Department of Pharmacy, College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea.
Int J Mol Sci. 2023 Mar 17;24(6):5774. doi: 10.3390/ijms24065774.
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 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 and V values of 0.197 mg/mL and 1.22 × 10 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.
蛋白酶是参与多种重要生理功能的重要酶，在工业应用中有很大的潜在用途。在这项工作中，我们报道了一种由 CSB55 产生的、具有去污稳定性、抗菌和抗生物膜潜力的蛋白酶（SH21）的纯化和生化特性。通过硫酸铵沉淀（40-80%）、Sepharose CL-6B 和 Sephadex G-75 柱层析，SH21 被纯化以获得均一性。通过 SDS-PAGE 和酶谱分析，确定其分子量约为 25 kDa。该酶的活性在 PMSF 和 DFP 的存在下几乎完全被抑制，这表明它是丝氨酸蛋白酶家族的一员。SH21 在广泛的 pH 和温度范围内表现出出色的活性，其最适 pH 为 9.0，最适温度为 55°C。该酶的 K 和 V 值分别估计为 0.197 mg/mL 和 1.22×10 U/mg。此外，它在存在不同有机溶剂、表面活性剂和其他试剂的情况下保持良好的活性。该酶表现出良好的抗菌活性，通过 MIC 对几种病原菌进行评估。此外，它表现出较强的抗生物膜活性，通过 MBIC 和 MBEC 测定和共聚焦显微镜研究分析表明其能够降解生物膜。这些特性表明 SH21 是一种潜在的碱性蛋白酶，可用于工业和治疗应用。
