Resumo

Bacteria and fungi are major sources of enzymes involved in the transformation of key compounds for the carbon fluxes on mangrove soils, characterized by the high prevalence of anaerobiosis, salinity and high content of organic matter. The decomposition of plant or animals residues under these conditions is very slow, acting as a selective pressure on the evolution of enzymes involved in the mineralization process. Metagenomics has provided access to the vast majority of the microbial diversity in the environment, through the generation of fosmid libraries, resulting in a promising scenario for bioprospection. In this study, we report the description and characterization of a novel enzyme of the family GH3, classified as a β-N-acetylhexosaminidase (EC 3.2.1.52), involved in the degradation of organic matter in mangrove soils contaminated by oil spill located in the city of Bertioga-SP. Through a screening of a metagenomic library (12,960 clones), a positive clone was detected and subjected to a next generation sequencing by Ion Torrent PGM platform. A total of 1,175,586 reads were generated, with an average size of 198pb. Sequences were trimmed based on quality, PHRED scores ≥30.0. Selected sequences were further curated by discarding those from the host cell E. coli and vector and assembled to a contig of 39.586Kb. The contig was submitted to annotation at RAST server, where ORF sequences were compared with sequences present at Protein Data Bank and SwissProt databases, revealing the occurrence of different genes. Among the annotated ORFs, a sequenced of 1,065 nucleotides was identified as coding for a β-N-acetylhexosaminidase. The annotated ORF was compared against the GenBank database (nr/nt), where low values of similarity were found with approximately 30% of identity. The enzyme was expressed and purified to a single band on SDS-PAGE gel, with estimated molecular mass of 43kDa. The activity characterization was performed for distinct substrates indicating an optimum temperature of 30°C, with the best pH values at 4.5 and 5.5 for the degradation of GalNac and GlcNac, respectively. The test for inhibition by salinity revealed a tolerance of this enzyme to values of 0.5M NaCl, while the stability analyses indicated a decrease in the enzyme activity after 3h of incubation at 30°C. The identification of new enzyme through metagenomics indicated the mangroves might be a reservoir of new enzymes with high potential use for biotechnological purposes.