多黏类芽孢杆菌抗菌肽的分离纯化及特性研究

试验旨在分离纯化多黏类芽孢杆菌（Paenibacillus polymyxa）BLCC1-0402代谢产物中的抗菌肽，为抗菌肽制备及其制品检测提供参考。采用离心、不同分子质量卷式膜超滤浓缩、Superdex peptide 10/300GL凝胶过滤层析对多黏类芽孢杆菌发酵上清液进行逐级分离纯化，对不同时段的收集液做抑菌试验，以大肠杆菌O78标准菌株为指示菌，采用打孔法进行抑菌活性检测，比较评价分步层析效果，以Tricine-SDS-PAGE进行分子质量检测。结果显示，通过5和3 ku卷式膜超滤获得的3~5 ku组分蛋白质样品抗菌活性较强；对于3~5 ku组分经凝胶过滤层析分离纯化，纯化后的抗菌肽A3抑菌活性最强，经Tricine-SDS-PAGE小分子多肽电泳检测，已达到电泳纯，分子质量为4 ku；抑菌活性检测结果显示，该抗菌肽A3对大肠杆菌O78标准菌株具有较强的抑菌作用。同时，抗菌肽A3表现出较好的耐热性，90~100 ℃处理15 min，抑菌活性可保持在96%左右；具有较好的酸碱稳定性，在pH 2.0~9.0下，抑菌活性保持在90%以上；经胃蛋白酶作用后抗菌肽A3抑菌活性降低20%，胰蛋白酶作用后抗菌肽A3抑菌活性降低18%，蛋白酶K对抗菌肽A3的抑菌活性几乎无影响。本研究结果表明，分离得到的抗菌肽A3是一种对大肠杆菌O78具有抑菌活性的新型抗菌肽，具有一定的开发潜力，可为下一步抗菌肽的结构分析、理化特性分析等深入研究提供一定参考依据。

Separation,Purification and Characteristics Analysis of Antibacterial Peptides from Paenibacillus polymyxa

The purpose of the study was to sepatate and purify an antimicrobial peptides from the metabolites of Paenibacillus polymyxa BLCC1-0402, and to provide a reference for the preparation of antimicrobial peptides and the detection of their products.Centrifugation, membrane filtration with different molecular weight, ultrafiltration and Superdex peptide 10/300GL gel filtration chromatography were used to separate and purify the fermentation supernatant of Bacillus polymyxa.Bacteriostatic test was carried out on the collected liquid in different periods to compare and evaluate the effect of step chromatography.The standard strain of Escherichia coli O78 was used as the indicator, and the antibacterial activity was detected by the agar diffusion method.Tricine-SDS-PAGE was used to detect the molecular weight.The results showed that, the 3-5 ku component protein samples obtained by 5 and 3 ku roll membrane ultrafiltration had strong antibacterial activity.The 3-5 ku fraction was purified by gel filtration chromatography.The purified antibacterial peptide A3 had the strongest antibacterial activity.The Tricine-SDS-PAGE small molecule peptide electrophoresis showed that the antibacterial peptide A3 had reached the electrophoresis purity and the molecular weight was 4 ku.The antibacterial activity test showed that the antimicrobial peptide had antibacterial effect on the standard strain of Escherichia coli O78.At the same time, antimicrobial peptide A3 showed good heat resistance, and its antibacterial activity could be maintained at about 96% when treated at 90-100 ℃ for 15 min.It had a good acid-base stability, and its antibacterial activity remains above 90% at pH 2.0-9.0.After pepsin treatment, the antibacterial activity of antimicrobial peptide A3 decreased by 20%, and trypsin treatment, the antibacterial activity of antimicrobial peptide A3 decreased by 18%.Protease K had almost no effect on the antibacterial activity of antimicrobial peptide A3.The results showed that the isolated antimicrobial peptide A3 was a new antimicrobial peptide with antibacterial activity against Escherichia coli O78, which had a certain development potential, and provided a certain reference for the further study of antimicrobial peptide structure analysis and physicochemical properties analysis.