CONICET | Buscador de Institutos y Recursos Humanos

The search of new enzyme immobilization techniques, being complementary approaches to genetic engineering, has been constantly pursued in order to obtain efficient biocatalysts. Research in this field is proving nowadays that amyloid-like protein fibrils are becoming interesting new biomaterials for development of nano/micro-scaffolds for different technological applications due to their remarkable mechanical, chemical and structural properties as well as biocompatibility. In the present work, we report the preparation and characterization of a new biocatalyst based on the photo-immobilization of a lipase onto hybrid amyloid nanofibrils formed with heparin and lysozyme. The highly sulfated glycosaminoglycan heparin was used to induce the amyloid-like aggregation of hen egg lysozyme without using extreme conditions, such as addition of organic solvents, low pH, or high temperature. Both fluorescence and infrared spectroscopies together with electronic microscopy demonstrated that structural and morphological characteristics of the insoluble aggregates are compatible with amyloid-like fibrils. Apparently, the heparin chain not only triggers the lysozyme aggregation, but also is a structural component of the obtained nanofibrils, and based on a biocomputational study, a heparin-lysozyme supramolecular arrangement is proposed.The new hybrid nanomaterial could be considered an efficient support for enzyme immobilization since: i) both the lysozyme antibiotic activity and its ability to induce changes in membrane permeability were eliminated, and ii) It can be easily functionalized, since reactive amino acid residues like tyrosine remain exposed to the solvent. We exploited this ability by using blue-light photosensitization of ruthenium (II) tris-bipyridine complex in the presence of ammonium persulfate to generate side-chain tyrosyl radicals, which by recombination attach lipases to the nanofibrils through dityrosine covalent bonds, as determined by fluorescence spectroscopy. Regarding to the free enzyme, our results show that the photo-immobilized lipase has better thermostability and higher organic solvent resistance. The procedure reported herein could be useful to design a new generation of insoluble biocatalyst by a single photo-click step in a way that appears cleaner and faster than conventional chemical crosslinked procedures.