Identificação e caracterização in silico e in vitro de celulases em bactérias do filo acidobacteriota

Abstract

Glycosyl Hydrolases (GH) are significant biotechnological enzymes, with applications ranging from the food industry to the production of biofuels. This family hydrolyzes glycosidic bonds and is therefore capable of deconstructing the most abundant land-based polymer on the planet, cellulose. Due to the constant demands to optimize processes and develop new technologies for the industry, discovering new cellulases, with new attributes, is a priority. Genomic studies of bacteria in the phylum Acidobacteriota have shown that they generally outnumber the known genes of lignocellulose-degrading bacteria. Many of these genomes do not have sufficient biological demonstration of the activity of these genes involved in cellulose degradation. In the present work we studied the cellulose degradation capacity of two strains of Acidobacteriota members using genomics and culturomics. The methodology included in silico analyzes for phylogeny, protein size, conservation and distribution of domains with homologues, together with growth assays on cellulose derivatives, using different associations of carbon sources, such as cellobiose and CMC, at different concentrations. In total, 32 probable GHs from 9 different families were identified. Among them, 5 GHs belonging to the GH5, 8, 9 and 44 families were prioritized, as they are enzymes noted as possible endo/exoglucanases. The enzymes present basic characteristics found in cellulases from other bacteria, although indel mutations were found, the enzymes may conserve its function. The results of in vitro analyses were similar between the two strains, with the formation of colonies on solid medium with 0.05% xylan, but not on CMC, MCC or Avicel®, unless they were combined with cellobiose, demonstrating that these substrates are not growth inhibitors at low concentrations. Although colonies did not grow on plates with CMC, Congo Red staining revealed a manipulation halo indicating partial exoglucanase activity. However, the manipulation assay could not provide information on endocellulase activity. This study demonstrates for the first time the growth of an Acidobacteria in a chemically defined medium enriched with CMC, but the results do not indicate that these strains are suitable for industrial applications as cellulolytic bacteria. However, adapted to different extreme environments, Acidobacteria represent a source of biological diversity that has not yet been explored, offering new opportunities for exploration that can help in obtaining other new bioproducts.