Purification, Characterization, Identification, and Anticancer Activity of a Circular Bacteriocin From Enterococcus thailandicus

Lamiaa A Al-Madboly; Nehal M El-Deeb; Amal Kabbash; Manal A Nael; Ahmed M Kenawy; Amany E Ragab

doi:10.3389/fbioe.2020.00450

Purification, Characterization, Identification, and Anticancer Activity of a Circular Bacteriocin From Enterococcus thailandicus

Front Bioeng Biotechnol. 2020 Jun 23:8:450. doi: 10.3389/fbioe.2020.00450. eCollection 2020.

Authors

Lamiaa A Al-Madboly¹, Nehal M El-Deeb², Amal Kabbash³, Manal A Nael⁴, Ahmed M Kenawy⁵, Amany E Ragab³

Affiliations

¹ Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Tanta University, Tanta, Egypt.
² Biopharmaceutical Products Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications (SRTA-City), Alexandria, Egypt.
³ Department of Pharmacognosy, Faculty of Pharmacy, Tanta University, Tanta, Egypt.
⁴ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Tanta University, Tanta, Egypt.
⁵ Nucleic Acids Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications (SRTA-City), Alexandria, Egypt.

Abstract

New anticancer agents are continually needed because cancerous cells continue to evolve resistance to the currently available chemotherapeutic agents. The aim of the present study was to screen, purify and characterize a hepatotoxic bacteriocin from Enterococcus species. The production of bacteriocin from the Enterococcus isolates was achieved based on their antibacterial activity against indicator reference strains. Enterococcus isolates showed a broad spectrum of antibacterial activity by forming inhibition zones with diameters ranged between 12 and 29 mm. The most potent bacteriocin producing strain was molecularly identified as Enterococcus thailandicus. The crude extracted bacteriocin was purified by cation exchange and size exclusion chromatography that resulted in 83 fractions. Among them, 18 factions were considered as bacteriocins based on their positive antibacterial effects. The anticancer effects of the purified bacteriocins were tested against HepG2 cell line. The most promising enterocin (LNS18) showed the highest anticancer effects against HepG2 cells (with 75.24% cellular inhibition percentages), with IC50 value 15.643 μM and without any significant cytotoxic effects on normal fibroblast cells (BJ ATCC® CRL-2522™). The mode of anticancer action of enterocin LNS18 against HepG2 cells could be explained by its efficacy to induce cellular ROS, decrease HepG2 CD markers and arrest cells in G0 phase. Amino acid sequence of enterocin LNS18 was determined and the deduced peptide of the structural gene showed 86 amino acids that shared 94.7% identity with enterocin NKR-5-3B from E. faecium. Enterocin LNS18 consisted of 6 α-helices; 5 circular and one linear. Model-template alignment constructed between enterocin LNS18 and NKR-5-3B revealed 95.31% identity. The predicted 3D homology model of LNS18, after circularization and release of 22 amino acids, showed the formation of a bond between Leu23 and Trp86 amino acid residues at the site of circularization. Furthermore, areas of positive charges were due to the presence of 6 lysine residues resulting in a net positive charge of +4 on the bacteriocin surface. Based on the above mentioned results, our characterized bacteriocin is a promising agent to target liver cancer without any significant toxic effects on normal cell lines.

Keywords: anticancer; bacteriocin; circular; protein sequence; purification.