Study On The Separation,Purification And Activity Of BT Lipopeptide

Antibacterial peptide is a kind of protein antibiotic produced inside the organism,which is a kind of small molecular peptide with biological activity.It exists widely in nature.Because of its broad-spectrum antibacterial activity,it has been closely concerned in various fields.In recent years,the problem of bacterial drug resistance caused by the abuse of traditional antibiotics has attracted worldwide attention and seriously threatened human health.Therefore,it has become one of the main tasks of many researches to find some new antibiotics with unique antibacterial mechanism as a supplement of traditional antibiotics.And antibacterial peptide has also become a research focus due to its unique antibacterial properties.In this study,a BT(Bacillus thuringiensis)lipopeptide,a kind of antibacterial peptide,was isolated from Texas Bacillus laterosporus.It is composed of many peptides,within which the content of BT1583 is the most abundant.Its sequence is Me2-4-methyl-4-[(E)-2-butenyl]-4,N-methyl-threonine-L-dO-I-V-V-dK-V-dL-K-dY-L-V-CH2 OH.The existence of Ornithine and D type residues inside it illustrated that the peptide was synthesized by non-ribosomal peptide synthetases(NRPS).There are few reports about the separation and purification of BT lipopeptide both at home and abroad.The purpose of this paper is to explore the separation and purification methods of BT lipopeptide and to study its antibacterial and anti-inflammatory activities.In this study,the BT lipopeptide was extracted from the fermentation product of Texas Bacillus laterosporus,and purified by ion exchange chromatography.The structure was identified by high performance liquid chromatography and mass spectrometry.Through the research of the activity of BT lipopeptide,it was found that the MIC value of BT lipopeptide to Gram-positive bacteria was significantly lower than that of Gram-negative bacteria,which indicated that BT lipopeptide had a higher activity to Gram-positive bacteria.It was found that BT lipopeptide could destroy thecell structure of bacteria and cause bacterial death via observing the cell morphology of bacteria with electron microscope.The anti-inflammatory effect of BT lipopeptide was investigated by cell experiment.During the experiment,the cell viability was detected by MTT first.The low,medium and high(2?g/mL ? 6?g/mL ? 10?g/mL)concentration dose and administration time of 18 hours of BT lipopeptide were preliminarily determined.Then,the macrophage RAW264.7 inflammation model induced by LPS was established,and BT lipopeptides of different concentrations were administered.The results showed that BT lipopeptide could inhibit the production of inflammatory cytokines such as tumor necrosis factor ?(TNF-?),interleukin-6(IL-6),nitric oxide(NO).The flow cytometry related to ROS was also investigated.At last,Western blot was employed to study the mechanism of BT lipopeptide on RAW264.7 cells.It was found that BT lipopeptide could activate the nuclear factor NF-? B in the downstream of RAW264.7 cells.The expression of JNK,p38,ERK and other related proteins showed that BT lipopeptide significantly inhibited the phosphorylation of JNK,p38,ERK.In conclusion,the results of this study showed that BT lipopeptide has an obvious antibacterial activity,especially as to Gram-positive bacteria;at the same time,BT lipopeptide has strong neutralization effect on LPS,which can effectively suppress the release of inflammatory cytokines and mediators induced by LPS.The results of Western blot also indicated that BT lipopeptide could significantly decrease the expression of NF-?B and inhibit the phosphorylation of ERK,JNK and p38,which are the main proteins in the MAPK signaling pathway.Based on the above experimental research,we came to the conclusion that as an antibacterial peptide,BT lipopeptide has a significant antibacterial activity and anti-inflammatory effect.