Purification and functional characterization of two basic proteases of South American Bromelia antiacantha fruit

Proteolytic enzymes from the bromeliad family are valuable biocatalysts with a broad range of biotechnological applications, thanks to their stability and broad substrate specificity. In this study, two novel cysteine proteases, AntB and AntC, were purified from ripe fruits of Bromelia antiacantha Bertol., using acetone precipitation and cation-exchange chromatography, thereby completing studies of the three main isoforms present in this plant. The functional characterization of both enzymes showed broad pH and temperature stability, with AntC retaining activity over pH 4.5-11 and both exhibiting optimal activity near 65 °C. Remarkably, AntB and AntC maintained activity in the presence of strong denaturants such as urea and guanidinium chloride, indicating high structural stability. Kinetic studies with synthetic substrates revealed differences in specificity and catalytic efficiency between the two enzymes and compared to stem bromelain, supporting their functional differences. Peptide mass fingerprinting further confirmed sequence homology with Fastuosain while highlighting structural divergences. Together, these findings establish AntB and AntC as robust and versatile proteases with promising potential for biotechnological, industrial, and biomedical applications and reinforce the value of underexploited bromeliad species as sustainable sources of biocatalysts.