PgTI, the First Bioactive Protein Isolated from the Cactus Pilosocereus gounellei, is a Trypsin Inhibitor with Antimicrobial Activity

Abstract

Aims: This work aimed to isolate, characterize and evaluate the antimicrobial activity of a trypsin inhibitor (PgTI) from the stem of Pilosocereus gounellei. Place and Duration of Study: Departamento de Bioquímica, Universidade Federal de Pernambuco between March 2013 and October 2018. Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro between June and July 2018. Methodology: PgTI was isolated from P. gounellei stem extract by gel filtration and ion exchange chromatographies. The inhibitor was characterized by isoelectric focusing, polyacrylamide gel electrophoresis, tryptic digestion followed by mass spectrometry analysis and for stability towards heating. Antibacterial and antifungal activities were investigated through broth microdilution assays. Viability of the microbial cells was also evaluated by flow cytometry analysis using thiazol orange and propidium iodide. Results: PgTI appeared as a single polypeptide band of 37.1 kDa and isoelectric point (pI) 5.88. The inhibition constant (Ki) for bovine trypsin was 14 nM and mass spectrometry analysis of PgTI did not reveal similarities with other plant proteins. Trypsin inhibitor activity was stable at temperatures up to 50ºC. PgTI inhibited growth of Gram-positive and Gram-negative bacteria (minimal inhibitory concentrations (MIC) from 7.5 to150 µg/mL) with bactericidal activity only against Escherichia coli (minimal bactericidal concentration: 75.0 µg/mL). PgTI also inhibited the growth of Candida krusei (MIC of 60 µg/mL). Flow cytometry confirmed that PgTI did not affect the viability of E. coli and C. krusei cells at the MIC. Conclusion: This is the first report on a bioactive protein purified from P. gounellei, which provides biotechnological value to this cactus.