Department of Biochemistry, Jain (Deemed To-Be) University, 18/3, 3rd Block, 9th Main Rd, Jayanagar, Bangalore, Karnataka, 560011, India.
Arch Microbiol. 2021 Jul;203(5):1995-2006. doi: 10.1007/s00203-020-02173-7. Epub 2021 Feb 5.
A bacteriocin from Bacillus subtilis (MK733983) originated from ethnomedicinal plant was purified using Preparative RP-HPLC. The HPLC fraction eluted with 65% acetonitrile showed the highest antimicrobial activity with Mycobacterium smegmatis as an indicator. Its specific activity and purification fold increased by 70.5% and 44%, respectively, compared to the crude bacteriocin. The bacteriocin showed stability over a wide range of pH (3.0-8.0) and preservation (- 20 °C and 4 °C), also thermal stability up to 80 °C for 20 min. Its proteinaceous nature was confirmed with complete loss of activity on its treatment with Trypsin, Proteinase K, and α-Chymotrypsin. Nevertheless, the bacteriocin retained up to 45% activity with Papainase treatment and was unaffected by salivary Amylase. It maintained ~ 95% activity on UV exposure up to 3 h and its activity was augmented by ethyl alcohol and metal ions like Fe and Mn. Most of the common organic solvents, general surfactants, preservatives, and detergents like Sulfobetaine-14, Deoxy-cholic-acid did not affect the bacteriocin's action. Its molecular weight was estimated to be 3.4KDa by LC-ESI-MS/MS analysis. The bacteriocin is non-hemolytic and exhibited a broad inhibition spectrum with standard strains of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Chromobacterium violaceum with MICs ranging 0.225 ± 0.02-0.55 ± 0.05 mg/mL. Scanning Electron Microscopy showed cell annihilation with pores in cell membranes of S. aureus and P. aeruginosa treated with the bacteriocin, implicating bactericidal mode of action. These promising results suggest that the bacteriocin is significant and has wide-ranging application prospects.
枯草芽孢杆菌（MK733983）来源的细菌素来源于民族药用植物，使用制备型反相 HPLC 进行纯化。用 65%乙腈洗脱的 HPLC 馏分对耻垢分枝杆菌显示出最高的抗菌活性。与粗细菌素相比，其比活性和纯化倍数分别提高了 70.5%和 44%。该细菌素在 pH 值（3.0-8.0）和保存（-20°C 和 4°C）范围广泛，以及热稳定性高达 80°C 20min 内保持稳定。用胰蛋白酶、蛋白酶 K 和α-糜蛋白酶处理完全丧失其活性，证明其为蛋白质性质。然而，细菌素在木瓜蛋白酶处理下保留了高达 45%的活性，不受唾液淀粉酶的影响。在暴露于 UV 下长达 3 小时内，其活性保持在 95%左右，并且乙基酒精和 Fe 和 Mn 等金属离子可增强其活性。大多数常见的有机溶剂、一般表面活性剂、防腐剂和清洁剂，如磺基甜菜碱-14、脱氧胆酸，均不会影响细菌素的作用。其分子量通过 LC-ESI-MS/MS 分析估计为 3.4KDa。该细菌素无溶血作用，对金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌、大肠杆菌和紫色色杆菌等标准菌株的抑制谱较宽，MIC 值范围为 0.225±0.02-0.55±0.05mg/mL。扫描电子显微镜显示，用细菌素处理的金黄色葡萄球菌和铜绿假单胞菌的细胞膜上出现孔，导致细胞死亡，表明其具有杀菌作用。这些有希望的结果表明，该细菌素具有重要意义，并且具有广泛的应用前景。
