BIOENGINEERING OF THE CIRCULAR BACTERIOCIN FROM ENTEROCOCCUS FAECIUM NKR-5-3 BY NNK-SCANNING TO ENHANCE ITS BIOACTIVITY

Bacteriocins are gene-encoded antimicrobial peptides that are traditionally appreciated as safe food preservative in the food industry and as possible alternative to conventional antibiotics in the pharmaceutical industry. Enterocin NKR-5-3B is a well characterized circular bacteriocin that possess exceptional stability due to the circular nature of its structure. In this paper, a mutant library of this bacteriocin was constructed through NNK-scanning whereby the presumed critical residues were targeted hoping to obtain bacteriocin derivatives with enhanced bioactivity. Thirteen (13) mutant phenotypes exhibited bioactivity enhancement relative to the native bacteriocin. The most notable bioactivity increases were observed from the phenotypes expressing V32C, V32A, and L40G that exhibited 233, 217, and 200% relative bioactivity, respectively. In-silico analyses of the resulting bacteriocin derivatives showed significant changes in the physico-chemical properties of the bacteriocin derivatives, particularly its hydrophobicity index, as a consequence of the introduced mutation. The V32C bacteriocin derivative which exhibited the strongest bioactivity enhancement was found to exhibit a reduction of the molecular surface hydrophobicity and isoelectric point. These changes may have contributed in the enhancement of its bioactivity. The identification of these critical mutations is highly valuable as basis for future studies on the rational design of bioengineered bacteriocins with enhanced bioactivity.