Experimental elucidation of an antimycobacterial bacteriocin produced by ethnomedicinal plant-derived Bacillus subtilis (MK733983).

A bacteriocin from Bacillus subtilis (MK733983) originated from ethnomedicinal plant was purified using Preparative RP-HPLC. The HPLC fraction eluted with 65% acetonitrile showed the highest antimicrobial activity with Mycobacterium smegmatis as an indicator. Its specific activity and purification fold increased by 70.5% and 44%, respectively, compared to the crude bacteriocin. The bacteriocin showed stability over a wide range of pH (3.0-8.0) and preservation (- 20 °C and 4 °C), also thermal stability up to 80 °C for 20 min. Its proteinaceous nature was confirmed with complete loss of activity on its treatment with Trypsin, Proteinase K, and α-Chymotrypsin. Nevertheless, the bacteriocin retained up to 45% activity with Papainase treatment and was unaffected by salivary Amylase. It maintained ~ 95% activity on UV exposure up to 3 h and its activity was augmented by ethyl alcohol and metal ions like Fe2+ and Mn2+. Most of the common organic solvents, general surfactants, preservatives, and detergents like Sulfobetaine-14, Deoxy-cholic-acid did not affect the bacteriocin's action. Its molecular weight was estimated to be 3.4KDa by LC-ESI-MS/MS analysis. The bacteriocin is non-hemolytic and exhibited a broad inhibition spectrum with standard strains of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Chromobacterium violaceum with MICs ranging 0.225 ± 0.02-0.55 ± 0.05 mg/mL. Scanning Electron Microscopy showed cell annihilation with pores in cell membranes of S. aureus and P. aeruginosa treated with the bacteriocin, implicating bactericidal mode of action. These promising results suggest that the bacteriocin is significant and has wide-ranging application prospects.