A nanostructured biomimetic system for enzymatic hydrolysis of xyloglucan

Abstract

Lignocellulosic materials are abundant agroindustry by-products that are a source of cheap, renewable carbon used in the production of biofuels fuels. One of the major challenges of using this material in second generation ethanol production is the high cost and difficulty of the enzymatic saccharification step due to the recalcitrance of the constituent polysaccharides. One of the most abundant polysaccharides in these materials is xyloglucan, which can be hydrolyzed by the combined action of glucanases, galactosidase, xylosidases and glucosidases enzymes. Protein engineering strategies offer the possibility of combining different enzymatic activities, and in the case of this project, using nanostructures through the co-immobilization of several enzymes active against xyloglucan. The use of nanomaterials in the immobilization of enzymes allows synergistic effect, due to the proximity between the catalytic centers. Therefore, this project proposes the development of biomimetic nanostructures, active in the depolymerization of xyloglucan. The main objectives of this project are the expression of enzymes endo-²-1,4-glucanase, ²-galactosidase, ±-xylosidase and ²-glucosidase derived from fungi and active against xyloglycan. These enzymes will be immobilized on magnetic nanoparticles derivatized with Chitosan / NTA, and the activity of these biomimetic nanostructures against xyloglucan will be characterized. The enzymes will be expressed heterologously in both Escherichia coli and Pichia pastoris, and subsequently immobilized, individually and in mixtures, with ferromagnetic nanoparticles, which will be obtained from magnetite (Fe3O4), whose particles will be coated with chitosan and crosslinked with Glutaraldehyde then functionalized with the addition of Ni-NTA, which region will bind the enzymes by means of their histidine (His-tag) extensions. The hybrid nanostructures will then be tested for their catalytic capacity against synthetic substrates, xyloglucan and crude biomass (AIR and sugar cane bagasse). The dosage of enzymatic activity will be done by colorimetric methods and the analysis of the products of the tests will be carried out by mass spectrometry. Tests of thermostability and reuse of the systems will also be performed. (AU)