Modeling and experimental validation of covalent immobilization of Trametes versicolor laccase in gold nanoparticles

Abstract

Introduction. Protein immobilization is one of the most used technologies to increase the enzyme operational stability and improve their usage in biotechnological applications. Usually, the synthesis of immobilized derivatives is performed by the trial and error method. Our strategy for the rational design of immobilized derivatives (RDID) allows to select the optimal conditions for their synthesis. Gold nanoparticles (AuNPs), as non-porous solid supports, represent excellent systems for protein immobilization, with successful applications in nanomedicine, biocatalysis and electronics. Methods. The objective of this work was to validate the RDID strategy for covalent immobilization of the enzyme laccase from the basidiomycete white-rot fungus Trametes versicolor on AuNPs, activated with mercaptoundecanoic acid (AuNP-MUA) and MUA-ethylenediamine (AuNP-MUAen), for the potential use of the resultant biocatalysts in bioconversion processes. Results. As a result, the laccase-AuNP-MUA immobilized derivative is better than laccase-AuNP-MUAen in predicted immobilized derivative functional competence (42,8 % vs 26,1 %, respectively). In addition, all clusters predicted for laccase binding on AuNP-MUA resulted in immobilized enzymes with accessible active sites. In contrast, the half clusters predicted for interaction of laccase with AuNP-MUAen resulted in enzymes with poorly accessible active sites. Activity prediction was confirmed by an experimental expressed enzymatic activity of 45 % in laccase-AuNP-MUA system. Experimental maximal protein load matches the theoretical value (12,7 ± 0,7 x 10-12 vs 12 x 10-12 protein mg / activated AuNP mL). The results obtained in this research suggest the possibility of using this biocatalyst on a large scale.