High-Throughput HTP selection of optimized Carbohydrate-Active enZYmes CAZYmes to improve the nutritive value of cereal-based diets and the degradation of ligno-cellulosic biomass

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High-Throughput production and characterization of Carbohydrate-Active enZYmes for animal nutrition

Publication . Lopes, Vânia Alexandra da Silva Cardoso; Fontes, Carlos Mendes Godinho de Andrade; Brás, Joana Luís Armada

The biodegradation of plant cell wall (PCW) carbohydrates is performed by microbial enzymes that are generally referred to as CAZymes. In animal nutrition, it is now well established that the monogastric animals produce a limited repertoire of CAZymes and as such cannot use efficiently some dietary ingredients that sometimes display antinutritional properties. The dietary supplementation with exogenous CAZymes improves the nutritive value of diets and increases animal’s performance. In particular, this study demonstrated that 1,3-1,4-β-glucanases and not cellulases improve the nutritive value of β-glucan-containing diets for monogastric animals. In addition, it was revealed that exogenous enzyme supplementation with β-xylanases improved the nutritive value of diets incorporating wheat lots with high viscosity and low endogenous endo-1,4-β-xylanase activity. In contrast, when the wheat lot showed lower viscosity and higher levels of endogenous endo-1,4-β-xylanase activity, broiler response was clearly diminished. Moreover, the data revealed that xylo-oligosaccharides released by xylanases acting on cereal arabinoxylans display a pre-biotic and positive effect in broiler chicks. However, although we observe an exponential accumulation of genomic and metagenomic information, knowledge on CAZYmes with potential to be used in animal nutrition is limited. This work also aimed to develop high-throughput (HTP) methodologies to isolate and characterize potentially important enzymes for animal nutrition. Thus, 1476 recombinant enzymes were selected and produced recombinantly. The data revealed that 79% of recombinant proteins were produced in the soluble form in Escherichia coli. Factors, such as, organism of origin, gene production strategy, fusion with solubility tags, protein molecular weight and amino acids composition of primary sequences may be used to justify and predict levels of solubility. The establishment of a high-throughput pipeline for recombinant enzyme production was used to obtain a library of feruloyl esterases (FAEs) and glucuronoyl esterases (GEs), enzymes which remove the side chains and break crosslinks between hemicellulosic carbohydrates and lignin. Thus 480 putative FAEs and 20 GEs were produced and biochemically characterized. Following gene isolation, 372 FAEs and 11 GEs were produced in a soluble form in E. coli. Activity results showed that 50% of the enzymes produced retained significant levels of activity and stability. The library of innovative FAEs and GEs produced during this project will be used to develop a novel generation of enzymes for animal nutrition, in particular to exploit the release of cellulose and hemicellulose from lignin.

2020-01-16Doctoral thesis

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