Directed Evolution of AtMP2 Peptide: Unlocking Enhanced Antibacterial Potential from Anabas testudineus

Previous studies have shown that AtMP1 and AtMP2, antimicrobial peptides found in the climbing perch (<i>Anabas testudineus</i>), possess antibacterial and anticancer properties. This study aimed to determine whether modified forms of the AtMP2 peptide could enhance its antimicrobial activity. In the research study, the epidermal mucus of <i>Anabas testudineus</i> was extracted and tested to contain antibacterial and anticancer properties. Two peptides were initially extracted from the freshwater fish <i>Anabas testudineus</i>; however, the focus was placed on AtMP2 to evaluate its potential for enhanced antibacterial activity. Systematic-directed evolution was used to generate AtMP2 varieties. The AtMP2 varieties were characterized using APD3, CAMP, and AMPFun. Based on the characterization, two peptides, AtMP2-1 and AtMP2-2, were selected for synthesis. AtMP2-1 and AtMP2-2 demonstrated higher antimicrobial activity against Gram-positive than Gram-negative bacteria compared to the AtMP2 peptide, based on Minimum Inhibitory Concentration (MIC) determination and Kirby-Bauer Disk Diffusion analysis. For the SRB cytotoxicity analysis using the HS-27 and Vero cell lines, both AtMP2-1 and AtMP2-2 are safe for human use at 20 μg/mL. ZDOCK and HPEPDOCK servers were used to evaluate the binding affinity between AtMP2-1 and AtMP2-2 peptides and proteins involved in the bacterial cell death cycle. The results showed varying docking scores, with more negative values indicating stronger binding interactions, particularly with proteins such as SecA, RpoB, GyrA, ClpP, and MetG. The study concluded that modified peptides derived from <i>Anabas testudineus</i> show enhanced antibacterial properties and minimal cytotoxicity, making them potential alternatives to traditional antibiotics. Future research should investigate the specific biochemical pathways affected by these AMPs to understand their mechanisms of action better and explore their potential as therapeutic agents, particularly in the treatment of bacterial infections, wound healing, and cancer therapy.