Resumo

Proteases are biochemically diverse enzymes that have as main characteristics their versatility for many applications, as well as their easy manipulation. In general, their activity is evaluated in isolated microorganisms using casein or albumin as substrates. This approach has some limitations, including the restrict nature of the amino acid sequence of the protein used as substrate for the enzymatic cleavage, as well as the number of evaluated microorganisms, usually only cultivated strains. The construction and screening of metagenomic fosmid clone libraries based on fluorescent probes for proteolytic activities can be an alternative and promising tool to search for new biocatalysts. In this context, the objective of this work was to explore the potential of microorganisms present in environments characterized by extreme conditions, such as mangrove sediments and petroleum reservoirs, using a metagenomic approach and high throughput screening tools to identify proteolytic activity. Two metagenomic libraries (from petroleum reservoir and mangrove sediments) were constructed using the “Cloning-Ready Copy Control pCC2FOS” kit (Epicentre®) and a total of 960 clones were subjected to high throughput screening for proteases, based on the use of fluorescence resonance energy transfer (FRET) peptide library probe, under three different pHs (4.0, 7.0 and 9.0). The hydrolysis of the FRET peptides was quantified using a Plate Spectrofluorimeter by measuring the fluorescence of Abz (ortho-aminobenzoic acid) as the fluorescence donor at ex 460 nm following emission at em 360 nm and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) as the fluorescence acceptor. Forty-five positive hits were obtained at pH 7.0, among which one showed significant fluorescence intensity, indicating a possible metalloproteinase. In addition, two clones were moderately active at pH 4.0, indicating possible cysteine proteases, which deserve further analysis since proteases of this class are rare and of great interest for application in biotechnology. The results obtained confirmed the potential of the microbiota from petroleum and mangrove environments as a source for novel biocatalysts with proteolytic activity. Further steps of the work will comprise clone insert digestion and ligation into an expression vector for activity analysis and further chemical characterization.