摘要
[目的]运用HPLC初步建立抗菌肽Cecropin-XJ发酵产物指纹图谱的测定方法,将抗菌肽发酵产物进行定性及定量分析。[方法]将基因工程菌扩大培养经纯化后得发酵产物并采用高效液相色谱法测定,Extend-C18色谱柱(4.6 mm×250 mm,5μm),流动相乙腈(A)-三氟乙酸(B)梯度洗脱,检测波长254 nm,柱温25℃,流速1 mL/min,对方法进行精密度、重复性考察,绘制标准曲线估算抗菌肽浓度,以及将色谱结果与抑菌圈大小对比。[结果]建立了抗菌肽发酵产物的HPLC指纹图谱,获得了8个共有峰,8个共有峰的相对保留时间及相对峰面积的RSD均<3%。通过标准曲线y=2E+07x-3E+06,R^(2)=0.994 7,得抗菌肽浓度约0.297 mg/mL。通过测量抑菌圈大小,表明抗菌肽样品活性较高。在一定范围内,各批次发酵产物抑菌圈大小与相应HPLC特征峰峰面积成正比。[结论]11批样品中共有的8个峰作为指纹图谱的共有峰,峰总面积大于总峰面积的90%,8个共有峰的相对保留时间及相对峰面积的RSD均<3%。抗菌肽浓度约0.297 mg/mL。实验结果说明抑菌圈大小与对应特征峰面积成正相关,表明特征峰出峰位置所示物质即为抗菌肽。
[Objective] Using HPLC to preliminarily establish a method for determining the fingerprint of the antimicrobial peptide Cecropin-XJ fermentation product,and perform qualitative and quantitative analysis of the antimicrobial peptide fermentation product.[Method] Enlarged culture of genetically engineered bacteria was purified to obtain fermentation products and determined by high performance liquid chromatography,Extend-C18 chromatographic column(4.6 mm×250 mm,5 μm),mobile phase acetonitrile(A)-trifluoroacetic acid(B) Gradient elution,the detection wavelength is 254 nm,the column temperature is 25 ℃,and the flow rate is 1 mL/min.The precision and repeatability of the method are investigated,a standard curve is drawn to estimate the concentration of antimicrobial peptides,and the chromatographic results are compared with the size of the inhibition zone.[Result] The HPLC fingerprint of the antibacterial peptide fermentation product was established,and 8 common peaks were obtained.The relative retention time and relative peak area RSD of the 8 common peaks were both <3%.Through the standard curve y=2 E+07 x-3 E+06,R~2=0.994 7,the concentration of antimicrobial peptide is about 0.297 mg/mL.By measuring the size of the zone of inhibition,it is shown that the antimicrobial peptide samples have higher activity.Within a certain range,the size of the inhibition zone of each batch of fermentation products is proportional to the peak area of the corresponding HPLC characteristic peak.[Conclusion] The 8 peaks shared by 11 batches of samples were taken as the shared peaks of the fingerprint spectrum,and the total peak area was greater than 90% of the total peak area.The relative retention time of the 8 shared peaks and the RSD of the relative peak area were both less than 3%.The concentration of antimicrobial peptide is about 0.297 mg/mL.The experimental results show that the size of the inhibition zone is positively correlated with the area of the corresponding characteristic peak,indicating that the substance shown in th
作者
傅子卓
魏锟
尹恒
程纯儒
刘忠渊
FU Zi-zhuo;WEI Kun;YIN Heng;CHENG Chun-ru;LIU Zhong-yuan(Sichuan University of Science&Engneering,Zigong 643000,China)
出处
《生物技术》
CAS
2022年第2期164-170,共7页
Biotechnology
基金
四川省应用基础研究项目(2019YJ0459)。
关键词
抗菌肽
发酵产物
高效液相色谱法
指纹图谱
抑菌实验
antimicrobial peptide
fermentation product
high performance liquid chromatography
fingerprint mapping
antibacterial experiments
