic activity, thermal stability and reutilization performance towards ABTS (2.2’-azino-bis(3-ethylbenzathiazoline-6- sulfonic) acid) oxidation was selected for being used for the biocatalytic oxidation of phenol. The best performance dealing with laccase immobilization was obtained for MWCNTs treated with EDC/NHS (N-ethyl-N-(3-(dimethylamino)propyl) carbodiimide hydrochloride/N-hydroxysuccinimide). The obtained values of immobilization yield and recovered activity were 99.8% and 20.4% respectively. Regarding reutilization tests, the best performing materials were also those treated with EDC/NHS, with an immobilized laccase activity of 65% after 5 cycles of reutilization. It was also noticed that the immobilization process using this treatment promoted an increase in laccase stability, protecting it from thermal denaturation comparing with free laccase. The characterization of MWCNTs using FTIR-ATR demonstrated that functionalization with EDC/NHS generated the introduction of functional groups, which promote the covalent immobilization of laccase on MWCNTs surface. The biocatalytic performance of the immobilized laccase was evaluated using phenol as model compound. Free laccase and immobilized laccase were able to degrade phenol. Using free enzyme, degradation process occurs faster, resulting in a degradation of 97% after 1 hour. For the immobilized enzyme, the process is slower, presenting 87% of phenol degradation after 4 hours of reaction. However, it was found that it is possible to reuse the enzyme-support complex obtaining a percentage of 70% of phenol degradation after reuse. Hereupon, the possibility of reutilization brings enormous economical and environmental advantages.