Metagenomic alkaline protease from mangrove sediment

Functional screening of metagenomic libraries is an important tool for the discovery of new molecules. The metabolic diversity of microorganisms enables survival in harsh environments and is related to the production of enzymes. In this study, we identified a protease-producing clone from a metagenomic library derived from mangrove sediment. The protease was purified by ammonium sulphate precipitation and gel filtration chromatography, with a yield of 77.27% and a specific activity of 8.57 U μg^-1 . It had a molecular weight of approximately 70 kDa. MS/MS in ESI-Q-TOF revealed nine peptides similar to a peptidase of Bacillus safensis. The aligned partial sequence showed 47.48% identity and 82.74% similarity to the conserved domains of a glutamyl aminopeptidase from the human gut metagenome and 32.12% total coverage. The protease had an optimal pH of 8.5 and optimal activity at 60°C. At pH 9-12, its activity was greater than 80%. It had moderate thermotolerance and thermostability at temperatures of 40 and 50 °C. The K_M and V_max values were estimated to be 0.92 mg ml^-1 , and 13.15 mmol min^-1 for azocasein. Substrate specificity analysis showed that PR4A3 was active on gelatin, blood, egg yolk, and milk. These results support the potential use of PR4A3 in biotechnological applications.