Expressão heteróloga e caracterização de mananases da família GH5

Abstract

Polysaccharides in plant cell walls are mainly composed of cellulose, a glucose homopolymer, and hemicellulose, a complex group of heterogeneous polymers and one of the largest sources of renewable organic matter, with mannan being one of the main constituents of hemicellulose. Mannanases are enzymes capable of hydrolyzing mannan, cleaving glycosidic bonds within the main chain and releasing short manno-oligomers. The main enzyme families that exhibit this activity are GH5, GH26, and GH113. They can be employed in both biomass pretreatment and the production of sugars for biofuel production. Additionally, mannan degradation can generate manno-oligosaccharides (MOS), which can be used as prebiotics by stimulating the growth of beneficial microorganisms in the intestinal flora. In this project, we successfully conducted the heterologous expression and purification of two mannanases from the GH5 family. Furthermore, we were able to define the main biochemical characteristics of the enzymes using the 3,5-dinitrosalicylic acid (DNS) method, such as enzymatic kinetics, substrate affinity, thermal stability, and optimal conditions. We also evaluated their application in mannan degradation and MOS production from lignocellulosic biomass. Finally, the main structural characteristics were studied based on three-dimensional structural models of the proteins generated from the amino acid sequence using the protein conformation prediction system. The obtained results significantly contribute to understanding the catalytic properties of these enzymes and their potential biotechnological applications.