Proteases are important enzymes that are engaged in a variety of essential physiological functions and have a significant possible use in industrial applications. In this work, we reported the purification and biochemical characterization of a detergent stable, antimicrobial, and antibiofilm potential protease (SH21) produced by Bacillus siamensis CSB55 isolated from Korean fermented vegetable kimchi. SH21 was purified to obtain homogeneity via ammonium sulfate precipitation (40–80%), Sepharose CL-6B, and Sephadex G-75 column. By analyzing the SDS-PAGE and zymogram, it was determined that the molecular weight was around 25 kDa. The enzyme activity was almost completely inhibited in the presence of PMSF and DFP, which indicated that it was a member of the serine protease family. SH21 showed excellent activity with a broad range of pH and temperature, with its maximum pH of 9.0 and temperature of 55 °C. The enzyme had estimated K_m and V_max values of 0.197 mg/mL and 1.22 × 10³ U/mg, respectively. In addition, it preserved good activity in the presence of different organic solvents, surfactants, and other reagents. This enzyme showed good antimicrobial activity that was evaluated by MIC against several pathogenic bacteria. Furthermore, it exhibited strong antibiofilm activity as determined by MBIC and MBEC assay and degraded the biofilms, which were analyzed by confocal microscopic study. These properties established that SH21 is a potent alkaline protease that can be used in industrial and therapeutic applications.

Antimicrobial peptides (AMPs) have attracted considerable attention as potential substitutes for traditional antibiotics. In our previous research, a novel antimicrobial peptide YS12 derived from the Bacillus velezensis strain showed broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria. In this study, the fractional inhibitory concentration index (FICI) indicated that combining YS12 with commercial antibiotics produced a synergistic effect. Following these findings, the combination of YS12 with an antibiotic resulted in a faster killing effect against bacterial strains compared to the treatment with the peptide YS12 or antibiotic alone. The peptide YS12 maintained its antimicrobial activity under different physiological salts (Na⁺, Mg²⁺, and Fe³⁺). Most importantly, YS12 exhibited no cytotoxicity towards Raw 264.7 cells and showed low hemolytic activity, whereas positive control melittin indicated extremely high toxicity. In terms of mode of action, we found that peptide YS12 was able to bind with LPS through electrostatic interaction. The results from fluorescent measurement revealed that peptide YS12 damaged the integrity of the bacterial membrane. Confocal laser microscopy further confirmed that the localization of peptide YS12 was almost in the cytoplasm of the cells. Peptide YS12 also exhibited anti-inflammatory activity by reducing the release of LPS-induced pro-inflammatory mediators such as TNF-α, IL-1β, and NO. Collectively, these properties strongly suggest that the antimicrobial peptide YS12 may be a promising candidate for treating microbial infections and inflammation.