Isolation,Purification,Structural Characterization,Activity Evaluation And Biosynthesis Regulation Of Antitumor Lipopeptides From Bacillaceae

Various activities of Bacillaceae-derived lipopeptides（BLP）,such as antitumor,antifungal,antibacterial,anti-inflammatory,antiviral and antiplatelet,have been reported.The families of surfactin,fengycin and iturin in BLP have showed antitumor activity and have the potential to be developed into antitumor agents.However,most of the BLP used in antitumor research are mixtures.It is difficult to synthesize pure BLP from microorganisms,which is mainly limited by the problems of low yield,complex coexisting components and difficult separation and purification.These problems also hinder the development of antitumor research of lipopeptide.Screening strains with high BLP yield,optimizing separation and purification methods,and regulating biosynthesis of BLP will effectively solve the above problems.With Bacillus strains in red vinasse,Chinese sauerkraut,plants rhizosphere soil and livestock manure as the starting strains,the lipopeptide was extracted by acid precipitation and alcohol extraction from the broth of the strains.And the composition of lipopeptide was analyzed by LC-ESI-MS.11 strains with strong ability to produce lipopeptide(lipopeptide yield>0.1 mg·m L^-1)were screened.The lipopeptides produced by these strains were mixture of iturin,fengycin and surfactin.MTT assay was used to evaluate inhibitory activities of lipopeptide peptides from 11 strains on breast cancer cell BT474.Combined with the ability of lipopeptide production,strain T701 with the highest tumor inhibitory activity and strong lipopeptide production ability was screened,and the strain was identified as Bacillus velezensis.Lipopeptide expression gene analysis showed that strain T701 had the ability to produce iturin and fengycin.It was found for the first time that B.velezensis strain could produce antitumor lipopeptide which could effectively inhibit the growth of cancer cells.To prepare pure antitumor lipopeptide more effectively,the yields of lipopeptide was improved by optimizing acid precipitation p H,acid precipitation time,centrifugation time,extraction temperature,methanol addition and extraction time.Then,the conditions for preparing lipopeptides by preparative liquid chromatography were established by investigating the effects of sample concentration,injection volume,mobile phase flow rate and elution gradient on the separation results of extracellular lipopeptides of strain T701.When the acid precipitation p H was 2,the acid precipitation time was 14 h,the centrifugation time was 25 min under 7000×g,the amount of methanol was 20 m L·g^-1,the extraction temperature was 50℃and the extraction time was 120 min,the corresponding yields of lipopeptides were 1189.89 mg·L^-1.When concentrating lipopeptide,the solvent could be effectively removed by the rotary evaporation method combined with the freeze-drying method to obtain lipopeptide solid.The best separation effect could be obtained when the lipopeptide sample concentration was 125 mg·m L^-1,the injection volume was 300μL,the flow rate 15 m L·min^-1,and the elution gradient was as follow:0～3 min,30%～40%A（0.1%TFA acetonitrile solution）;3～5 min,40%A;5～7 min,40%～60%A;7～9 min,60%A;9～10min,60%～70%A;10～12 min,70%A;12～17 min,70%～80%A;17～20 min,80%A;20～21min,80%～100%A;21～30 min,100%A.A total of 15 components were collected.The inhibitory activities of these components on BT474 cells were determined by MTT assay.The results showed that component A had the highest antitumor activity.After separation and purification by preparative chromatography,129 mg of component A was finally obtained,and its purity was more than 99.5%.The preparation method of component A by preparative liquid chromatography system was established and optimized,and the milligram scale preparation of component A was realized.The molecular structure of component A was characterized by HRMS,MS/MS,¹H-NMR,¹³C-NMR,DEPT 135°NMR and HSQC-DEPT.And the antitumor activity of component A was determined using cervical cancer cell He La,breast cancer cell MCF-7 and BT474,human gastric cancer cell NCI-N87 and MGC 803 as subjects.The results of HRMS showed that the[M+Na]⁺value of component A was 1065.5435 and the molecular formula was C₄₈H₇₄N₁₂O₁₄.The results of MS/MS suggested that the peptide sequence of component A was Asn-Tyr-Asn-Gln-Pro-Asn-Ser.Combined with the results of ¹³C-NMR,DEPT 135°NMR and HSQC-DEPT,component A was determined as n-C₁₄iturin A-2.The IC₅₀ values of iturin A-2 on the above five tumor cells were（66.8±2.9）,（77.5±3.5）,（95.0±2.0）,（51.7±5.7）and（41.2±4.3）μg m L^-1,respectively.It was reported for the first time that iturin A-2had good inhibitory activity against tumor cells MCF-7,He La,BT474,NCI-N87 and MGC803.It is necessary to obtain a large number of iturin A-2 to meet the needs of animal experiments to study the antitumor mechanism of iturin A-2.To increase the yield of iturin A,especially the proportion of iturin A-2 in iturin A,by regulating and controling carbon source,nitrogen source,inorganic salt,fermentation temperature,fermentation time and other factors,the optimum conditions for biosynthesis of iturin A-2 by strain T701 were acheived.The medium composed of sucrose 25 g·L^-1,peptone 10 g·L^-1,sodium chloride 0.5 g·L^-1,ferrous sulfate 0.7 g·L^-1,sodium dihydrogen phosphate 0.2 g·L^-1 and sodium dihydrogen phosphate0.4 g·L^-1 was used.When the initial p H of fermentation was 7.0,the bottling amount was 100m L/250 m L,the inoculation amount was 10%,the fermentation temperature was 35℃,the agitation of the shaking table was 180 r/min and the fermentation time was 96 h,the yield of iturin A-2 and iturin A were 1055.6 mg·L^-1and 1317.9 mg·L^-1,respectively.Under the optimal fermentation conditions,the yield ratio of iturin A-2 to iturin A was 80.1%,which is conducive to the downstream separation and purification.In this paper,the antitumor lipopeptides isolated from Bacillus were studied.B.velezensis strain T701 with high antitumor lipopeptides was screened.A method for the isolation and preparation of iturin A-2 in milligram scale from the extracellular lipopeptides of strain T701 was established.The inhibitory activity of iturin A-2 against five kinds of tumor cells was reported for the first time.The regulation of the biosynthesis of iturin A-2was conducted.This study laid theoretical foundation for scale-up production of iturin A-2,and further antitumor research of iturin A-2.