Kyung Bin Nam

Global public health is facing a major issue with emerging resistance to antimicrobial agents. Antimicrobial agents that are currently on the market are strong and efficient, but it has not been ruled out that these medications will eventually cause resistance to bacteria. Exploring novel bioactive compounds derived from natural sources is therefore, crucial to meet future demands. The present study evaluated the mode of action of the antimicrobial potential protease enzyme SH21. Protease SH21 exhibited antimicrobial activity, strong heat stability (up to 100 °C), and pH stability (pH 3.0 to 9.0). In terms of mode of action, we found that protease SH21 was able to disrupt the bacterial cell membrane as the results of the nucleotide leakage and cell membrane permeability assay. In addition, we also checked inner membrane permeability by PI uptake assay which suggested that protease SH21 has the ability to enter the bacterial cell membrane. Our results revealed that the antimicrobial protease SH21 might be a promising candidate for treating microbial infections.

Nowadays, the abuse of antibiotics has led to the rise of multi-drug-resistant bacteria. Antimicrobial peptides (AMPs), with broad-spectrum antimicrobial activity have attracted considerable attention as possible alternatives to traditional antibiotics. In this work, we aimed to evaluate the antimicrobial and anti-biofilm activity of an antimicrobial peptide designed as YS12 derived from Bacillus velezensis CBSYS12. The strain CBSYS12 was isolated from Korean food kimchi and purified followed by ultrafiltration and sequential chromatographic methodology. Hereafter, Tricine SDS-PAGE revealed a single protein band of around 3.3 kDa that was further confirmed in situ inhibitory activity of the gel. A similar molecular weight (~ 3348.4 Da) protein also appeared in MALDI-TOF confirming the purity and homogeneity of peptide YS12. Intriguingly, YS12 revealed a strong antimicrobial activity with a minimum inhibitory concentration (MIC) value ranging from 6 to 12 μg/ml for both Gram-positive and Gram-negative bacteria, such as E. coli, P. aeruginosa, MRSA 4–5, VRE 82, and M. smegmatis. We also determined the mode of action of the peptide against pathogenic microorganisms using different fluorescent dyes. In addition, the anti-biofilm assay demonstrated that peptide YS12 was able to inhibit biofilm formation around 80% for both bacterial strains E. coli and P. aeruginosa at 80 µg/ml. Notably, YS12 exhibited a greater biofilm eradication activity than commercial antibiotics. In summary, our study proposed that peptide YS12 may be used as a promising therapeutic agent to overcome drug and biofilm-related infections.

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.