Chemical modification of proteins, applications in inmobilization, catalytic performance and fluorescent labeling

Abstract

ABSTRACT This doctoral thesis entitled "Chemical modification of proteins, applications in immobilization, catalytic performance and fluorescent labeling." aims to develop methodologies of chemical modification of proteins and applications thereof. The thesis has been divided into four sections in each of which a methodology of protein modification is addressed and one or more applications of the methodology are proposed. In a first section, is proposed to improve the "carrier-free" immobilization technology: CLEAs (cross-linked enzyme aggregates) using diepoxide reagents to bind proteins together. The epoxy-CLEAs manufacturing method was studied and optimized with the response surface methodology, obtaining stable and active epoxy-CLEAs. The expoxy-CLEAs were photographed by tomographic microscopy. The manufacturing method was also applied to the fabrication of magnetic CLEAs. The aggregates also retain the catalytic activity and have the advantage of being remove from the reaction medium using a neodymium magnet, facilitating the purification of the final product and the reuse of the biocatalyst. In the second section the laccase from Trametes versicolor was immobilized on silica supports or inside the pores of the supports, both commercial and synthesized in the laboratory. A laccase biocatalyst was thus prepared which was applied to the synthesis of two phenoxiazones: Curie 22 and cinnabarinic acid. In a third section a direct modification of magnetic nanoparticles (9-12 nm) is proposed. These particles are functionalized directly by nucleophilic substitution with organic halides. Using the functionalized nanoparticles a sustainable method to obtain a semi-natural benzyl acetate in a solvent-free system is proposed, using lipases immobilized on the particles. The fourth and final section is the application of fluorescent derivatives synthesized in the laboratory for application in molecular and cellular biology. Compounds incorporating fluorescent molecules and compounds with some kind of affinity with proteins have been synthesized. The compounds have been used to label proteins in liquid media, to stain acrylamide gels, to quantify proteins and to stain cells, obtaining encouraging results.