There is an increasing need to find novel and robust biocatalysts with promising features that
compete with those currently available on the market. Composting is an extreme habitat of high
microbiological diversity that represents a suitable source of lignocellulose-degrading
enzymes, such as cellulases, hemicellulases and ligninases, properly active under harsh
conditions. These enzymes can convert the recalcitrant structure of lignocellulose into valuable
bioproducts with great biotechnological potential. -Glucosidases are glycoside hydrolases
responsible for degrading cellulose, namely in the disruption of the final glycosidic bonds of
short-chain oligosaccharides to obtain glucose. Metagenomics is an emerging cultureindependent
technique that has proven effective in identifying new biocatalysts with better
catalytic activity through the analysis of DNA extracted from a vast number of environments.
The metagenomic analysis is divided into two main technologies: sequence- and functionalbased
approach. Function-based screening aims to discover and identify new genes capable
of producing biocompounds/biomolecules with new or improved functions. This screening is
based on the detection and isolation of clones with a positive response to the desired
phenotype when activity-based techniques are applied. In this study, high-molecular-weight
DNA extracted from a compost sample was used to construct a fosmid metagenomic library.
This library was evaluated through a functional screening to identify clones that expressed
cellulase activity, specifically -glucosidase activity. The enzymatic activity was unravelled
using esculin as substrate through the formation of a brown halo as a positive response (Figure
1). The functional screening was performed in 96-well microplates and the detection of -
glucosidase activity was evaluated at different temperature (25-60 °C) and pH (4.5-9.5)
conditions. It was possible to identify clones with the enzymatic activity of -glucosidase in
almost all tested conditions, except at 60 °C. The best conditions for clone growth occur in a
longer initial incubation time (3 days, 37 °C). On the other hand, the lower pH and incubation
temperature favoured a faster detection of -glucosidase activity.The study received financial support from the Portuguese Foundation for Science and
Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the
Project LIGNOZYMES (POCI-01-0145-FEDER-029773).info:eu-repo/semantics/publishedVersio
