Abstract
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.
Highlights
Bacteriocins belong to a huge and diverse family of ribosomally synthesized bioactive peptides more commonly known as ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by a wide range of bacterial species (Arnison et al, 2013)
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
The previously reported three-dimensional solution structure of enterocin NKR5-3B revealed that the residues at the 2nd helix region and the inter-helix region (V36 to A43) were found to be highly dynamic which is indicative that these regions are potentially critical for its bioactivity (Himeno et al, 2015)
Summary
Bacteriocins belong to a huge and diverse family of ribosomally synthesized bioactive peptides more commonly known as ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by a wide range of bacterial species (Arnison et al, 2013). The introduction of site-directed mutagenesis into the gene encoding the bacteriocin precursor molecule could serve as a potential biological template for the production of novel peptides with improved functionalities (Cotter et al, 2006). Enterocin NKR-5-3B is a well-characterized circular bacteriocin It is synthesized from an 87-amino acid residue precursor peptide consisting of a 64-amino acid core peptide attached to a 23-amino acid N-terminal leader peptide. The resulting plasmid was termed pNK-B1234 (Perez et al, 2016) In this present study, we attempted to bioengineer some potentially critical residues of this novel circular bacteriocin guided by the information from its three dimensional NMR solution structure (Himeno et al, 2015). Our findings here provide information on the critical mutations in relation to bioactivity that could be used as basis for future studies on the rational design of enhanced bioactive peptides utilizing this circular bacteriocin as a scaffold
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