Expression and purification of epinecidin-1 variant (Ac-Var-1) by acid cleavage.

The demand for massive quantities of therapeutic active antimicrobial peptides (AMPs) is high due to their potential as alternatives to antibiotics. However, each antimicrobial peptide has unique properties, necessitating distinct synthesis and purification strategies for their large-scale production. In this study, we bio-synthesized and purified a functional enhanced variant of the AMP epinecidin-1, known as Ac-Var-1 (acid-cleavable variant-1). To generate the active peptide, we cloned the gene for Ac-Var-1 with acid-cleavable site (aspartic acid-proline) into the pET-32a expression vector, purified the fusion protein by His tag enrichment chromatography, and performed acid cleavage to release the active Ac-Var-1 peptide. After acid cleavage, the active Ac-Var-1 was purified and characterized by SDS-PAGE and mass spectrometry. The results from both techniques provided confirmation of the intactness of the purified Ac-Var-1. The Ac-Var-1 inhibited the growth of pathogenic Escherichia coli and Staphylococcus aureus. KEY POINTS : • Epinecidin-1 is a well-known antimicrobial peptide having multipotential bioactivities. • Epinecidin-1 variant is developed via the site-directed mutagenesis method to improve its structural stability and bioactivity. • AC-Var-1 development is an economical and easy method to remove peptide from tag protein.

Phenotypic assessment of safety and probiotic potential of native isolates from marine fish Moolgarda seheli towards sustainable aquaculture.

Aquaculture is a highly productive and fast-growing agricultural sector. The occurrence of epidemic or sporadic disease outbreak is a major limiting factor in this sector, thus better alternatives are the need of the hour. Use of indigenous probiotics is a promising strategy to control infectious diseases. Thus, the present study was aimed to screen and characterize potent indigenous probiotics from marine fish, Moolgarda seheli, towards enhancing sustainable aquaculture production. Totally 347 bacterial isolates were obtained from M. seheli gastrointestinal tract, out of these, four isolates (KAF121, 124, 135, 136) were confirmed as potent probiotics in terms of biosafety, highly resistant to acidic pH, gastric juice, bile salt, high hydrophobicity to solvents, auto and co-aggregation potential. These four isolates also exhibited virtuous antioxidant activity. Further the isolates, KAF124 and 135 proved their efficiency in growth and survival of fish after challenged againt Aeromonas hydrophila. The isolates were identified based on their 16S rRNA gene sequence and the data were submitted to Genbank as Pseudomonas aeruginosa KAF121 (MH393516), Bacillus cereus KAF124 (MH393226), Bacillus thuringiensis KAF135 (MH393230), and Pseudomonas otitidis KAF136 (MH393230). The results conclude that two isolates, KAF124 and KAF135 are highly safe and potent probiotics which are first time isolated from the marine fish M. seheli. The two Bacillus strains could be used as better alternatives to antibiotics and other chemical-based drugs to prevent/control infectious diseases in aquaculture.

Bacteriocin-a potential antimicrobial peptide towards disrupting and preventing biofilm formation in the clinical and environmental locales.

Biofilm, a consortium of microbial cells, protected by extracellular polymeric matrix, is considered a global challenge due to the inherent antibiotic resistance conferred by its lifestyle. Besides, it poses environmental threats causing huge damage in food industries, fisheries, refineries, water systems, pharmaceutical industries, medical industries, etc. Living in a community of microbial populations is most critical in the clinical field, making it responsible for about 80% of severe and chronic microbial diseases. The necessity to find an alternative approach is the need of the hour to solve these crises. So far, many approaches have been attempted to disrupt the initial stage of biofilm formation, including adherence and maturation. Bacteriocins are a group of antimicrobial peptides, produced by bacteria having the potential to disrupt biofilm either by itself or in combination with other drugs than antibiotic counterparts. A clear understanding on mechanisms of bacterial biofilm formation, progression, and resistance will surely lead to the development of innovative, effective biofilm control strategies in pharmaceutical, health care industries and environmental locales.