针对1株分离自甘肃河西盐碱土壤的具有产抗菌肽潜质的枯草芽孢杆菌B3菌株(Bacillus subtilis B3)，拟构建一种有效的抗菌肽分离纯化方法，旨在为获得该抗菌肽纯品及其结构信息，并为抗菌肽制备及其制品检测提供方法参考。采用由阳离子交换层析、疏水层析和尺寸排阻层析单元组成的串联柱层析系统对B3菌株液态发酵产物进行初步分离，对不同时段的收集液做抑菌实验，比较评价分步层析效果；再通过反相HPLC进一步分离纯化后用质谱分析检测，以获得具有抑菌活性多肽的分子质量及结构信息。经串联层析法分离的具有抑菌活物质是一种小分子富脯氨酸抗菌肽，其氨基酸序列Pro-Tyr-Ile-Pro-Gln-Pro-Arg-Pro-Pro-His-Pro-Arg-Leu，分子质量为1 567.86 Da，等电点为10.84。经美国国立生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库比对，发现其为一种未报道的多肽。经抑菌活性检测表明，该抗菌肽对大肠杆菌 (Escherichia coli)和沙门氏杆菌(Salmonella enterica subsp. enterica)均有抑菌作用，其最小抑菌质量浓度均为0.020 312 μg/mL。同时，该抗菌肽表现出较强的耐热性、耐酸碱性和光照稳定性，是一种对供试靶标菌具有抑菌活性的新抗菌肽。
As a kind of potential antibiotic alternatives, antimicrobial peptides possesses great development potential. In this paper, an effective method for extracting and purifying antimicrobial peptide from a Bacillus subtilis B3 strain isolated from the saline soil of Gansu was established to provide a reference for the preparation of antimicrobial peptides and analysis of product. In this experiment, a chromatography system composed of cation exchange chromatography, hydrophobic chromatography and size exclusion chromatography unit was used to extract antibacterial substance from the fermentation broth of Bacillus subtilis B3. The antibacterial experiments were carried out to evaluate the effect of step-by-step chromatography, and then the antibacterial active peptide was further purified by RP-HPLC and analyzed by ABI-4800 chromatography to obtain the molecular weight and structure information. The antimicrobial active substance isolated by tandem chromatography was a kind of small proline-rich antimicrobial peptide. The amino acid sequence was Pro-Tyr-Ile-Pro-Gln-Pro-Arg-Pro-Pro-His-Pro-Arg-Leu, its molecular weight was 1 567.86 Da, and its isoelectric point was 10.84. By NCBI database comparison, it was found to be an unreported peptide. It was proved that this peptide had good antibacterial activity against Escherichia coli and Salmonella enterica subsp. eterica, and the minimum inhibitory concentration was 0.020 312 μg/mL. Meanwhile, the antibacterial peptide exhibited strong heat resistance, acid and alkali resistance and light stability. We have obtained a novel peptide with antibacterial activity against Escherichia coli and Salmonella enterica subsp. eterica from Bacillus subtilis B3 strain.
