A nanostructured biomimetic system for the enzymatic hydrolysis of xyloglucan

Author(s)

Jéssica de Moura Soares

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-11-07

Examining board members

Marcelo Damario Gomes; Carmen Lúcia Cardoso; Gustavo Orlando Bonilla Rodriguez

• Advisor: Richard John Ward

Abstract

Lignocellulosic materials are abundant agroindustrial byproducts and constitute a renewable carbon source that can be used for biofuel production, such as second-generation bioethanol. Xyloglucan is a hemicellulose and one of the most abundant polysaccharides in lignocellulose and it is entirely hydrolyzed by the combined action of at least four different enzymes. The approach of protein immobilization on nanomaterials offers the possibility of colocalisation of different enzymes on structures designed to mimic biological reactions, and even entire metabolic pathways. In addition, the use of nanomaterials in enzyme immobilization may increase the synergistic effect due to the proximity effect between the catalytic centers. In this work, the immobilization of four enzymes for xyloglucan depolymerization was studied, with the purpose of developing biomimetic nanostructures. The main objectives were: the expression of the enzymes - xyloglucan specific endo-?-1,4-glucanase, ?-galactosidase, ?-xylosidase and ?-glucosidase, and their immobilization on magnetic nanoparticles and consequent characterization of the activity of these nanostructures against xyloglucan. The heterologously expressed enzymes were covalently immobilized, individually and in combination, on ferromagnetic iron oxide (Fe3O4) nanoparticles coated with chitosan and cross-linked with glutaraldehyde. These hybrid nanostructures were then tested for their catalytic capacity against synthetic substrates and xyloglucan extracted from Jatobá (Hymenaea courbaril) seeds. Enzymatic characterizations demonstrated differences for optimal pH and temperatures determined between free and immobilized enzymes. All enzymes remained active after co-immobilization on the nanomaterial, however specific activity was reduced for all enzymes after immobilization. The optimum temperature of immobilized ?-xylosidase YicI increased by ten degrees compared to the free enzyme. Binding efficiency on the particles ranged from 5 to 92% between enzymes. The enzyme ?-galactosidase Bga1 demonstrated the best immobilization efficiency as well as the highest reuse capacity, maintaining up to 50% of activity after ten cycles. In addition, assays with different enzymatic proportions were performed to optimize xyloglucan hydrolysis by the four enzyme-containing nanosystems. The capacity of xyloglucan hydrolysis by the nanosystems was determined by measuring glucose released after the reactions and by mass spectrometry of the reaction products. Mass spectrometry results indicated the presence of the xylose and glucose/galactose monosaccharides in the xyloglucan hydrolysis products, showing the depolymerization activity of the hybrid nanostructures containing the four enzymes. (AU)

• FAPESP's process: 17/14452-1 - A nanostructured biomimetic system for enzymatic hydrolysis of xyloglucan
• Grantee: Jéssica de Moura Soares
• Support Opportunities: Scholarships in Brazil - Master