Proteases da biomassa microalgal de Chlorococcum infusionum: obtenção, caracterizações físico-químicas e enzimáticas

Abstract

The biodiversity of microalgae remains a relatively underexplored resource. Its biomass represents a rich source of natural bioactive compounds, such as proteins and lipids, which hold potential applications in human and animal nutrition, as well as in the cosmetic, pharmaceutical, and fine chemical industries. Moreover, they exhibit promise in wastewater treatment and the bioremediation of harmful compounds. Specifically, microalgae belonging to the Chlorococcum genus are considered promising sources of macromolecules; however, their potential as protein sources remains inadequately investigated. In this context, the objective of this study was to extract, isolate, and evaluate the physicochemical and enzymatic characteristics of proteases present in the biomass of Chlorococcum infusionum microalgae produced in a photobioreactor. To achieve this, the microalgae were initially cultivated in Bold's Basal Medium (BBM) in an autotrophic closed-flat plate photobioreactor system. The resulting biomass was characterized and utilized in the preparation of aqueous extracts through a combination of lysis methods (EBCi). The microalgae cultivation yielded an average fresh biomass of 2.35 ± 0.03 g.L-1 after the 12th day of cultivation, with a composition rich in lipids (37.82%) and proteins (27.32%). For the isolation of proteases, EBCi underwent purification steps involving subsequent gel filtration chromatography using a Sephadex G-100 column and ion exchange chromatography using a DEAE-Sepharose™ Fast Flow column. The predominant peak obtained (designated as FPCiT-P3) was subjected to proteolytic activity assays against a nonspecific substrate (azocasein) and through zymograms. Optimal parameter determination assays, stability assays against temperature and pH variations, and structural stability evaluation through circular dichroism spectroscopy were conducted. FPCiT-P3 exhibited an optimum pH for activity around 7.0-9.0 and an optimum temperature of 50°C, being partially inhibited by PMSF and EDTA. The fraction showed low stability at acidic pHs and temperatures above 60°C after a 30-minute incubation. The circular dichroism spectrum of FPCiT-P3 indicated a predominance of β-sheet structure.